In silico model of infection of a CD4(+) T-cell by a human immunodeficiency type 1 virus, and a mini-review on its molecular pathophysiology
IIn silico model of infection of a CD4(+) T-cellby a human immunodeficiency type 1 virus, and amini-review on its molecular pathophysiology.
Vivanco-Lira, A. ([email protected]) and Nieto-Saucedo,J.R. Department of Medicine and Nutrition, University of Guanajuato,Leon, Mexico. Department of Exact Sciences and Engineering, Open andDistance Learning University of Mexico, Mexico City, Mexico.Submitted on February 2021
AbstractIntroduction . Infection by the Human Immunodeficiency Virus canbe defined as a chronic viral infection which mainly affects T-cells; thisvirus displays a set of both structural and regulatory proteins that aid inits survival within the host’s cell, through these proteins, can the virusalter the host’s gene expression pattern and with it, signaling involvedin cell cycle control, cytokine response, differentiation, metabolism, andothers; therefore by hijacking the host’s genetic machinery there exists apromotion in viral fitness, and the ground is cemented for changes in thehost’s cell differentiation status to occur.
Methods . We will considertwo stochastic Markov chain models, one which will describe the T-helpercell differentiation process, and another one describing that process of in-fection of the T-helper cell by the virus; in these Markov chains, we willconsider a set of states { X t } comprised of those proteins involved in eachof the processes and their interactions (either differentiation or infectionof the cell), such that we will obtain two stochastic transition matrices( A, B ), one for each process; afterwards, the computation of their eigen-values shall be performed, in which, should the eigenvalue λ i = 1 exist,the computation for the equilibrium distribution π n will be obtained foreach of the matrices, which will inform us on the trends of interactionsamongst the proteins in the long-term. Results . The stochastic pro-cesses considered possess an equilibrium distribution, when reaching theirequilibrium distribution, there exists an increase in their informationalentropy, and their log-rank distributions can be modeled as discrete betageneralized distributions (DGBD).
Discussion . The equilibrium distri-butions of both process can be regarded as states in which the cell is well-differentiated, ergo there exists an induction of a novel HIV-dependent a r X i v : . [ q - b i o . M N ] F e b ifferentiated state in the T-cell; these processes due to their DGBD dis-tribution can be considered complex processes; due to the increasing en-tropy, the equilibrium states are stable ones. Conclusion.
The HIV viruscan promote a novel differentiated state in the T-cell, which can give ac-count for clinical features seen in patients (decrease in na¨ıve T cell counts,and the general T-cell count decline); this model, notwithstanding doesnot give account of YES/NO logical switches involved in the regulatorynetworks.
Contents
N C ∈ gag ): viral genome package andfacilitator. . . . . . . . . . . . . . . . . . . . . . . . . . . . 101.2.4 p6 ( p ∈ gag ): incorporating vpr into salient viral particles. 111.2.5 p24 (capsid protein, CA ∈ gag ): protector of the viralgenome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111.2.6 p17 (matrix protein, M A ∈ gag ): multifunctional protein. 131.2.7 p10 (protease, P R ∈ P OL ): catalyzer of viral and host’sproteins breakdown. . . . . . . . . . . . . . . . . . . . . . 141.2.8 p51 (reverse transcriptase, RT ∈ P OL ): loader of nucleicacids and structural support. . . . . . . . . . . . . . . . . 171.2.9 p15 (RNase H, ( p ∨ p ∈ pol ): converter of RNA intodsDNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181.2.10 p32 (Integrase, IN ∈ pol ): immersing viral DNA into thehost’s genome. . . . . . . . . . . . . . . . . . . . . . . . . 201.2.11 vpr (virus protein r): nuclear importer of viral genome. . 211.2.12 vpu (virus protein unique, p16): CD4 downregulator. . . 221.2.13 nef (negative regulating factor, p27): downregulator ofhost’s proteins. . . . . . . . . . . . . . . . . . . . . . . . . 241.2.14 tat (transactivator protein, p14): promoting viral genomeexpression. . . . . . . . . . . . . . . . . . . . . . . . . . . 251.2.15 rev (RNA splicing regulator, p19): exporter of viral RNA. 261.2.16 vif (viral infectivity protein, p23): evading APOBEC3Gresponse. . . . . . . . . . . . . . . . . . . . . . . . . . . . 271.2.17 tev (tat/env/rev protein, p26): fusion protein. . . . . . . 281.3 T-cell differentiation. . . . . . . . . . . . . . . . . . . . . . . . . . 281.4 Role of TOX in thymocyte differentiation . . . . . . . . . . . . . 292 Materials and methods. 29 in vivo pro-cesses of T-cell differentiation, and HIV-cellular infection. . . . . 353.4 Evolution of distributions until equilibrium. . . . . . . . . . . . . 363.5 The Th-cell differentiation and the HIV-infection processes tendtowards an increasing entropy, S ≤ S ≤ ... ≤ S n when reachingits equilibrium distribution. . . . . . . . . . . . . . . . . . . . . 36 π n . 394.2 Th-differentiation, and HIV-infection processes as martingales. . 404.3 Th-cell differentiation, and HIV-infection equilibrium distribu-tions as discrete generalized beta ones. . . . . . . . . . . . . . . . 41 Human Immunodeficiency Virus infection and the spectrum of related disordersare relatively new to the human being, with the first known displays of thisdisease occurring in the decade of the 1980s, however, efforts were made todetermine a more precise temporal origin of the virus, with some estimatescomputing that it appeared as early as in the 1920s decade [311]; while it becameclearer a bit later that HIV may have originated by means of zoonoses fromprimates in Africa [252]. During the first-ever recorded outbreak in the 1980s,the epidemic was said to be limited to the population of the 4 Hs: homosexuals,haemophiliacs, haitians, heroin addicts [88] nevertheless, was this soon proved tobe a far too narrow assumption, and that the actual boundaries of the infectionwere not these, for the set of susceptible population was indeed a larger one.HIV was later discovered to exhibit two basic genotypes: HIV-1 and HIV-2 withdifferences described both in the clinical outcome and course of the disease [236].HIV is known to exhibit affinity for the receptors in T cells CD4, CCR5, CXCR4,as well as other cofactors necessary for entry into the cell [302], then using thesecells for replication, inducing later apoptosis in these ones, this apoptosis inthe CD4 (+) T cells (and as well in CD8(+) T cells) is responsible for theimmunodeficiency seen in the HIV(+) patients, in which, when untreated, may3esult in the development of opportunistic infections, including infection byhuman herpesvirus type 8, being able this virus to promote the establishmentof a malignancy, say Kaposi’s sarcoma.
Both HIV-1 and HIV-2 are species belonging to the
Lentivirus genus whichcontains other notable viruses, such as
Simian immunodeficiency virus , Felineimmunodeficiency virus and others; this genus belongs itself to the subfamily
Orthoretrovirinae , part of the
Retroviridae family which also contains the sub-family
Spumaretrovirinae in which we may find those endogenous retroviruseswhich have incorporated into the host’s genome and may take part in the geneticdiversification. The
Retroviridae family is part of those reverse transcribingRNA and DNA viruses [262]. The viral particle is about 100 nm in diameter,with an outer membrane as envelope, this envelope contains 72 knobs, each knobcomprised by trimers of Env proteins, the Env protein possesses in its structurethe gp120 and the gp41 (gp160) proteins in its stead, where the gp41 anchors tothe lipid membrane and gp120 binds to gp41 finally protruding from the mem-brane. In its envelope, the viral particle also contains MHC pertaining to thehost. Within the particle, we find another membrane, comprised by the matrix(p17) and the protease (p10) proteins; attached to the matrix protein membranewe find the lateral bodies, which are polarized bodies inside the particle. Finally,the capsid lies in the core of the viral particle, the shell being composed of thep24 protein (capsid protein) and attached to the matrix protein by means of thep6 protein (link protein). In the innards of the capsid lies the genome, wherethe reverse transcriptase complex is found attached to the genome, along withthe nucleic acid binding protein (p7) [1]. The viral particle’s bilayer is enrichedin specific lipids: aminophospholipids, dyhydrosphingomyelin, plasmenyl phos-phoethanolamine, phosphatidylserine, phosphatidylethanolamine and phospho-inositides; being the lipid composition bearers of importance in HIV fusion andinfectivity (chol-chelating compounds inhibit these actions), interaction withT-cell immunoglobulin and mucin domain proteins block release of HIV frominfected cells, interaction with bavituximab (which targets phosphatidylserine)suppresses productive HIV infection [111]. The clues leading to the thought ofHIV being a retrovirus came when in the 1980s outbreak, HIV-2 was shown toalso cause AIDS in patients, HIV-2 was related to HIV-1 but also to a simianvirus which caused immunodeficiency in macaques. It has been determined thatthe SIV from the Sooty mangabey originated the HIV-2 (groups A-H), while theSIV from the chimpanzee originated both the HIV-1 (groups M, N and probablyO) as well as the SIV from Western gorillas, which in turn gave place to HIV-1(groups P and probably O) [252]; it has been proposed as well that HIV origi-nated from SIV by means of several genetic mutation transitions and that theuse of unsterile injections may have promoted the increase in transmission [161],other mechanisms have been proposed, such as: population growth, changingsexual practices, migration, increased hunting and deforestation in post-colonialAfrica [196]; however, retroviruses have been computed to appear as far back4s 460 to 550 million years ago (during the Palaeozoic Era), along with theradiation of
Vertebrata , nevertheless it remains unclear whether
Retroviridae originated from
Metaviridae or whether
Retroviridae gave place to
Metaviridae [106]. HIV-1 displays three major groups: major (M) with a transmission eventoccurring between 1912 and 1941, outlier (O), and nonmajor and nonoutlier (N);the virus may have commenced with the HIV-1 O group in gorillas, then spreadamong humans from the Congo River into Kinshasa, Zaire, with the earliestdocumented case of HIV-1 infection (M group) in humans dating from 1959.The M group is the predominant circulating group, and it has been divided intosubtypes: A1, A2, A3, A4, B, C, D, F1, F2, F, H, J, K; further recognition ofsubtypes by means of full-genome sequencing has been made (CRFs and URFs)[274]. Meanwhile, HIV-2 displays A-H grups, with the A group possessing twosubdivisions: A1 and A2 [1]. The life of those patients infected with HIV gavea very important turn with the advent of the antiretroviral drugs, and has ap-proached the amount of years to be lived at age 20 years two thirds that ofthe general population [49], other measurements have been made estimatingexpected average ages of death in those patients receiving antiretroviral ther-apy, finding 67.6 years for men and 67.9 years for women [50]. In addition, themutation rate of the HIV-1 genome in vivo has been shown to be rather high,i.e., 4 . ± . × − per base per cell, this rate is contributed 2% by the reversetranscriptase and 98% by the host’s cytidine deaminases of the A3 family [56]. gp120 owes its name to its molecular weight ranging from 110 to 120 kDa[55], being this protein a portion of the gp160 complexes (due to the fact thatgp120 matures from a larger, gp160 peptide), the gp120 associates with thegp41 molecule assemblying into a heterodimer, which will trimerize to form themature Env protein, in its turn bound to the viral bilayer membrane. Theprotein is comprised by five variable regions and five conserved regions, fold-ing into a globular structure with an inner and an outer domain bound by abridging sheet. gp120 is glycated mostly by high-mannose glycans, and com-plex glycans fucosylated and containing multiple antennas, along with a vari-able amount of sialic acid ; the high-mannose oligosaccharides were found to be( GlcN Ac ) ( M an ) and ( GlcN Ac ) ( M an ) [218]; deficiencies in glycosylation(as they have been induced in bacteria-produced gp120) result in unsuccess-ful binding of gp120 to CD4 [78]. gp120 binds to CD4 by means of a pocketfound above the bridging sheet [313], this pocket is known to be hydrophobic ingp120 and when bound to CD4 a phenylalanine residue caps this pocket, thisresidue has proved essential for a successful binding, the whole complex (gp120hydrophobic pocket + phenylalanine residue) is termed the Phe43 cavity [65];moreover, the affinity with which CD4 interacts with gp120 has been determined5o be K d = 4 × − M [275] while the entropy of this process has been seento be ∆ S = 220 ± kJmol − and an enthalpy of ∆ H = − ± kJmol − (at 310 K) (in which we find that this binding of CD4 to gp120 is an irre-versible (due to ∆ S >
0) and spontaneous (because ∆
G <
