Natalia Sharova

Persistence of episomal HIV-1 infection intermediates in patients on highly active anti-retroviral therapy

Treatment of HIV-1-infected individuals with a combination of anti-retroviral agents results in sustained suppression of HIV-1 replication, as evidenced by a reduction in plasma viral RNA to levels below the limit of detection of available assays. However, even in patients whose plasma viral RNA levels have been suppressed to below detectable levels for up to 30 months, replication-competent virus can routinely be recovered from patient peripheral blood mononuclear cells and from semen. A reservoir of latently infected cells established early in infection may be involved in the maintenance of viral persistence despite highly active anti-retroviral therapy. However, whether virus replication persists in such patients is unknown. HIV-1 cDNA episomes are labile products of virus infection and indicative of recent infection events. Using episome-specific PCR, we demonstrate here ongoing virus replication in a large percentage of infected individuals on highly active anti-retroviral therapy, despite sustained undetectable levels of plasma viral RNA. The presence of a reservoir of ‘covert’ virus replication in patients on highly active anti-retroviral therapy has important implications for the clinical management of HIV-1-infected individuals and for the development of virus eradication strategies.

Macrophages archive HIV-1 virions for dissemination in trans

Viruses have evolved various strategies in order to persist within the host. To date, most information on mechanisms of HIV‐1 persistence has been derived from studies with lymphocytes, but there is little information regarding mechanisms that govern HIV‐1 persistence in macrophages. It has previously been demonstrated that virus assembly in macrophages occurs in cytoplasmic vesicles, which exhibit the characteristics of multivesicular bodies or late endosomes. The infectious stability of virions that assemble intracellularly in macrophages has not been evaluated. We demonstrate that virions assembling intracellularly in primary macrophages retain infectivity for extended intervals. Infectious virus was recovered directly from cytoplasmic lysates of macrophages and could be transmitted from macrophages to peripheral blood lymphocytes in trans 6 weeks after ongoing viral replication was blocked. Cell‐associated virus decayed significantly from 1 to 2 weeks post infection, but decreased minimally thereafter. The persistence of intracellular virions did not require the viral accessory proteins Vpu or Nef. The stable sequestration of infectious virions within cytoplasmic compartments of macrophages may represent an additional mechanism for viral persistence in HIV‐1‐infected individuals.

Primate lentiviral Vpx commandeers DDB1 to counteract a macrophage restriction.

Primate lentiviruses encode four “accessory proteins” including Vif, Vpu, Nef, and Vpr/Vpx. Vif and Vpu counteract the antiviral effects of cellular restrictions to early and late steps in the viral replication cycle. We present evidence that the Vpx proteins of HIV-2/SIVSM promote virus infection by antagonizing an antiviral restriction in macrophages. Fusion of macrophages in which Vpx was essential for virus infection, with COS cells in which Vpx was dispensable for virus infection, generated heterokaryons that supported infection by wild-type SIV but not Vpx-deleted SIV. The restriction potently antagonized infection of macrophages by HIV-1, and expression of Vpx in macrophages in trans overcame the restriction to HIV-1 and SIV infection. Vpx was ubiquitylated and both ubiquitylation and the proteasome regulated the activity of Vpx. The ability of Vpx to counteract the restriction to HIV-1 and SIV infection was dependent upon the HIV-1 Vpr interacting protein, damaged DNA binding protein 1 (DDB1), and DDB1 partially substituted for Vpx when fused to Vpr. Our results indicate that macrophage harbor a potent antiviral restriction and that primate lentiviruses have evolved Vpx to counteract this restriction.

MODULATION OF HIV-1 INFECTIVITY BY MAPK, A VIRION-ASSOCIATED KINASE

Infection of a cell by human immunodeficiency virus type 1 (HIV‐1) results in the formation of a reverse transcription complex in which viral nucleic acids are synthesized. Efficient disengagement of the reverse transcription complex from the cell membrane and subsequent nuclear translocation require phosphorylation of reverse transcription complex components by a virion‐associated kinase. In this study, we identify the virion‐associated kinase as mitogen‐activated protein kinase (ERK/MAPK). Upon density gradient fractionation, MAPK, but not its activating kinase MEK, co‐sedimented with viral particles. Expression of a constitutively active, but not kinase‐inactive, MEK1 in virus producer cells was able to activate virion‐associated MAPK in trans. Stimulation of virion‐associated MAPK activity in trans by the mitogen phorbol myristate acetate (PMA) increased viral infectivity. Conversely, suppression of virion‐associated MAPK by specific inhibitors of the MAPK cascade markedly impaired viral infectivity. These studies demonstrate regulation of an early step in HIV‐1 infection by the host cell MAPK signal transduction pathway.