0, considering ∆ G as the free energy) process [157]) with an intermolecular hydrogen-bond net-work of 166 atoms (for CD4) and 130 atoms (for gp120) [109]. The bindingof gp120 to CD4 induces conformational changes in gp120, and promotes thecreation of a high-affinity binding site for CCR5, as well as the exposure ofgp41 in order to induce membrane fusion; furthermore, has it been shown thatthe binding of the soluble form of CD4 (sCD4) to gp120 promotes the dissoci-ation of gp120 from gp41, and some variable loops (V1, V2, V3) change theirconformation or become more more exposed; the exposure of these loops (V1and V2) could be potentially recognized by the monoclonal antibodies 17b and48d (this nomenclature is due to the epitopes exposed when the conformationalchanges are induced) [264], where the monoclonal antibody 17b is an anti-HIV-1gp120 monoclonal antibody which is obtained from EBV transformation of Bcells (let us recall that these transformed B cells are those infected by EBV inwhich EBNA1, EBER1 and EBER2 are expressed, inducing the translocation ofMYC into the immunoglobulin loci, activating permanently this transcriptionfactor [134]) of an asymptomatic HIV-1 infected individual [226]; however, dueto steric hindrance and conformational masking, these epitopes remain obscuredor unaccesible to the antibodies directed against gp120, therefore, by means of achimeric protein comprised of a soluble CD4 bound to the monoclonal antibodydirected towards 17b, the necessary conformational change occurs and the epi-topes are exposed, method through which, the monoclonal antibody may thenbind to the epitope [136]; nevertheless, there exist also entropic barriers in thegp120 core which may decrease the potential binding of antibodies [217]. Thebinding of CD4 to the heterohexamer (( gp ( gp ) happens in a stoichiom-etry of ( CD ( gp ( gp this because two CD4 molecules may be as closeas 19 . nm in the cell membrane context, that is, the root of the CD4 moleculesfor these molecules protrude from the cell membrane and be closer in the extra-cellular space, the maximum distance at which they can be approached one fromthe other has been measured to be 3 . nm in the extracellular space and withoutsteric interference [135]. After the binding of CD4 to gp120, we have describedthat there exist some conformational changes which promote the interaction ofCCR5 with gp120 (used in early infection) and with CXCR4 (in late infection),it has been displayed that in the absence or changes in sequence of the loops V1and V2, there could be a loss in the ability of gp120 of binding to CD4, then be-ing able to infect CD4(-) cells with CCR5(+) or CXCR4(+); the conformationalchanges of gp120 by means of CD4 diminish the entropy of the protein [217];this binding of gp120 to the chemokines happens through the V3 loop which ispredominantly composed of 35 residues, connected by a disulfide bridge betweenresidues 1 and 35, being those residues essential for the binding with the core-ceptors those from 13 to 21, additionally, gp120 binds with near equal energeticproperties to CCR5 and CXCR4 [269]; the binding of CD4 to gp120 induces adiminishment in the distance of the trimer to the target cell membrane by 2 nm p ∈ [0 . , . Igλ in immatureB cells; mature B cell survival and population restoration is dependent on theNFKB canonical pathway [241]. gp120 also exerts actions down- and upregu-lating cell cycle- or transcriptional regulation-related genes in T cells (as seenwhen T cells are treated with the V3 loop of gp120), some of these upregu-lated genes are: NOC2L (inhibitor of a histone acetyltransferase independent ofHDAC, which is seen upregulated) [173], SEPT9 (member of a family of GTP-binding proteins, which exhibit roles in cytokinesis, cytoskeleton, cell cycle con-trol; whose hypermethylation is related to several cancers [254]), IFI6, SPIN,HNRNPM; while some other downregulated genes are: ABCG1, PGPEP1, PT-PLA, SPATA21; overall, the V3 loop of gp120 affects the following sets of geneswithin T cells: cell cycle (62 genes), cellular development (function, mainte-nance, compromise, morphology) (54 genes), aminoacid or lipid metabolism (36genes), gene expression (65 genes), DNA metabolism (replication, recombina-tion, repair) (27 genes), cellular assembly and organization (30 genes), cellularmovement (25 genes), hematopoiesis (25 genes), immune response (32 genes),cellular death (growth and proliferation) (100 genes), infection mechanism (34genes); therefore we witness a hijacking of the genetic machinery in T cells whichmay compromise by all means their further function and proliferation [173]. Var-iois gp120 inhibitors have been described, such as: BMS-378806, BMS-488043,BMS-626529, BMS-663068, NBD-556, JRC-II-191, and 18A [150], BMS-378806does not display action against HIV-2, SIV, and other viruses, whose metabolismis cythochrome-dependent [310] and thus major drug-drug interactions are ex-pected [294]. BMS-488043 is an analog of the BMS-378006 compound and hasbeen seen to display in vivo efficacy against HIV-1 in a monotherapy regime[150]. BMS-626529 or temsavir is administered as a prodrug (BMS-663068),8 methyl phosphate prodrug (fostemsavir) which is hydrolized by an esterase,whose metabolism is equally contributed by the cytochromes [328], the recordedadverse effects include: headache, rash, and micturition urgency [180]; addition-ally, this prodrug has shown a better performance in the inhibition of duallytropic viruses [192]; fostemsavir has very recently been suggested to be used asa drug in multidrug-resistant HIV-1 infection [130].
This protein mediates fusion with the host’s cell membrane [39], portion of theenv protein pertaining to HIV; this protein displays C-terminal helices whichback around N-terminal helices, forming a six-helix bundle [166], this structureorganizes into an extracellular ectodomain, anchoring gp120 to the cell surface,a transmembrane domain (promoting further anchoring to the lipid bilayer),and a cytoplasmic tail, this tail contains a highly conserved endocytosis motif,and three alpha helical motifs known as lentiviral lytic peptides [80]; where thetransmembrane region of gp41 is followed by a region of high hydrophilicity (thecytoplasmic domain), containing a highly immunogenic region and a C-terminalregion with two amphipathic segments (the lentiviral lytic peptides , LLPs), aswell as a leucine zipper motif between LLP2 and LLP1; the cytoplasmic domainof gp41 has been suggested to confer conformational stability to the Env pro-tein, but it has been determined that the cytoplasmic domain interacts with hostproteins: AP1, AP2, CAM, CTNNA, luman, MA, TP115, perilipin-3, plasmamembrane, PRA1, prohibitin 1/2, TAK1. Furthermore, the cytoplasmic domainhas been proved essential for both replication and incorporation into several celllines, such as: monocyte-derived macrophages, peripheral-blood mononuclearcells, B cells, epithelial carcinoma-derived cells; however, the replication andincorporation processes have proved independence of the cytoplasmic domain ofgp41 in other cell lines, mostly CD4(+) T cells [208]. Furthermore, gp41 fusionprotein works as an inhibitor of T cell activation by different mechanisms. Theimmunosupressive (ISU) sequence impairs T cell activation through the interac-tion with the T cell receptor (TCR) complex and the direct inhibition of proteinkinase C mediated phosphorylation; gp41 fusion peptide inhibits antigen-specificT cell activation by binding to the TCR α transmembrane domain; gp41 trans-membrane domain can enhance the overall immunosupressive effect through aninteraction with the fusion peptide; and the recent described immunosupressiveloop-associated determinant (ISLAD) is another inhibitor of antigen-specific Tcell proliferation and proinflammatory cytokine release by interacting with theTCR α [12].Peptides derived from gp41 N-terminal heptad repeat (NHR) and C-terminalheptad repeat (CHR) sequences can inhibit HIV-1 infection by interaction withtheir counterparts in gp41. Some of these peptide fusion inhibitors are: DP178,later named T20 (generic name: enfuvirtide) was the first fusion inhibitor ap-proved by the U.S. FDA., but the high cost and inconvenience of twice dailyinjection, prevents it from being considered as a regular drug [34]; FB006M(generic name: albuvirtide) was approved in 2018 by the Chinese FDA.; SFT9generic name: sifuvirtide) is a novel and potent gp41 inhibitor that has shownpromising results; other gp41 fusion inhibitors under development and studyare: T1249, 2F5, 4E10, C52L, VIR-576, among others [210]. N C ∈ gag ): viral genome package and facil-itator. This protein derives from the Gag precursor which is cleaved into p17, p24,and p7 proteins [164]. Nucleocapsid protein 7 (NCp7) is the major internalcomponent of the HIV virion core, it has been seen to be highly conserved(however not so in spumaretroviruses), displays RNA-binding properties, con-taining two zinc fingers (ZF) [62], reminding us that these domains are main-tained by a zinc ion, coordinating a cysteine and a histidine molecule, thesemotifs have two β -sheets and one α -helix; it has been seen that these proteinsbind to DNA but may as well bind to RNA [38]. p7 possesses functions in-volved in the selection and packaging of the viral genome, as well as otherswhich play a role in viral replication [164]; NCp7 interacts with the viral RNAand is required for its dimerization, encapsidation, and initiation of its reversetranscription, where NCp7 enhances the reverse transcriptase processivity andRNase H activity [143]; p7 additionally binds to proviral DNA (by means ofits basic residues), and protects it from nuclease digestion [140]. Interestingly,zinc ejection or mutations affecting the zinc finger folding and conformation ofthe nucleocapsid hydrophobic plateau, lead to non-infectious viral particles [94].The importance of its conserved structure is the low probability of mutationsfound in treatment-resistant strains [127]. Thus, NCp7 represents a promisingtherapeutic target for an effective next-generation antiretroviral therapy. NCp7inhibitors are divided into covalent and non-covalent inhibitors. Covalent in-hibitors, also referred to as irreversible inhibitors or zinc ejectors, which canrecognise NCp7 among cellular proteins containing ZFs, some examples of themare 3-Nitrosobenzamides (NOBAs); disulfide-substituted benzamides (DIBAs);thioesters and pyridinioalkanoyl derivatized thioesters (PATEs); benzisothia-zolone (BITAs); azodicarbonamide (ADA); thiocarbamates (TICAs); S-acyl 2-mercaptobenzamides (SAMTs); transition metal complexes; diselenobisbenza-mides (DISeBAs); and more recently, thioether prodrugs. All of these covalentinhibitors are structurally characterised by a weak electrophilic group that isattached by the distal ZF domain, after this covalent complex has been formed,the zinc is ejected, causing the loss of the tertiary protein structure and con-sequently, all of its functions [282, 238]. Non-covalent inhibitors and nucleicacid (NA) binders are another therapeutic target against NCp7. These com-pounds have weaker antiviral potency when compared with covalent binders,and they have not been approved for clinical trials. Several interactions in theNA/NCp7 complexes are involved with the W37 hydrophobic plateau residue,offering a chance to develop competitive inhibitors. Some of these are pseudod-inucleotides, HTS-derived small molecules, thiadiazoles, and thiazolidinones.All of these molecules are characterized by a π -rich area capable of interactingwith W37, seeking to avoid the interaction of NCp7 with NAs. On the other10and, NA-binding NCp7 inhibitors include stem loop structure-binders and an-thraquinones, which block the NCp7-RNA-DNA complex formation, but mostof them are unable to disrupt a preformed complex [117]. p ∈ gag ): incorporating vpr into salient viral particles. This protein, also a byproduct of the Gag precursor cleavage, promotes virusparticle budding, and the incorporation of the vpr protein into the viral particles[122]; inclusively, p6 is a factor involved in the capsid maturation and virus coreformation processes, this by means of its phosphoprotein features; these func-tions are performed due to the close Euclidean distance which is encounteredbetween p6 and the plasma membrane of the cells, this protein may be adsorbedonto the inner surface of the plasma membrane and promote, for instance, theincorporation of vpr into the viral particles. This protein possesses two α -helicaldomains which are connected by a flexible region, this structure is more pro-nounced in hydrophobic conditions; its C-terminal region contains vpr-bindingresidues; the Ser40 residue has been seen to be a potential protein kinase Cphosphorylation site [194]. TSG101 (tumor susceptibility gene 101) is a key cel-lular protein as part of the endosomal sorting complexes required for transport(ESCRT), which is recruited to viral assembly sites via p6, where the Pro-Thr-Ala-Pro (PTAP) motif in p6 acts as a docking site for TSG101. This processis critical for HIV release. The duplication of this PTAP motif has shown anenhanced replication advantage of HIV-1 subtype C by engaging TSG101 witha higher affinity [250]. Furthermore, the deletion of the YPx n L motif, whichbinds to ESCRT component ALG-2-interacting protein X (ALIX), is associatedwith a decrease in virus release from infected cells (as seen in HIV-1 subtypeC), and conversely, PYxE motif insertion can reconstitute the p6 binding toALIX and consequently, viral budding mediated through the ESCRT pathway[68]. Depletion of the ESCRT components have shown a powerful block to HIVparticle release [259], for this reason, therapeutic targets are principally focusedon disrupting the p6-TSG101 interface, such as peptoid hydrazones, cyclic pep-tide 11, F15 (esomeprazole) and N16 (tenatoprazole), these last two are able tojoin to the ubiquitin E2 variant domain of TSG101, highlighting the possibilityof interfering with previously unknown therapeutic targets and expanding thefuture perspectives of TSG101 inhibitors [66]. CA ∈ gag ): protector of the viral genome. Capsid protein (CA) is member of the subset of proteins derived from the Gagpolyprotein cleavage, a [24 , kDa protein which may be detected before sero-conversion, this protein may assemble into a protective shell around the viralRNA [99] which is a spontaneous process [35], this has been described as a”fullerene cone”, hexamers of p24 link into an hexagonal surface lattice, with 12capsid-protein pentamers, finally possessing around 1500 p24 monomers. Thisprotein is comprised by seven α -helices, a β -hairpin (N-terminal region), a C-terminal region with four α -helices, and a flexible linker [321], when assembled11nto the cone, the N-terminal domain is located on the outer surface of the coneand the C-terminal domain is oriented towards its interior; inside this cone, theRNA genome, and the POL proteins are located (that is the integrase, protease,reverse transcriptase and others); the cone may act in order to shield the geneticcontent from a host response [35], such as those induced by STING, DDX14 orIFI16 acting as foreign DNA sensors [289]. Several models have been proposedin regards to the viral uncoating, that is, the dissociation of the capsid coneinto monomers or simpler polymers: immediate uncoating, cytoplasmic uncoat-ing and nuclear pore complex uncoating; there exist studies which support eachof these models, in the first case, the uncoating occurs rapidly after the entryinto the host’s cell has been performed, however, this model has lost its predic-tive power due to the fact that the core provides protection against the host’sforeign-DNA sensors, furthermore, the capsid possesses a pocket to which thereverse transcriptase complex binds to and allows it to be imported into thenucleus. The second model, the one of cytoplasmic uncoating proposes the dis-assembly of the capsid after a certain time interval has been spanned withinthe host’s cell, this model may be nevertheless challenged as well by the factorswhich we have already commented (foreign DNA host’s response), moreoversome additional cellular factors may protect the viral’s genome from the host’sresponse, it may occur by means of the HMGA1 protein (high mobility groupAT-hook, also called HMGIY), which has been recollected from the preintegra-tion complexes, that is the reverse transcriptase complex before they integrateinto the host’s genome [75]. The third model refers to the nuclear pore complex(NPC) uncoating, in which when the intact capsid reaches the nuclear pores,and disassembles in situ, while the reverse transcriptase complex is importedinto the nucleus; this model displays another range of problems which are notcompatible entirely with the experimental data, ergo a more suitable modelshould be provided in following years. Both viral and host’s factors take part inthe uncoating process: PPIA (or CYPA, a peptidylpropyl isomerase A, with anative function of accelerating the folding of proteins and isomerize the prolineimidic bonds [188]), dynein (cytoplasmic trafficking), CPSF6 (nuclear import,which is a cleavage and polyadenylation specific factor, interacts with RNA[186]), TNPO3 (nuclear import, this protein is member of the importin- β fam-ily of proteins, has been seen to bind to the viral integrase, and as well interactswith the capsid protein), NUP358, NUP153 (nuclear import) [35]. Maturationinhibitors are a novel class of antiretroviral drugs targeting the cleavage sitebetween the C-terminal portion of CA and the spacer peptide 1 (SP1), thiscleavage site usually triggers a conformational switch that destabilizes the im-mature Gag and the mature core formation. Maturation inhibitors cause anaccumulation of CA-SP1 precursor, which eventually leads to the loss of viralinfectivity [66]. The first reported maturation inhibitor was Bevirimat (BVM),which causes an abnormal virion morphology and inhibition of viral replication,but failed in the phase IIb trial due to resistance mutations in CA-SP1. A sec-ond compound named PF-46396 was identified, it shows structural differencescompared with BVM, but it induced resistance mutations at different locations,this lead to the identification of second-generation maturation inhibitors, such12s GSK3532795, which successfully overcame the inconvenience of drug resis-tance, but showed a high rate of adverse gastrointestinal events and frequencyof treatment-emergent nucleoside reverse transcriptase inhibitor (NRTI) resis-tance, reason why its evaluation was interrupted [172]. However, the promisingresults obtained support the continued development of drugs against this thera-peutic target. Small molecules and peptide-based antivirals designed to disruptCA-CA interactions in the immature Gag lattice, the mature core, or both havebeen studied during the last years. CAP-1 was the first small molecule developedto target the CA protein, it produced abnormal core morphologies, and conse-quently noninfectious particles. Besides small molecules compounds, 12-merpeptide CA inhibitor (CAI), binds in a hydrophobic CA dimerization interface,but it can not penetrate cell membranes, limiting its clinical use. Some morestable α -helical peptides, such as NYAD-1 and NYAD-13, showed a strongeraffinity for the binding site than CAI. Other classes of CA inhibitors are ben-zodiazepienes and benzimidazoles. PF74 and the pyrrolopyrazolones BI-1 andBI-2, seem to compete with CPSF6 and Nup153 for CA binding, disruptingnuclear import. The main problem of the compounds described so far, is thelow clinical relevance due to their pharmacological characteristics. Recently, anew type of CA inhibitors has been described, this group includes GS-CA1 andits derivative, GS-6207, are promising drugs that have showed higher potencythan PF74 [66]. M A ∈ gag ): multifunctional protein. The HIV-1 p17 protein (matrix protein, MA) is associated with the inner sur-face of the viral envelope, and may primarily function as an anchor of the gp41protein on the virion surface. When in solution it is mainly encountered ina monomeric form, while in a solid state it trimerizes [163]; it displays five α -helices, and a three-strand β -sheet, whereby the C-terminal domain, exposescarboxyl-terminal residues which aid in the early states of HIV infection, and ba-sic residues promote membrane binding and nuclear localization (the residueswhich take part in this process are located in a cationic loop connecting β -strands one and two). This protein additionally functions in RNA targeting tothe plasma membrane, incorporation of the envelope into virions and particleassembly, and aids in the transport of the reverse transcriptase complex throughthe nuclear pores [162], it also acts as a viral cytokine, by binding to a cellularreceptor, namely p17R [83], this receptor has been seen to be expressed in RajiB cells, and activates AP-1, as well as ERK1/2 and downregulates AKT, thisby means of maintaining PTEN in its active state through the serine/threoninkinase ROCK [45]; the identity of this p17R protein is a bit obscured in theliterature, nonetheless through a non-exhaustive search in regards to Raji cells’receptors we came up with a list of potential proteins which may play such role,bearing in mind as well that the H9 cell line lacks this p17R protein in its surface,if we consider that the surface proteins of the Raji cells are members of the set R = { p , ..., p n } and those of the H9 cell line pertain to the set H = { r , ..., r m } ,therefore ∃ p i ∨ r i : p i ∈ R, p i / ∈ H [231]; those proteins in the set R ∩ H are:13VRL1, LILRB1, DRD4, CRLF3, and ADGRD2; such that these receptorsmay not be the p17R protein; now, those proteins which are exclusively ex-pressed in Raji cells and take part in PI3K/AKT/PTEN/AP1/GPC (G-proteincoupling) are: NTSR2, TACR3, FOLR2, MC5R, OR2J3, TNFRS13B, PTAFR,CCR8, GPR173; this sets are shown in Figure 1; the activation of this pathway(by means of this p17R) may lead to a promotion in proliferation and releaseof proinflammatory cytokines from T-cells [83]; but if p17R is also expressedin B cells, it ought indeed to promote B cell growth and tumorigenesis [45].MA protein has a fundamental role in virion assembly, because of its highlyconserved PI[4,5]P /nucleic acid binding site, it has become an attractive sitefor the development of new antiretroviral drugs, nevertheless, other drugs havebeen described targeting the nuclear localization signal of MA or the MA-RNAinteraction. Thiadiazolane based compounds where first described, they targetthe MA-RNA interaction, but showed significant levels of toxicity. In contrast,PI[4,5]P binding site inhibitors were not associated with cytotoxic effects, butwork is actively ongoing in optimizing the affinity/potency of this type of chemo-types. New targeting sites are highly desirable, one of them could be the in-volvement of MA in Env incorporation, where MA trimerization is importantfor the recognition of Env cytoplasmic tail (gp41-CT) and virus assembly [66]. P R ∈ P OL ): catalyzer of viral and host’s pro-teins breakdown.
This protease is encoded by the pol gene being an aspartic protease, and asin other retroviruses, displays its function when in its homodimeric form; eachmonomer is an aspartic peptidase with four elements: two hairpin loops, widecatalytic aspartic acid loop, and an α helix [70], the dimeric interface compriseseight N- and C-terminal residues of each chain, some of these are exposed tothe solvent, and some others (the hydrophobic ones) are oriented towards theinterior of the enzyme [320]. This protein displays two states (when in the ho-modimer form), either open or closed, depending on the presence of a ligand(open when in the free state, and closed when in the bound state) [308] wherethe open form is more stable than the closed one (considering the free energyof the process ∆ G ) [320]. The HIV protease cleaves the polyproteins Gag andGag-Pol creating protein subunits [167]; all the same the viral protease displaysproteolytic abilities of the host’s proteins, such as those belonging to: the cy-toskeleton: vimentin, desmin, GFAP [256], actin, troponin [277], laminin [182];immune system: proIL1B; and transmembrane proteins: APP [277]; cytosolicproteins: BCL2, CASP8 [182]. Vimentin plays a role in the diapedesis pro-cess of T cells, whereby this protein, T cells migrate across the endothelium,and in genotypes vim − / − , there is a diminished capacity of T cells to home tomesenteric lymph nodes and spleen, this may be mediated by changes in ex-pression or distribution of ICAM1 and VCAM1 [183]; while actin polymerizesor depolymerizes when the T cell becomes active, this T cell activation leadstowards the formation of a distal-pole complex which is an actin-rich structure;disruptions in the cytoskeleton might as well induce changes in the organization14igure 1: Venn diagram of the sets representing the receptors expressed in Rajicells (Raji) and H9 cells (H9), where we regard at their cardinalities, that is, | R | = 28, | H | = 68, and R ∩ H = 6; in this case the p17R protein lies in the R − H set. 15f the supramolecular activation clusters in T cells (SMACs) [21] which takesa central part in the immunological synapse, being the SMAC a nanoclustercomprised of an actin mesh, transmembrane proteins (members of the TCRcomplex), and cholesterol-enriched nanodomains [89]; these disruption in thecytoskeletal proteins might be added to the one induced by the gp120 protein,as this protein increases ICAM1 expression [221, 273], inducing the formation ofa type of SMAC but in this case gp120-dependent (and likely protease-aided),in which gp120 clusters in the centre, and LFA1 and ICAM1 concentrate inthe periphery, this type of SMAC is termed the virological synapse, throughoutwhich the virus may spread between T cells without the need of being exter-nalized from the cell (no need of viral budding) [159]; this SMAC recruits thehost’s proteins which we may as well find in the immunological synapse, suchas: TCRZ, ZAP70, LAT, SLP76, ITK, PLCG, and only a weak recruitmentof: CD3E, and ABTCR; however when this protein nanocluster was assembled,there was an abnormal signaling (distinct to the one induced by the immuno-logical synapse), where no PKC recruitment, no calcium mobilization nor CD69upregulation were observed [284]. By means of the cleavage of both BCL2 andCASP8 can the protease induce cell death [182, 142]. HIV-1 protease inhibitors(PI) play a key role in the antiretroviral treatment. First generation of PIs werebased on hydroxyethylene and hydroxyethylamine isosteresstarted. Saquinavirwas the first PI approved by the FDA in 1995, since then many other PIs havebeen developed. Ritonavir was found to be a potent inhibitor of cytochromeP450 3A, a major metabolic enzyme for PIs [133], due to this finding, ritonaviris more frequently used as a PI pharmacokinetic booster than as a PI itself.Other first generation antiretrovirals are indinavir, nelfinavir, and amprenavir.Due to the major problems of first-generation PIs (high metabolic clearance, lowhalf-life, poor oral bioavailability, gastrointestinal distress, and the emergence ofdrug-resistance strains), second-generation PIs were developed. The balance ofhydrophobicity and hydrophilicity allowed for a longer half-life. Some of thesedrugs are lopinavir, atazanavir, tipranavir (reserved as a salvage therapy), anddarunavir (high potency against multidrug-resistance strains) [92]. Despite theprogress in PIs therapy, some drug-resistance variants have emerged, they areclassified in two groups: primary mutations, which involve changes in residuesdirectly involved in substrate binding and manifest themselves in the active siteof the enzyme; while secondary mutations are located away from the active siteand are usually compensatory mutations to mitigate the deleterious effects ofprimary mutations [171]. Even, secondary mutations may not manifest in theprotease itself but instead in the protein cleavage site on the Gag-Pol and Gagsubstrates [202]. One of the main strategies to avoid drug resistance, is thedesign of PIs by promoting hydrogen bonding interactions with the backboneatoms in the HIV-1 protease active site, since mutations that cause drug re-sistance cannot significantly alter protease active site backbone conformation,as an example we have darunavir, that showed extensive binding interactionswith backbone atoms and maintained a potent antiviral activity against panelsof clinically relevant multidrug-resistant HIV-1 variants [92, 93]. Recently, anew pharmacokinetic enhancer, cobicistat, which does not have anti-HIV activ-16ty, has been developed, offering the advantage over ritonavir, where cobicistatwill not contribute toward the emergence of drug-resistant HIV-1 variants [305].Many other new classes of PIs with innovative ligands are under preclinical de-velopment for the next generation, molecular design efforts have focused on thesynthesis of P2-ligands promoting enhanced backbone binding and non-peptidePIs containing different structural scaffolds distinct from hydroxyethylsulfon-amide isosteres [92]. RT ∈ P OL ): loader of nucleic acidsand structural support.
The reverse transcriptase’s main function is to convert the viral RNA which hasjust entered the host’s cell after membrane fusion into double-stranded DNA,this occurs in the host’s cell cytoplasm, this DNA will later be translocated to-wards the nucleus in order for it to be finally integrated into the host’s genome.It is a heterodimer comprised of p66 (or p15 or RNase H) and p51, both ofwhich derive from a common Gag-Pol polyprotein, cleaved by the viral protease(p10, PR); these subunits (p66 and p51) share a common amino terminus; thedetails of p15 will be addressed in the following section 1.2.9. p51 possesses4 subdomains (which are homonymous to those of p15 or p66): fingers, palm,thumb, and connection [240]. The heterodimerization process is dependent onfactors such as: mutations (e.g., the L289K mutation, the L289 residue in thep51 subunit makes various hydrophobic contacts with p66 but the mutationdoes not yield towards an ill-heterodimerization or no heterodimerization at all;contrarywise, the same mutation but occurring at the protein p66 yields towardsa lack of dimerization), nucleotide substrates, temperature, magnesium, and so-lution conditions [324]. It has been noted that the catalytic site of the reversetranscriptase lies in the p15 (p66) domain, however p51 may take part in: struc-tural support and facilitating the loading of nucleic acids onto p66, which wouldexplain why the N348I mutation to p51 may confer resistance to reverse tran-scriptase inhibitors (both nucleoside and non-nucleoside), as well, p51 has beenseen to contribute to the architecture of the RNase H primer grip/phosphatebinding pocket, finally, deletions to the C-terminal sequence of p51 may alterchanges in the RNase H activity [44]. The p51 subunit in itself may undergohomodimerization under certain conditions, this was observed by small-angle X-ray scattering (SAXS), this phenomenon occurs dependent on the concentrationof the p51 monomer, and may coexist with the homodimer p51/p51 in equilib-rium, furthermore it has been noted that p51 may exist in two forms: p51E andp51C, the latter is the form in which it may be encountered when bound to thep66 in the heterodimer, and the former is an extended form in which it mayresemble the structure of p66, the homodimerization may occur with one p51Especies and one p51C species assemblying into p51E/p51C, this could happendue to the stabilizing effect of p51C on the structure of p51E. p51 interactswith non-nucleoside reverse transcriptase inhibitors (NNRTIs) forming two dif-ferent species: p51E-NNRTI and p51E/p51C-NNRTI, this due to the fact thatp51E is in possession of a p66-like structure [324]; the monomeric form of p5117s favored by low concentration, low salt, and low temperature conditions [325].Reverse transcriptase inhibitors are divided into nucleoside/nucleotide and non-nucleoside reverse transcriptase inhibitors, NRTIs and NNRTIs, respectively.NRTIs inhibit viral replication through competition with purines and pyrim-idines, avoiding adding new nucleotides and consequently, finishing viral DNAreplication. In contrast, NNRTIs bind to an hydrophobic pocket in p66, thisbinding leads to a stereo-chemical change in the protein, preventing the additionof new nucleosides and blocks the cDNA elongation. Typically, NRTIs consti-tute the backbone of the antiretroviral therapy, among them we can find teno-fovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), which areadenosine-derivated; emtricitabine (FTC) and lamivudine as cytosine analogs;abacavir as guanosine analog; zidovudine and stavudine as thymidine analogs;and didanosine as an inosine derived. An advantage of the current NRTIs is thelow clinically significant drug-drug interactions but they have the disadvantageof significant side effect profiles and problems with resistant HIV-1 variants [216].All of them can be affected by selected resistance mutations, either by mutationsin the N-terminal polymerase domain of the enzyme, where the most commonare K65R, L74V, Q151M, and M184V; or by thymidine analog mutations, whereare included M41L, D67N, K70R, L210W, T215Y/F, and K219Q/E [216, 155,11]. Among NNRTIs, we can find the first generation (efavirenz and nevirap-ine) and second generation (rilpivirine and etravirine). Resistance mutationsagainst NNRTIs are based on the inhibited drug interaction with the NNRTIbinding pocket (as seen with K103N and K101E), disruption between the drugand NNRTI binding domain residues (Y181C and Y188L), or changes in theconformation or size of the NNRTI binding pocket (Y188L and G190E)[4, 61,60, 220]. Unlike NRTIs, NNRTIs have a higher plasma half-life, so they can actas perpetrators of drug interactions additionally to their significant side-effectprofile [216]. New NRTIs and NNRTIs are focused on overcoming resistancesand reduce side effects [215]. Islatravir (MK-8591) is a nucleoside nonobligatechain terminator that inhibits the enzyme by preventing its translocation, andtherefore it has been categorized as a nucleoside reverse transcriptase transloca-tion inhibitor (NRTTI), demostrating a robust antiviral activity in vivo againstHIV-1 [156]. Doravirine (MK-1439), a novel NNRTI, has showed its potencyat low doses against common resistant strains and has less potential drug in-teractions than its counterparts because it does not have appreciable inductiveor inhibitory effects on CYP enzymes [23]. Elsulfavirine is the prodrug of theactive compound VM-1500A, it has shown a high genetic barrier to the develop-ment of resistant drug mutations [318]. A study showed that elsulfavirine wasnot inferior to efavirenz combined with TDF/FTC in virologic control, and inaddition, elsulfavirine was better tolerated [176]. ( p ∨ p ∈ pol ): converter of RNA into dsDNA. We have previously commented that the reverse transcriptase protein is com-prised of two subunits: p51 and p66 (or p15), where the enzymatic activity ofthe protein is performed by the p15 subunit, this enzymatic activity is performed18y both the polymerase and by the RNase H, the polymerase domain contains 4subdomains: fingers, palm, thumb, and connection [240]. The RNase H belongsto a nucleotidyl-transferase superfamily, this family includes enzymes such as:transposase, retroviral integrase, Holliday junction resolvase, and RISC nucle-ase Argonaute. In retroviruses, this RNase H converts an ssRNA into dsDNA,removes the template RNA after the DNA synthesis and produces a polypurineprimer for the second-DNA-strand synthesis. In reactions catalyzed by theRNase H, the nucleophile is derived from a water molecule or a 3’-OH from thenucleic acid; the active site of these enzymes is dependent on magnesium ormanganese to bind to the substrate and catalyze the reactions, these reactionsoccur in a one-step bimolecular nucleophilic substitution; two metallic ions arerequired for the reactions, in which one metal ion activates the hydroxyl nu-cleophile and the other one stabilizes the pentacovalent intermediate. VariousRNases display a conserved ∼
100 residue core structure: five stranded β sheetand three α helices (A,B,D). Specificity for the RNA strand is determined bycontacts between the RNase H and five consecutive 2’-OH groups, and when theRNA complexes (around 6 bp) with the enzyme, around 100 nm of the enzymaticsurface becomes buried, a groove which protrudes near the site of the buried sur-face is the active site, where catalytic carboxylates (E109 and D132) make hydro-gen bonds with 2’-OH groups. The DNA binding site is located in the alpha he-lices (polymerase), which fold into a groove, a phosphate binding pocket accom-panies this binding site (comprised of the amino acids T104, N106, S147, T148);the pocket when not in enzymatic activity is occupied by a sulfate ion, which isreplaced by the DNA’s phosphate [193]. It has been noted that the manganesemay specifically promote a specific hydrolytic activity (hydrolysis of dsRNA)to the enzyme, rather than the more general endonuclease and directional pro-cessing activities, divalent metal binding may be mediated by the p66 residueE478 [46]. The initial RNAse H inhibitors were described almost 30 years ago,however, the first inhibitor with a relevant effect was N -(4- tert -butylbenzoyl)-2-hydroxy-1-naphthaldehyde hydrazine (BBNH), although it was also an inhibitorof the RT DNA polymerase [26]. After, a derivative, ( E )-3,4-dihydroxy- N ’-((2-methoxynaphthalen-1-yl)methylene)benzohydrazide (DHBNH), which has itsbinding site between the RT polymerase active site and the polymerase primergrip, alters the trajectory of the template-primer, so the RNAse H cannot cleavethe RNA strand in RNA/DNA complexes [108]; metalchelating RNase H active-site inhibitors, such as 1-hydroxy-pyridopyrimidinone, pyridopyrimidinone, di-hydroxycoumarin, diketo acids, β -keto acids, 3-hydroxypyrimidine-2,4-dionesand hydroxypyridonecarboxylic acids, might sequester Mg +2 ions required forRNAse H activity [279, 293]. An alternative to metalchelating inhibitors are al-losteric inhibitors that interfere with the RNA-DNA binding and induce changesin the RNAse H active site, examples of these are the cycloheptathiophene-3-carboxamide (cHTC) derivatives [293]. Several other inhibitors have been pro-posed and developed such as the dual inhibitors RDS1643 (co-targeting HIV-1 IN and RNAse H), EMAC2005 (co-targeting HIV-1 RNA-dependent DNApolymerase and RNAse H), and RMNC6 (co-targeting RT and RNAse H) [293].However, despite extensive research and improvement in RNAse H inhibitors,19he efficacy of these molecules remains low [279]. IN ∈ pol ): immersing viral DNA into thehost’s genome. After the dsDNA has been produced in the reverse transcriptase enzyme, thisDNA is part of a large nucleoprotein complex, the reverse transcription complex[54], this DNA shall later transite from the reverse transcriptase towards thepre-integration complex (PIC) which contains the integrase enzyme, as well asthe viral proteins: vpr, capsid, and matrix, and the host proteins: INI1 andPML [116], moreover, it has been noted that vpr-defective viral particles failfor their PICs to be effectively translocated to the nucleus, whereas matrix-defective viral particles are only partially effective in the transport process, thiscould be related to the finding that vpr binds to KPNA (karyopherin alpha),the ideal concentration of the vpr protein in order for this to occur is < nM ,for when concentrations of the vpr protein such as > . nM happen, the trans-port mechanism is inhibited [207]; this pre-integration complex will afterwardsbe transported towards the nucleus by active mechanisms (due to the size im-pediment of the PIC, being this complex around 28 nm wide, versus 9 nm asthe limit of passive transport towards the nucleus), the active transport mech-anism is dependent on the capsid protein (CA) from the HIV-1 particle, aswell as various nuclear transport proteins: NUP153, NUP358, CPSF6, TNPO3,and ADAM10 [73]. The integration of the viral genome into that of the host’sseems partially non-random, due to the fact that the viral genome has beenseen to prefer transcriptionally active regions of the host’s genome; the inte-grase enzyme also displays preference towards chromatinised DNA substratesrather than naked ones, i.e., the viral genome integrating process can be de-scribed by means of a stochastic process { S x } , where the x represents the host’sDNA fragment, the probability of the viral DNA being bound to the genome ina position near the nucleosome increases 4-fold, and when this position shiftstowards naked DNA, the probability distribution becomes a uniform one. Theefficiency of integration has also been computed to increase when the chromatinis found in an open fashion (euchromatin) and not in its closed one (heterochro-matin) [168]; furthermore, not only does the viral DNA prefers transcriptionallyactive sites, but as well as intronic genetic ones. The viral DNA integration isaided partially by the host, through a protein product of the PSIP1 gene, saythe LEDGF/p75 protein which belongs to the hepatoma-derived growth factor-related proteins, which contains a conserved N-terminal PWWP (this domainis a member of the Royal superfamily, functions as a chromatin methylationreader, by recognizing methylated DNA or histones, through its high contentof basic residues, thus raising its isoelectric point and facilitating DNA interac-tions [228]) and A/T hook domains (this is a DNA binding peptide motif, witha Gly-Arg-Pro tripeptide, where we may find in its sides basic amino acids,these DNA binding motifs, that might as well bind to RNA [82]) [5]. Availableintegrase inhibitors include bictegravir, cabotegravir, dolutegravir, elvitegravir,and raltegravir, that may be used in combination with other antiretrovirals (co-20icistat, emtricitabine, tenofovir, rilpivirine, and others) [243]; bictegravir tendsto be used along with emtricitabine and tenofovir alafenamide, (BET) in whichcase bictegravir displays a high genetic barrier in HIV-1 resistance development,this combination of antiretrovirals was similar or superior to the one of dolute-gravir, abacavir, and lamivudine (DAL); where in na¨ıve adults, BET establishedvirological suppression in treatment during 96 weeks [63]; bictegravir inhibitsthe integrase with an IC = 7 . ± . nM [280]. Cabotegravir, dolutegravir,and bictegravir have been seen to most efficiently inhibit B HIV-1 subtypes,compared to non-B subtypes [179]. This protein is a 96 amino acid, 14 kDa protein, which is conserved amongother retroviruses [128]. The structure of the vpr protein is featured by three α -helices, with the N- and C-terminal domains surrounding these helices [95],this protein has been seen to exists as an oligomer being the amino acids 36-76 of essence for this polymerization to occur [322], in vivo both dimers andhexamers have been observed, where in the dimeric state only helices II andIII of the protein’s structure are involved, while in the hexameric state, helixI also takes part [285]. Its functions include: the nuclear import of the prein-tegration complex [281], induction of G2 cell cycle arrest, modulation of T-cellapoptosis, transcriptional activation of both viral and host genes, regulationof NFKB activity [128]; these functions are regulated by distinct domains ofthe vpr protein, the N-terminal domain may take part in nuclear localizationand virion packaging, the LR domain is involved in the nuclear localizationof vpr, and the C-terminal domain regulates cell cycle arrest [153]. Cell cy-cle arrest (in G2-M phase) is described as one of the most dramatic functionsof vpr, process occurring with the aid of some host’s proteins, for instance:DCAF1, CRL4, which includes CUL4A, RBX1, and DDB1 [303]; DCAF1 canpromote ubiquitylation of proteins through CRL4 and another E3 ligase, as-semblying later with another protein into a complex, DCAF1-DDB1-DYRK2-EDD; it has been determined that DCAF1 regulates cell-cycle progression andapoptosis either with TP53 or without it, where DCAF1 negatively regulatesTP53, as well as some TP53 target genes (TP21, TP27), while another E3 lig-ase, MDM2 can promote TP53’s ubiquitylation, inducing then its degradation;DCAF1 not only acts directly on TP53 but can additionally alter its epigeneticlandscape through an interaction with HDAC1, then recruiting this deacetylasetowards TP53-dependent promoters [244]; nonetheless TP53 is not the only pro-tein whose expression is affected by DCAF1, for MUS81 and MCM10 are alsoaffected by it [303], where MUS81 is a Holliday junction resolvase, regulatingcell growth and meiotic recombination [102], and MCM10 is a protein essen-tial for the initiation of chromosome replication containing a single-strandedDNA binding domain, this protein complexes with CDC45 and GINS proteinswhich togetherly display helicase activity [297] (ATP-dependent DNA unwind-ing enzyme [214]). Interestingly, vpr can alter the host’s transcriptome, thisdone through ubiquitination of HDAC1, HDAC2, HDAC3, and HDAC8, being21DAC3 the most affected one; this has a two-fold response: enhance the viralgenome expression and that of the host [227], some of the host’s proteins whoseexpression has been seen to become altered by vpr are: ANT, VDAC, PMCA,GLUD2 (mitochondrial), HK1 (glucose metabolism), G6PDH (pentose phos-phate pathway), GAPDH (glycolysis), ATR (stress response), DCAF1 (protea-some), NFAT, NFKB, CEBP, AP1, HIF1A, HDAC1, GR, SP1 (transcription),SLX4, HLTF, and UNG2 (DNA metabolism) [95]; therefore, vpr possesses thepotential to radically modify the host’s T-cell transcriptome. Some naturallyoccurring compounds have been recorded to exhibit inhibitory activity againstvpr, such as isopimarane diterpenoids and picrasane quassinoids [300]; whilecoumarin compounds in in silico experiments have yielded information thatthese compounds may bind to the hydrophobic pocket in vpr and could inhibitthe protein [42]; a novel hematoxylin derivative was developed, this compoundbinds to vpr and can inhibit HIV-1 replication, this hematoxylin derivative canbe produced by reacting the hematoxylin molecule with 2,2-dimethoxypropanein acetone with p-toluene sulphonic acid and phosphorus pentoxide, with an in-termediate molecule being produced, the acetonide-protected hematoxylin, thisis reacted with allyl bromide with K CO in dimethylformamide, where wemight then obtain the stable hematoxylin derivative [103]. This protein is an 81-amino acid type I transmembrane protein, pertains to theviroporin family of proteins; this protein displays certain functions: downregu-lates CD4 receptor expression, promotes release of virions from infected cells;this protein is translated from the gene which also encodes for the env com-plex [97], and it has been suggested that the expression of env and vpu arecoordinated; the vpu gene lies between the first exon of the tat and env genes,this gene is absent in HIV-2. Vpu may assemble into homooligomers, and themonomers exhibits an N-terminal domain (luminal), a single transmembranedomain, and C-terminal hydrophilic domain (into the cytoplasm); where thistransmembrane domain appears essential for the oligomerization of Vpu, and astoichiometry of (
V pu ) n , n = 5 is required for the formation of the pore [69];there have been experimental suggestions in regards to the ion channel activityof this protein, such as that result of a selective conductance for cations of thisprotein (where vpu has been expressed in frog oocytes), this protein then hasbeen compared with the M2 protein of the influenza virus, because vpu pos-sesses a similar transmembrane topology, an uncleaved transmembrane domain,similar length, and casein kinase II phosphorylation sites; features whereby theM2 protein has been seen to transport protons, in this protein a motif withinthe transmembrane domain has been proved essential for its ion channel activ-ity, the His-X-X-X-Trp motif, in this case, His is the proton sensor and Trp isthe pore; when drugs which targeted the transmembrane domain of vpu, therelease of virions could be decreased, additionally, the vpu protein displays anAla-X-X-X-Trp motif in a very similar position as the previously mentionedmotif does in the M2 protein; nonetheless, these discoveries while they indeed22roof the necessity of this protein by the virus in releasing the virions mainlythrough its transmembrane domain, its role as an ion channel has not yet beenfully proved [234], but this protein may alter potassium transport, with a pos-sible role as an ion channel and additionally with an increased TASK1/KCNK3protein degradation (KCNK3 is a member of the superfamily of potassium chan-nel proteins, it is activated by halothane and isoflurane [187]) which can leadto membrane depolarization and viral release [97]. The mechanism throughwhich vpu promotes CD4 downregulation is by means of targeting newly syn-thesized CD4 molecules for proteasomal degradation, promoting the bindingof CD4 to the SKP1-CUL1 E3 ubiquitin ligase which allows for the ubiqui-tination of CD4 on lysine, serine, and threonine residues, then enabling theretention of the CD4 protein in the endoplasmic reticulum, being afterwardsrecruited the VCP-UFD1-NPL4 dislocase complex (NPL4 tends to recognizeLys48-linked polyubiquitinylated substrates [242], VCP extracts ubiquitinatedproteins from lipid membranes [189]), finally leading to degradation by meansof the proteasome. Downregulation of CD4 is not accomplished purely by theaction of the vpu protein, but instead it is a constructive result of vpu, nef,and env proteins [232]. If the viral particle uses CD4 as a main receptor for itsentry into the host’s cell, why would it downregulate this molecule? Becausethe unlimited binding of the viral particle to the CD4 molecules would leadto superinfection (acquisition of different viral strains infecting a single T-cell[20]) of a T-cell, and superinfection of T-cells leads to increased apoptotic rate,by diminishing the available CD4 molecules for the viral particle to bind to,this superinfection is in its stead decreased, and consequently, the likelihood ofincreased T-cell apoptosis, which can lead to a more everlasting viral infection[298]. Downregulation of the CD4 molecule expression on the membrane surfacecould also serve the virus in promoting the virion release from the cell surface(due to the finding that CD4 may bind to the env protein in virions, as well asCD4 prevents the env protein from inserting itself into the virion surface) [270].In vpu-defective virions, where the viral release was still allowed, when treatedwith type 1 interferons, these viral particles were no longer released, and weretethered onto the host’s cell surface; it was later observed that tetherin (BST2)was one of the proteins involved in this process [69], whose expression is pro-moted by type I interferons [209]. Vpu aids virion release and avoidance of viriontethering onto the cellular surface by ubiquitinating BST2 at the cytoplasmiclysine residues 18 and 21, likely promoting subsequently proteasomal degrada-tion, nevertheless, lysosomal degradation is also possible and has been docum-mmented (because lysosomal inhibitors have been seen to inhibit vpu-mediatedBST2 degradation) [69]. Tetherin displays this restraining activity in regardsto viral release from the host’s cell not only in HIV1, but as well in: HIV2,SIV (simian immunodefficiency virus), alpha-, beta-, and gammaretroviruses;filoviruses (Marburg and ebola viruses), arenaviruses (Lassa virus), herpesvirus(type 8 or associated with Kaposi’s sarcoma); thereof can tetherin be regarded asan antiviral molecule [131]. A molecule BIT225 (2-naphthalenecarboxamide, N-(aminoiminomethyl)-5-(1-methyl-1H-pyrazol-4-yl)) has been described as a vpuinhibitor regarding its viroporin function, and can also work as a hepatitis C23irus protein 7 inhibitor [132], SM111 (1-(2-(azepan-1-yl))nicotinoyl)guanidine)has also been described as a potential vpu inhibitor, for HIV-1 replication dimin-ishes when SM111 comes into play even with protease, reverse transcriptase, andintegrase inhibitors-resistant strains [177]; another class of molecules which showpromise in inhibiting vpu function, are phlorotannins (brown-seaweed derivedmolecules, with a structural unit of polyphenols [286]), where these moleculeshave been tested in silico, one advantage of these molecules is the finding thatthey can as well inhibit the reverse transcriptase and the protease proteins, re-ducing the likelihood of HIV replication within the cell, one point, nonetheless,to be considered is the hydrophyllicity of these molecules which may reduce oralbioavailability [138]. Nef is a 27 to 35 kDa N-myristoylated protein, in which the myristoylation pro-cess is mediated by the N-myristoyl transferase (NM1), and is further cleavedby the viral protease, in a site near a hydrophobic groove, which stabilizes aPxxP loop [91]; structurally, this protein is highly plastic, and many conforma-tions have been determined [123]. Through its N-myristoylation, is nef able tolatch onto the inner surface of the host’s cell membrane and can therefore inter-act with the clathrin-coated vesicle machinery, redirecting some transmembraneproteins from the cell surface. Nef can interact with AP2 (heterotetramer com-prising the monomers α , β µ
2, and σ
2) which is a bridge between a membraneprotein substrate (which will be later cargoed onto the clathrin-mediated endo-cytosis process towards its degradation) and clathrin, throughout this AP2-Nefinteraction the latter protein can downregulate the host’s proteins: CD4, CD8,CD28, CD3, SERINC3, SERINC5, CXCR4, CCR5; Nef also interacts with AP1(a heterotetrameric adaptin, which mediates the recruitment of clathrin andrecognition of sorting signals of transmembrane receptors [184]) being able sub-sequently to downregulate both tetherin and MHCI [31]. We can witness theimpact of the Nef-induced downregulation of proteins, such as CD3, essentialfor TCR signaling; and CD28 with vital roles in T-cell proliferation, and Th2cell development [185]. The small molecule 2c (2,4-dihydroxy-5-(1-methoxy-2-methylpropyl)benzene-1,3-dialdehyde [67]) has been seen to inhibit the downreg-ulation of MHC-1 mediated by the action of nef through disrupting the interac-tion of nef with a Src family kinase [64]; 4-amino substituted diphenylfuropyrim-idines have also been described as nef inhibitors, and subsequently inhibiting thenef-dependent HCK activation, the values of the inhibitory concentration IC range the micromolar order; one of these compounds relates to the tyrosine ki-nase type of inhibitors (which are built around a 5,6-biarylsulfo[2,3-d]pyrimidinepharmacophore [72]. 24 .2.14 tat (transactivator protein, p14): promoting viral genomeexpression. This protein has a weight which may be found within the interval of w ∈ [14 , kDa [211], which uses the alternative splicing machinery of the host inproducing this set of possible tat translated isoforms [248]. It has been observedthat the tat protein displays what could be considered as antagonistic propertiesto other viral proteins, such as vpu or nef, in regards to the fact that tat pro-motes CD4 surface expression, as well as it promotes cellular survival (throughBCL2) [211], however it has been observed that the interplay between the pro-teins tat and nef, which would seem initially antagonistic, instead provide opti-mal viral infection, which by binding nef to tat, induction of transcription oc-curs, leading to a specific cellular fate determined by the viral fitness [124]. Thisprotein promotes gene expression (both viral and those of the host cell) by bind-ing to a nascent RNA structure termed transactivator response region (TAR)which results in the transactivation of the long terminal repeat (LTR) promoter;tat can also recruit histone acetyltransferases, inducing histone acetylation inthose genes whose expression is mediated by the LTR; these LTR-induced ex-pressed genes may not only be promoted by means of TAR, but as well tat mayinduce these genes’ expression by binding to the NFKB enhancer [59]. Further-more, tat regulates a set of cellular genes by various mechanisms: by means ofbinding to TAR-like sequences, binding to promoter region or interaction withtranscription factors; these mechanisms regulate then the expression of genessuch as: IL6, TNFB, MAP2K6, IRF7, PTEN, PPP2R1B, PPP2R5E, REL, IL2,IL2RA, OGG1, LMP2, CD69, VAV3, ADCYAP1, and FAM46C [47]; as it is pos-sible for us to regard, these genes are related to cytokine signaling, cell cycle,TCR activation, amongst other processes. Tat can be inhibited by various com-pounds: peptide-based, oligonucleotide-based, and small molecules. Peptide-based compounds are arginine-enriched and can assemble into complexes withthe Transactivation response element RNA (TAR RNA), both of which can com-pete for binding to the tat protein; peptoids are another type of peptide-basedcompounds which have been seen to inhibit the assembly of the tat-TAR RNAcomplex, peptoids are peptide isomers, in which the branching amino acids areall bound to nitrogens in the backbone. In regards to oligonucleotide-based com-pounds we can find TAR decoys, which resemble the TAR RNA sequence andcan bind to the tat protein, however with a limited impact on HIV-1 replication;antisense sequences to that of the TAR RNA one can also inhibit tat activity;small interfering RNAs can also be used in inhibiting tat activity by promotingthe degradation of the target mRNA sequence. Interestingly, in regards to thesmall molecule compounds, the quinolones have displayed anti-tat activity atthe nanomolar range by means of chelating M g +2 in the TAR RNA sequence,one specific quinolone compound, WM5 has been tested (6-amino-1-methyl-4-oxo-7-[4-(2-pyridyl)piperazin-1-yl]quinoline-3-carboxylic acid) [224], the use ofquinolones in tat inhibition can aid in the treatment of bacterial coinfections,although by all means can also may give rise to quinolone-resistant bacteria,which poses a challenge in its clinical use.25 .2.15 rev (RNA splicing regulator, p19): exporter of viral RNA. This protein is a 18 kDa, 116 amino acid phosphoprotein, and binds to rev-responsive elements, assemblying into multimers [205]; rev is phosphorylated bythe kinases CSNK2A1 and MAPK, and it has been seen that the residues Ser8 aswell as Ser 5 are phosphorylated by CSNK2A1, and CSNK2A1-induced phospho-rylation may induce downregulation of the rev protein [165]. These multimers,when in solution, display a hollow fiber-like configuration (with around 20 nmin diameter), and when interacting with RNA, the RNA molecule might be en-gulfed into the hollow volume in order to protect it from nucleic acid metabolism,promoting its cytoplasmic translation [301]. HIV-1 mRNA nuclear export hasbeen seen to be one of the main functions of the rev protein, but it may as wellpromote the translation of rev-responsive mRNAs (RRE), and is able also todownregulate its own expression as well as that of other viral genes [205]; theRRE is found within env -coding region, it spans around ∼
350 nucleotides inlength, and when transcribed, this region binds to rev, where the complex rev-mRNA (ribonucleoproteic in nature) then promotes the recruitment of XPO1and RAN [79], where this multimeric ribonucleoproteic complex shall subse-quently be exported from the nucleus, XPO1 is an exportin (also termed CRM1or chromosome region maintenance 1), member of the importin beta superfam-ily of nuclear transport receptors, required for the export of many RNAs (butis enabled to export proteins by all means), CRM1 interacts with a nuclear ex-port signal (NES) which is leucine-enriched (rev possesses a NES domain [17]),but can additionally be a hydrophobic domain; thus, the rev-mRNA-CRM1-RAN-GTP complex found in the nucleus binds to the RAN binding protein 3(RANBP3) which will then attach to the nucleoporin complex comprised by theNUP214 and NUP88 proteins (found in the nuclear membrane), hatched to theNUP358 protein with its cytoplasmic ending bound to the RAN GTPase acti-vating protein 1; resulting in the export of the rev-mRNA-CRM1-RAN-GDPcomplex and release onto the cytoplasm [112]. Cellular splicing tends to oc-cur within the nucleus [81, 37] (however there do exist reports of cytoplasmicintronic unspliced mRNA [268], some of these unspliced mRNAs are: APP,ATF4, CACNA1H, CAMK2B, FMR1, HP, IL1B, and others which may pro-mote in increasing the variability of the transcribed message [29]), and exportof mRNAs tends to be that of spliced mRNAs, nevertheless, rev promotes theexport towards the cytoplasm of both spliced and unspliced viral RNA [76].A small molecule PKF050-638 (Ethyl (Z)-3-[5-(2-amino-5-chlorophenyl)-1,2,4-triazol-1-yl]prop-2-enoate) is recorded to disrupt the XPO1-NES interaction,thereof being able to inhibit rev-mediated nuclear export, this compounds bindsto the Cys539 residue of XPO1, and exhibits similar effects to those inducedby the XPO1 inhibitor, leptomycin B [58]. Three alternate compounds werefound to suppress HIV-1 gene expression of tat and rev, these compounds are apyrimidin-7-amine, a diphenylcarboxamide, and a benzohydrazide [15].26 .2.16 vif (viral infectivity protein, p23): evading APOBEC3G re-sponse.
This is a basic ∼ kDa phosphoprotein, required by the HIV-1 particle in or-der to replicate in nonpermissive cells, such as lymphocytes, macrophages andleukemic T-cells; but vif is redundant in permissive cells (T-cells) [230]. Vif’sphosphorylation occurs within the C-terminus: Ser144, Thr155, and Thr188,which may occur through protein kinase C or cGMP-dependent kinases [309](serine-threonine protein kinase family of proteins). Interestingly, vif-defectiveviral particles when infecting a permissive cell, may continue to replicate, andproduce infectios vif-defective viral particles which may then infect nonpermis-sive cells; when these vif-defective viral particles derived from nonpermissivecells infect either a permissive cell or a nonpermissive cell, viral replicationceases, this is due to negative imprinting mechanisms applied onto vif [230]. Ithas been observed that the permisiveness feature of the cell relies on the expres-sion pattern of the APOBEC3G protein which can be found in T-cells, B-cells,macrophages, and myeloid cells; in T-cells, APOBEC3G’s expression is pro-moted by T-cell activation [129], this protein is member of a family of cytosinedeaminases which play a role in innate immunity by restricting viral replica-tion through inducing deamination and mutation of viral genes, these enzymesdisplay one or two zinc-binding motifs, one of the members of this family is es-sential for antigen-driven B-cell differentiation (AICDA); the APOBEC3 genesare clustered in chromosome 22; and the expression of the gene APOBEC3Gin monocytes has been seen to be linked to IFNA, IFNB, IFNG, TNFA, andIL4 [53]. APOBEC3G is able to deaminate ssDNA cytosines to uracils, whoseability is not constrained merely to HIV1, but as well to other retroviruses,and inclusively retrotransposons which effectively reduces the viral capabilityof replicating in the absence of vif [195], due to vif being able to induce ubiq-uitination and degradation ov APOBEC3G through complexing with CUL5,and KITLG (vif-CUL5-KITLG) [316]. How can vif-defective viral particlesderived from permissive cells infect nonpermissive cells which indeed containAPOBEC3G? This could be evaded by action of both the viral core and thenucleocapsid proteins which can then esterically protect reverse transcriptionfrom the deleterious actions of APOBEC3G [230, 30]. The compound RN-18which is a small molecule containing three benzene rings, has been reportedto diminish vif activity, and increasing APOBEC3G levels [178]; another smallmolecule compound (IMB-301) inhibits vif-mediated APOBEC3G by bindingto APOBEC3G and inhibiting the binding of vif to APOBEC3G [151], an-other study reported that the small molecule compound, Redoxal, inhibits theDHODH enzyme (dihydroorotate dehydrogenase), thereof diminishing the poolof available pyrimidine molecules, which has been seen to increase APOBEC3Gprotein stability [203]; these three molecules, RN-18, IMB-301, and Redoxal,aid then in decreasing HIV-1 replication rate.27 .2.17 tev (tat/env/rev protein, p26): fusion protein. This is one of the fusion proteins present in the HIV1 proteome, comprised ofportions from three genes: env, rev, and tat, displaying a molecular weight of26 kDa ; this protein is able to transactivate viral transcription (through its tatportion) at an activity of 70% relative to that of the tat protein, however itsrev activity is weak or nonexistant. This protein localizes to the nucleolus, andmay be phosphorylated [139].
Based on the one cell multiple fate theory, several T cell subsets can be differen-tiated from a common T cell precursor (either a na¨ıve T-cell or already primedT-cell) [87]. Each subset of T helper cells is involved in immunity against spe-cific pathogens. It is well known that the cytokine-mediated signal traducersand activators of transcription (STATs) activation is critical for Th-cell-fatedetermination [327].For Th1 differentiation, T-cell receptors (TCRs) are stimulated by IL12through the STAT4 pathway, promoting TBX21 expression, which promotesthe transcription of the IFNG gene, and its own via STAT1 activation. TBX21inhibits GATA3, the principal regulator of Th2 cells. Th1 cells mainly expressCXCR3, INFG, IL2, and TNFA [87, 327, 245].Th2 cells are essential in the control of helminth infections. Their effectorcytokines are IL4, IL5, and IL13, while IL2 and IL4 mediate their differentiation.These cells express CCR4 and STAT6 (mediated by IL4), which induce GATA3expression, the master transcriptional regulator of Th2 cells differentiation, itdirectly binds to the
Il4/Il13 gene locus and through this binding, it induces IL5and IL13 transcription. Retroviral expression of GATA3 is sufficient to induceendogenous GATA3 [327]. In addition to IL4, IL2 activates STAT5, critical forTh2 differentiation, inducing IL4RA and maintaining GATA3 expression. Otherproposed transcriptional regulators are NLRP3 and IL25 [87, 245].Th17 cells are principally located in the gastrointestinal tract. Their primaryfunction is to respond to extracellular bacteria or fungi infections. Their differ-entiation is mediated by TGFB, IL1B, IL6, and IL23. Through IL6-mediatedSTAT3 activation, TGFB induces IL23R and RORC (the principal regulatorof Th17 differentiation), leading to the production of both IL17A, and IL17F.IL21 and IL23 have a similar function as IL6, inducing STAT3 activation. Th17cells also can produce IL22 and TNFA. IL2 activates STAT5, which inhibitsSTAT3 and consequently reduces IL17A expression. FOSL2, MINA, FAS, andPOU2AF1 promote Th17 differentiation, against TSC22D3, which negativelyregulates it. NFIL3 (induced by melatonin) blocks RORC [87, 327].FOXP3(+) Treg cells are essential to regulate auto-reactive T cells. Theyare characterized by the constitutive expression of the IL2 receptor alpha chain(CD25). These cells can be generated within the thymus (tTreg) or in peripherallymphoid organs (pTreg). Their master transcriptional regulator is FOXP3. IL2and TGFB1 are their main differentiation factors, through the IL2-mediated28TAT5 activation, which also suppresses Th17-cell production. In the absenceof IL2, IL2RA, or IL2RB, Treg cells are downregulated [87, 327].Transcriptional factors usually cross-regulate the expression of factors in-volved in the development of other lineages. For example, TBX21 suppressesGATA3 through direct protein-protein interaction; GATA3 suppresses the ex-pression of STAT4 and the production of IFNG; RORC and FOXP3 antagonizeeach other through a protein-protein interaction [327]. Interestingly, all T-cellsubsets have demonstrated some degree of plasticity, adapting their phenotypeto the other T-cell subsets under certain conditions [87].
Thymocyte selection-associated high-mobility group box (TOX) is a DNA-bindingfactor that regulates transcription by producing specific changes in DNA struc-ture and allowing the formation of multi-protein complexes [152].TOX was initially identified as a thymic transcript; it is transiently upregu-lated during β -selection and positive selection of developing thymocytes. Thisupregulation is mediated by TCR-mediated calcineurin signaling. Expression ofTOX induces the factor RUNX3, resulting in CD4 downregulation and CD8 sin-gle positive cell formation, but TOX stimulus alone is insufficient to substitutethe complete TCR signaling during positive selection. Mice deficient in TOX re-vealed a requirement for TOX in CD4 T cell lineage development. RUNX3 andZBTB7B are key nuclear factors for CD8 and CD4 T cells differentiation in thethymus, respectively. It has been suggested that TOX is an upstream regulatorof ZBTB7B. TOX is required also for the development of NK, LTi and CD4(+)cells, probably by modulating E protein activity thought the upregulation ofID2, an E protein inhibitor [6]. The stochastic model, further computations, and graphs were performed bymeans of RStudio: Integrated Development Environment for R [233].
The data was acquired from two main databases: The BioGrid [22], and STRING[2], as well as from various papers: [120, 208, 34, 306, 326, 118, 304, 276, 41,213, 239, 40, 107, 267, 261, 204, 323, 74, 201, 31, 212, 158, 8, 110, 290, 237,292, 113, 14, 260, 121, 154, 90, 219, 57, 148, 257, 251, 258, 206, 175, 197, 314,7, 125, 235, 288, 115, 255, 291, 315, 33, 32, 149, 271, 52, 86, 35, 18, 265, 246,36, 191, 317, 3, 307, 141, 144, 27, 296, 295, 278, 272, 137, 104, 263, 312, 48,126, 139, 229, 145, 10, 9, 19, 105, 165, 96, 100, 101].29 .3 Stochastic model.
The interactions pertaining to the T-cell differentiation molecular network wereassessed, 562 proteins were included, a Markov chain was then associated tothese interactions, the stochastic process { X t } displays a space of states S , suchthat S = { p , p , ..., p n } where n = 562 and p i stands for the protein or genethat the system encounters itself in; the space of the temporal parameter is T = { , , ...m } , the units of this temporal parameter shall be defined subse-quently. The space of states of the stochastic process { X t } may be divided,in order to analyse the selection of the molecules taking part of it, into twosubsets, the TOX-related process subset A ⊂ S , and the Th-cell-related processsubset B ⊂ S , such that, A + B − ( A ∩ B ) = S due to the fact that somemolecules in each subset are repeated, A ∩ B (cid:54) = ∅ . The TOX-related process iscomprised of three major networks, A = { C, D, E } , where the C network standsfor one of the roots of the process: the TOX protein; the D network stands forthose proteins which interact with TOX and amongst themselves (KRTAP12-1,KRTAP26-1, DMRTB1, ZDHHC17, TOX2, BANP, GRAP, FUS); the E net-work stands for those proteins which interact with TOX-related proteins andamongst themselves. The Th-cell-related process subset is in its turn comprisedof ten networks, B = { K, L, M, N, O, P, Q, R, S, T } , the K network stands forthose proteins involved in the Tfh-differentiation process, the L network standsfor the proteins involved in the Th1-differentiation process, the M networkstands for those proteins involved in the Th2-differentiation process, the N net-work stands for those proteins involved in the Th17-differentiation process, theO network is composed of those proteins involved in the Treg-differentiationprocess; we may regards that the set U ⊂ B , U = { K, L, M, N, O } is a set ofroots of the process (TBX21, GATA3, STAT4, STAT5, STAT6, RUNX3, IRF1,HLX, EOMES, EST1, FI1, ASCL2, TCF7, BCL6, IRF4, CMAF, JUNB, DEC2,IKAROS, RORC, FOXP3, BATF1, RORA, RUNX1, HELIOS, FOXO). The Pnetwork stands for those proteins interacting with the Tfh-related ones, the Qnetwork is comprised of proteins interacting with the Th1-related ones, the Rnetwork stands for those proteins interacting with the Th2-related ones, the Snetwork stands for those proteins interacting with the Th17-related ones, and fi-nally the T network is comprised of proteins which interact with the Treg-relatedones; the set v = { P, Q, R, S, T } is thus a secondary network. The adjacencymatrix and corresponding graph summarising this data is shown in . Thestates comprising the processes forecommented are not mutually exclusive, i.e.,one protein may be involved in various networks.We may describe the probabilities of going from one state to another in aMarkov chain by means of a transition probability matrix A = ( a ij ) where a ij is the probability of transiting from the i-th state to the j-th state, the existenceof a probability between two molecules in this model is given by the followingcondition, ∃ R | p i Rp j → ∃ a ij (1)Should the relationship R between two elements of the space of states ( p i p j ) exist, the probability of transiting from the i-th state to the j-th stateexists ( a ij ), the probability is computed by the amount of states the processcan transite to from a given initial state; say we start our process at the p molecule, this molecule interacts with q molecules, these molecules in the vector Q = ( p , ...p q ), Q ⊂ S , then the probability of transiting from p to p q shall be,( a q ) = 1 q (2)in this case, we have considered that all molecules involved interact withthemselves, i.e., a ii >
0. And the condition for the stochastic matrix is thusfulfilled, k (cid:88) i =1 ( a ik ) = 1 (3)The row is normalised. The transition matrix corresponding to the moleculesinteracting in the T-cell differentiation process is shown in 3.The eigenvalues were also computed in order to obtain a matrix Λ, where λ ∈ Λ is an eigenvalue of P; since λ = 1 ∈ Λ, we may intuit that the equilibriumdistribution π n exists, such that, π n = πP n , n → ∞ (4)The equilibrium distribution has been computed taking in consideration thefollowing linear system, π (1)( p −
1) + p π (2) + ... + p n π ( n ) = 0 p π (1) + π (2)( p −
1) + ... + p n π ( n ) = 0... p n π (1) + p n π (2) + ... + π ( n )( p nn −
1) = 0 n (cid:88) i =1 π i ( n ) = 1 (5)Where π ( i ) stands for the i-th state, p ii stands for the probability of tran-siting from the i-th state to the i-th state when the transition matrix is P .The eigenvalues’ matrix Λ is shown in section 3, as is as well displayed theequilibrium distribution.In regards to the T-cell differentiation network when the HIV particle entersthe cell, the network was extended to 2874 molecules (including those that werepreviously in the T-cell differentiation network). The most connected node inthis network was the one of the TP53 molecule with an adjacency k T P of k T P = 169, the transition matrix is shown in figure 4.31 Results.
The eigenvalues for the T-cell differentiation process matrix were obtained, fur-ther assembled into a set Λ, this set contained two eigenvalues of special interest,the presence of λ = 1 which, as has been commented, permits us to intuit theexistence of an equilibrium distribution π n [225], which has been computed; thecomputation of this equilibrium distribution has been performed by means of anumerical method yielding results ˆ f ( π ) where ˆ f is the approximation methodwhich differ from the theoretical ones by a factor (cid:15) a = | f ( π ) − ˆ f ( π ) | where (cid:15) a is the absolute error from the process; considering that the events are subjectto a σ -algebra F and that there exists a measurement P of the probabilityover F , we may consider that (cid:80) π = 1. In this case, (cid:15) a = 5 . × − and we are able to correct our probabilities, therefore we performed a mapping g : ˆ f ( π ) → f ( π ). These corrected probabilities were then the argument of thebase-10 logarithm (in order to more vividly represent the computed probabil-ities, where some probabilities were p <<
0) that is, h ( π ) = log ( p ), this isshown in 5a; from this we can regard that 2 . × − of the probability iscontained in 555 molecules, while only 3 molecules span p = 1 − . × − of the probability, should we exclude for a moment these 3 molecules, the log-arithmic probability would be seen as is displayed in 5b. The correspondingeigenvalues for the HIV+Th-cell differentiation network were computed, andpoured into a matrix Λ , where we come to find λ i ∈ Λ : λ i = 1, thereforeshould we start intuiting about the existence of an equilibrium distribution forour compound network; the second largest eigenvalue in the network is λ j (cid:54) = λ i :mod ( λ j ) = 0 . π nHIV , this is shown in figure 6. Graph 5b may be considered a rank-ordered relation, where the rank is themolecule and the probabilities are logarithmic frequencies, Zipf showed thatwhen plotted, the logarithmic frequencies fall on a slope m = −
1, which maysuggest a power law, we may try and fit the data by means of the followingfunction, x ( r ) = C ( N + 1 − r ) b r a ≡ log ( p ) (6)Where r is the rank value, N is the maximum value, C is a normalizationconstant and a, b ∈ R are two constants to be determined [160], furthermore,it has been observed that many types of data follow this distribution: ranks of32rticles and citations in journals, linguistic data, citation profile, English, andSpanish letter frequency distribution, and more; this distribution, dependingon the value of the constants a, b , may give rise to other common probabilitydistributions, that is, when a = b = 0, it yields a constant random variable,when a = 0 , b = 1 we may obtain a uniform distribution, when b = 0 it becomesa Pareto distribution, when a = b it yields a Lavalette distribution [85]. Herewe will determine the constants a, b for the equilibrium distributions of both theT-cell differentiation and the HIV infection processes, as well as the R fittingvalues for said functions. This we may do by means of firstly obtaining thelogarithm of the function X ( r ) (in order to ease the computations),ln( x ( r )) = ln( C ) + b ln( N + 1 − r ) − a ln( r ) (7)Then, the first moment of the logarithmic function is ( E [ln( x ( r )] ≡ E [ln(log ( p ))]), E [ g ( r )] = (cid:90) ∞−∞ g ( r ) dF X ( x ) → E [ln( x ( r ))] = (cid:90) ln( x ( r )) · drdx dx (8)= (cid:90) ln( x ( r )) dr We can substitute the function ln( x ( r )), E [ln( x ( r ))] = (cid:90) [ln( C ) + b ln( N + 1 − r ) − a ln( r )] dr = [ r ln( C ) + b ( N + 1 − r )(1 − ln( N + 1 − r )) − ar (ln( r ) − r )] (9)= ln( C ) + a + b [ N (1 − ln( N )) − ( N + 1)(1 − ln( N + 1))]When N = 0, E [ln( x ( r ))] = ln( C ) + a − b (10)Which goes in line with [85]; now, when we plug in the values from thesimulation ( N = 2 . { ln(log ( p )) } ), we obtain, E [ln( x ( r ))] = ln( C ) + a + 1 . b (11)Let us compute the variance now, V ar [ln( x ( r ))] = E (cid:2) [ln( x ( r ))] ] (cid:3) − E [ln( x ( r ))] (12)We have already computed the expression for the first moment E [ln( x ( r ))],we are missing now the computation of E [[ln( x ( r ))] ],ln ( x ( r )) = (ln( C ) + b ln( N + 1 − r ) − a ln( r ))
33 ln ( C ) + 2 ln( C )(ln( N + 1 − r ) − a ln( r )) + ( b ln( N + 1 − r ) − a ln( r )) (13)The integral that we are looking for then is, I = ln ( C ) (cid:90) · dr + 2 ln( C ) (cid:90) ln( N + 1 − r ) dr − a ln( C ) (cid:90) ln( r ) dr + b (cid:90) ln ( N + 1 − r ) dr − ab (cid:90) ln( N + 1 − r ) ln( r ) dr + a (cid:90) ln ( r ) dr (14)Which equals, I = ln ( C ) + 2 ln( C )[( N + 1) ln( N + 1) − N (ln( N ) − a ln( C )+ b [( N + 1) ln( N + 1)(ln( N + 1) − − N ln( N )(ln( N ) −
2) + 2] − ab (cid:20) ( N + 1) (cid:18) ln( − N ) − ln( − N − − Li (cid:18) N + 1 (cid:19)(cid:19) − ln( N ) + 2 (cid:21) + 2 a (15)Where Li ( . ) is the polylogarithm function. The variance equals (we makeln( C ) = φ ), V ar (ln( x ( r ))) = φ + 2 φ [ln( N + 1)( N + 1) − N ( l ln( N ) − aφ + b [( N + 1)ln( N + 1)(ln( N + 1) − − N ln( N )(ln( N ) −
2) + 2] − ab (cid:20) N (cid:18) ln( − N ) − ln( − N − − Li (cid:18) N + 1 (cid:19) − ln( N ) + 2 (cid:19) + 1 (cid:21) + 2 a − ( c + a + b [ N (1 − ln( N )) − N (1 − ln( N + 1)) − (16)In the case of N = 2 . V ar [ln(log ( x ( r )))] = a − . b − . ab + 7 . bφ − aφ + 9 . φ (17)The median is where r = 0 . M ed (ln( x ( r ))) = φ + b ln( N + 1 / − a ln(1 /
2) (18)= φ + 1 . b + 0 . a (19)the values of the first moment, variance, and median are, E [ln(log ( p ))] = Z = 2 . × ar [ln(log ( p ))] = V ar ( Z ) = 6 . × − (20) M ed (ln( x ( r ))) = M ed ( Z ) = 2 . × Therefore we possess a system of three variables and three equations, Z = φ + a + 1 . b = 2 . V ar ( Z ) = a − . b − . ab + 7 . bφ − aφ + 9 . φ (21) M ed ( Z ) = φ + 1 . b + 0 . a = 2 . (cid:18) . . . . . (cid:19) (22)Which yields the partial solution, a = 3 . − . φb = 0 . φ − . φ , 2 . φ + 3 . φ − . φ , φ ) = (2 . , − . φ i ∈ Φ, φ , (cid:18) ˆ a ˆ b (cid:19) = (cid:18) . − . (cid:19) φ , (cid:18) ˆ a ˆ b (cid:19) = (cid:18) . − . (cid:19) (25)Whose graphs are shown in figure 7. in vivo processes of T-cell differentiation, and HIV-cellular infection. The second eigenvalue of interest is one which allows us to compute the rate ofconvergence, should it exist, and in this case, we have already commented onits existence; this rate is dependent on the second maximum eigenvalue µ [283], µ = max { λ i ∈ Λ : µ (cid:54) = λ = 1 } µ = 0 . T , that is, the necessary stepsfor the process to achieve equilibrium, T = 1 log ( µ − )= 1148 . u (27)The process, in this case a T-cell, may reach equilibrium after 1148 . in vivo or in vitro data collectedin the past, this achievement of equilibrium may be regarded as the completelifespan of a T-cell, and has been reported to be as high as 17 years [114] andas low as 6 days [25, 98], thus this unit may range in the following interval,1 u ∈ [4 . × , . × ] s (28)In regards to the HIV infection process, we may regard that the coupledset of viral particle and T-cell reach equilibrium when there begins to exist adecline in the Th-cell count, moment in which we can start to perceive thatthe genetic/proteic machinery of the virus has effectively hijacked that of thecell towards another more deleterious, that of more massive apoptosis, this ismore generally seen after five years of infection [77], then the time to reachequilibrium T , will depend on the second maximum eigenvalue ν , T = 1 log ( ν − )= 6656 . u (29)Now, 6656 . u ≡ y → . u/y . The probability distributions π i for i ∈ [1 , T ] where T is the time when theprocess has converged towards equilibrium π n have been computed and areshown in 8. S ≤ S ≤ ... ≤ S n when reaching its equilibrium distribution. Entropy in a thermodynamic setting may relate to the concepts of reversibil-ity or order in a system, more specifically, should we have a protein with athermodynamic state π ( x ) = exp (cid:2) − β H ( x ) Z (cid:3) where H ( x ) is the Hamiltonian( H = T + V , T is the kinetic energy and V is the potential energy), β = kT Z is the partition function, we can thus compute the entropy S ( π ) of thesystem by means of, H ( π ) = − k N (cid:88) i =1 π ( x ) ln π ( x ) (30)[198] this could be therefore considered as the summation over all the parti-cles of each of their stochastic entropies s t , s t = − ln π ( x t , t ) (31)The probability is to be computed by means of solving the Fokker-Planckequation [247], which should we recall, describes the evolution of the probabili-ties π from an ensemble of particles when this particles start their trajectories in t = 0, we would call this distribution the initial vector π [16] that we have pre-viously commented on, thus we may regard the solution to this Fokker-Planckequation in our case as a discrete one (which comes as no surprise, due to thefact that the continuous probability considered in the Fokker-Planck equationis a continuous Markovian one). Should we divide the expression 30 by 1 /k , wewould obtain, S ( π ) = − N (cid:88) i =1 π ( x ) ln π ( x ) (32)This is the Shannon entropy of the system [198], which will help us in theunderstanding of the changes of the microscopical states π in our system, thesestates are then the thermodynamic states pertaining to each particle in theregarded system. We have thereof computed S ( π i ) for i ∈ [1 , N ] where N = 1148in this case, due to this being the time of convergence for the Th-differentiationprocess, which may be seen in 9, the entropy S ( π HIV ) was also computed forthe HIV-infection process, and is seen in 10We may regard that there exists a feature for each S i computed, S < S < ... < S n (33)Therefore, the process tends towards an increasing entropy value S n , whichaccording to Boltzmann, systems tend towards increases in entropy when theyreach equilibrium due to this being the state in which there exist the mostpossible configurations [13]. However we may notice two aspects in this entropycomputation:1. ∆ S > ∆ S > ... > ∆ S n − → (∆ S n → S t asa measurement of the information or the number of states in the system37t any time t ∈ T , therefore, the set of states Z ts in the system accountedfor by the entropy are defined by, t = 1 , Z s = { Z , Z , ..., Z m } (34)Then Z s is a measurement of events π ∈ Ω (some molecules may have arepeated value of probability) over P = [0 , Z s is a σ -algebra; now, letus establish a relationship between the cardinalities of the sets of states, | Z s | < | Z s | < ... < | Z ns | (35)And, ∀ Z m ∈ Z s , Z m ∈ Z s ∀ Z m + i ∈ Z s , Z m + i / ∈ Z s → Z s ⊆ Z s ∧ Z s ⊃ Z s (36)In our simulation we can state thus, Z s ⊆ Z s ⊆ ... ⊆ Z ns (37)And we may say that there exists a collection of σ -algebras for every step t = 1 , , ..., N in the process, this collection is then a filtration { Z ns } n ≥ such that the following is fulfilled, Z ns ⊆ Z n +1 s (38)The process { X n } can be Z -measurable, and we even may couple theentropy with the filtration { Z ns } n ≥ and, the acquisition of informationthroughout the Th-cell differentiation process may be considered a mar-tingale.2. The Euclidean distance δ ∈ R between the initial entropy S and the finalentropy S f is δ ( S ) = S f − S >
0, therefore this model describes a possibleprocess, and moreover an irreversible one: When reaching equilibrium thechange in entropy, δ ( S ) > Discussion.
HIV-1 transmission results from viral exposure at mucosal surfaces or from per-cutaneous inoculation. Since direct analysis of these mechanisms is inaccessible,understanding HIV-1 infection has been done by indirect methods [253].The initial period between the moment when the first cell is infected and themoment when the virus can be detected in blood, is called the eclipse phase (es-timated at 7-21 days). During this phase, clinically silent, HIV-1 is propagated.Once virus becomes detectable in blood plasma, it increases exponentially [253,222].Most dynamic models of HIV infection do not usually take into account theearliest stage of infection, where stochastic models play a key role [200]. π n . One of the motivating questions for this paper was the question: can the HIVchange the differentiation status of a T-cell? In order to answer such conundrumone would ask oneself how would the modeling for the differentiation processin the T-cell ever occur, we have addressed this problem in a two-fold fashion,one in a biological manner, i.e., a differentiated state in a cell may be under-stood as a committed state in the cell, one in which the cell cannot returntowards a totipotent or pluripotent cell and has a narrower, more defined setof functions [170]; and another in a more physical manner, i.e., this differenti-ated state as a state which is in equilibrium, or that it would require energyfrom its environment in order to de differentiate and return to a less differenti-ated state (as it may occur in certain processes, such as neoplasms [190]), thisenergy E is not available readily for such endeavours for any cell, requiring aspecial effort for it to happen, this equilibrium state can be regarded as theequilibrium reached for a Markov process, a state which is independent of anyinitial state of the system for it will be reached notwithstanding the initial con-ditions; a Markov process can model as well the behaviour of the differentiatingcell due to the stochastic nature of interactions between signaling, structural,and energy-related molecules. Through this simulation we have observed thatin both stochastic processes (Th-cell differentiation, and HIV-infection) thereexist equilibrium distributions, providing us with in silico evidence that theseprocesses indeed lead to differentiated states of the cell, the Th-cell differentia-tion may lead to the various phenotypes (Th1, Th2, Th17, Treg, etc), and theHIV-infection-dependent differentiation may lead to a novel differentiated state, T h
HIV
11, this state may be featured by an altered immunological synapse, andpromotion of induction of the virological synapse, expression of antiapoptoticfactors in early phases of infection, but lately would it be overrun with promo-tion of proapoptotic factors leading to the decline of T-cell count in a chronicphase of the infection. This has been seen to occur in an in vivo setting, whereCD45RA(+) (PTPRC(+)) T cells decline over time, with a more rapid decreasein the na¨ıve phenotype type of T cells than those which bear a T-cell memory39henotype [51], this molecule was included in our simulation, and displayed − log ( p ) = 13 . in vivo or clinicalsettings, for we can track the decline in the CD45RA(+) T-cells in the courseof the HIV-1 infection, and establish a relation with the adimensional temporalunits in this model. Here, we have commented on a method through which one may be able to provean stochastic process { X t } is a martingale by a non-conventional path, for wehave proposed that a martingale can be regarded as such set of random variableswhich are coupled with filtrations, these filtrations are steps in the Markov chainin which by each step, the process gains information, therefore increases itsinformational Shannon entropy. Both stochastic processes considered here havebeen proved to gain entropy when t → ∞ , this increase in entropy can also beseen as a way in which the systems tend towards equilibrium, both the T-celldifferentiation process, and the HIV-infection process. A connection betweenShannon’s entropy and the more thermodynamic sense of it can be performedshould we remind ourselves that the increase in the states in which the systemmay be encountered (the increase in information) are molecules within suchsystem, and probabilities the system may find itself in given molecule for a givenamount of time. Interestingly, our simulation has yielded results, in which theprobabilities of the system are very unequally divided between the members ofthe system, that is, there is a small set of molecules { S } ⊂ { X } : | S | << | X | ,while (cid:80) i p Si >> (cid:80) j p Xj , these molecules contain most of the probability withina very small set of them, this has been observed in other simulations whichdo not pertain to the biological realm, but to the economical one, in which insimulations of continuous bids within a given population, a very small amountof people possess most of the wealth, and the majority of the population end upwith a very small amount of wealth [84, 24], in the yard-sale model trying to giveaccount for wealth distribution inequality and the formation of oligarchies, it isalso of interest to note that should we consider { X n } as the wealth of a givenplayer in the model in the n-th moment, the conditional expectancy equals, E [( X in +1 − X in ) | X in ] = a min ( X in , − X in ) (39)where X ∞ ∼ Ber ( X ), that is the stochastic process { X n } is also a non-negative martingale which converges to the random variable X i ∞ [43]. In ourmodel, we can consider that there exists the formation of molecular ”oligarchies”which possess most of the wealth (time spent on those molecules) which can playcentral roles in both stochastic processes considered.40 .3 Th-cell differentiation, and HIV-infection equilibriumdistributions as discrete generalized beta ones. One additional result which astounded the authors of this paper is that of thelog-rank equilibrium distribution of the stochastic processes, for they display themorphology of the discrete generalized beta distributions, this distribution hasbeen encountered in various processes: distribution of impact factor in journals[71], letter frequency distribution in political speeches [147], k-mer distributionsin the human genome [146], traffic networks [181], however it extends to art,and genetic regulatory networks [160]; all these phenomena might be regardedas complex networks, which is the case we display here, and its importance of anHIV-infection towards a single cell (as well as the T-cell differentiation process)as complex ones, that is, that the gross phenomena which arise from it mightdiffer from the individual products of its constituents, i.e., while we might notbe able to explain the clinical course of the HIV infection by means of trackingthe course of a single molecule included in the process, should we regard thenetwork and the behaviour it displays as a whole, then can we establish thosemappings from the molecular realm towards the clinical and more macroscopicone.
Three main findings may be exposed: the T-cell differentiation process tendstowards an equilibrium state which can ensure its differentiated state (for itcould be required an energetic stimulus in order to exit from it), and the HIV-infection of a T-cell leads to another, novel T-cell differentiated state, which canbe antiapoptotic at first, but when time passes by and equilibrium is reached,there is a tendency towards a proapoptototic state. These processes lead to-ward ”oligarchic” states, that is, states in which only a few molecules possessthe wealth of the process (time spent on those molecules), this can be seen ashow some molecules can play central roles in the process. Finally, these twoprocesses can be considered complex ones, their phenomenology correspondsto that in which the behavior in whole cannot be directly explained by theindividual behaviour of its constituents. This model can serve as scaffold forfuture simulations of pathogen/cell infections, the interaction of this systemswith additional pathogens can be considered, for instance, what occurs whenan opportunistic virus such as herpes virus type 8 infects the body? What isthe nature of the interaction of this virus with the T-cells and their novel HIV-induced differentiated state? Other questions which can be addressed are thosewhich correspond to the treatment: what happens when we block gp120? Canthe path towards equilibrium continue? If so, how does this path look?One of the clearest limitations of this study is the lack of the logical gatesYES and NO, that is, whether a protein with a higher probability to be inter-acted with represents an inhibition or a promotion of its function, this can beaddressed in further studies; another limitation is the one of the heterogeneity41n the nature of the information treated with, that is, the levels of evidenceincluded in the interactions, The BioGrid database already displays a systemof levels of evidence regarding the interactions of every protein, however in thedata-acquisition process, we have not only considered interactions derived fromthis database, for we have considered interactions recorded in individual papers,as well as in the STRING database, all of them with distinct levels of evidence.Additionally, the interactions considered do not consider the very nature of theinteraction, it does not distinguish whether the interaction is genetic, physical,or else. Finally, this model does not take into account mutations which maylead to dysfunctional protein products of both the host and the virus, as wellas it does not consider the compound effect of the infection in various cells, butmerely in one cell.
To my friend and colleague Luz Fernanda Jim´enez Mu˜noz for her input inregards to section 3.5.
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