Small-molecule inhibition of HIV-1 Vif

The HIV-1 protein Vif, essential for in vivo viral replication, targets the human DNA-editing enzyme, APOBEC3G (A3G), which inhibits replication of retroviruses and hepatitis B virus. As Vif has no known cellular homologs, it is an attractive, yet unrealized, target for antiviral intervention. Although zinc chelation inhibits Vif and enhances viral sensitivity to A3G, this effect is unrelated to the interaction of Vif with A3G. We identify a small molecule, RN-18, that antagonizes Vif function and inhibits HIV-1 replication only in the presence of A3G. RN-18 increases cellular A3G levels in a Vif-dependent manner and increases A3G incorporation into virions without inhibiting general proteasome-mediated protein degradation. RN-18 enhances Vif degradation only in the presence of A3G, reduces viral infectivity by increasing A3G incorporation into virions and enhances cytidine deamination of the viral genome. These results demonstrate that the HIV-1 Vif-A3G axis is a valid target for developing small molecule–based new therapies for HIV infection or for enhancing innate immunity against viruses.

In-Depth Analysis of the Interaction of HIV-1 with Cellular microRNA Biogenesis and Effector Mechanisms

ABSTRACT The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4+ peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. IMPORTANCE MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms. MicroRNAs (miRNAs) are a ubiquitous class of small regulatory RNAs that serve as posttranscriptional regulators of gene expression. Previous work has suggested that HIV-1 might subvert the function of the cellular miRNA machinery by expressing viral miRNAs or by dramatically altering the level of cellular miRNA expression. Using very sensitive approaches, we now demonstrate that neither of these ideas is in fact correct. Moreover, HIV-1 transcripts appear to largely avoid regulation by cellular miRNAs by adopting an extensive RNA secondary structure that occludes the ability of cellular miRNAs to interact with viral mRNAs. Together, these data suggest that HIV-1, rather than seeking to control miRNA function in infected cells, has instead evolved a mechanism to become largely invisible to cellular miRNA effector mechanisms.

Identification of Flavopiridol Analogues that Selectively Inhibit Positive Transcription Elongation Factor (P-TEFb) and Block HIV-1 Replication

The positive transcription elongation factor (P‐TEFb; CDK9/cyclin T1) regulates RNA polymerase II‐dependent transcription of cellular and integrated viral genes. It is an essential cofactor for HIV‐1 Tat transactivation, and selective inhibition of P‐TEFb blocks HIV‐1 replication without affecting cellular transcription; this indicates that P‐TEFb could be a potential target for developing anti‐HIV‐1 therapeutics. Flavopiridol, a small molecule CDK inhibitor, blocks HIV‐1 Tat transactivation and viral replication by inhibiting P‐TEFb kinase activity, but it is highly cytotoxic. In the search for selective and less cytotoxic P‐TEFb inhibitors, we prepared a series of flavopiridol analogues and evaluated their kinase inhibitory activity against P‐TEFb and CDK2/cyclin A, and tested their cellular antiviral potency and cytotoxicity. We identified several analogues that selectively inhibit P‐TEFb kinase activity in vitro and show antiviral potency comparable to that of flavopiridol, but with significantly reduced cytotoxicity. These compounds are valuable molecular probes for understanding P‐TEFb‐regulated cellular and HIV‐1 gene transcription and provide potential anti‐HIV‐1 therapeutics.

Synthesis and Structure–Activity Relationship Studies of HIV‐1 Virion Infectivity Factor (Vif) Inhibitors that Block Viral Replication

The human immunodeficiency virus 1 (HIV‐1) virion infectivity factor (Vif) protein, essential for in vivo viral replication, protects the virus from innate antiviral cellular factor apolipoprotein B mRNA‐editing, enzyme‐catalytic, polypeptide‐like 3G (APOBEC3G; A3G) and is an attractive target for the development of novel antiviral therapeutics. We have evaluated the structure–activity relationships of N‐(2‐methoxyphenyl)‐2‐((4‐nitrophenyl)thio)benzamide (RN‐18), a small molecule recently identified as an inhibitor of Vif function that blocks viral replication only in nonpermissive cells expressing A3G, by inhibiting Vif–A3G interactions. Microwave‐assisted cross‐coupling reactions were developed to prepare a series of RN18 analogues with diverse linkages and substitutions on the phenyl rings. A dual cell‐based assay system was used to assess antiviral activity against wild‐type HIV‐1 in both nonpermissive (H9) and permissive (MT4) cells that also allowed evaluation of specificity. In general, variations of phenyl substitutions were detrimental to antiviral potency and specificity, but isosteric replacements of amide and ether linkages were relatively well tolerated. These structure–activity relationship data define structural requirements for Vif‐specific activity, identify new compounds with improved antiviral potency and specificity, and provide leads for further exploration to develop new antiviral therapeutics.

1,2,3-Triazoles as Amide Bioisosteres: Discovery of a New Class of Potent HIV ‑ 1 Vif Antagonists

RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G) expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC50 = 6 μM) by isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, 1d (IC50 = 1.2 μM). We identified several potent HIV-1 inhibitors from a 1d based library including 5ax (IC50 = 0.01 μM), 5bx (0.2 μM), 2ey (0.4 μM), 5ey (0.6 μM), and 6bx (0.2 μM).

Ubiquitin-independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity

Recent findings indicate that the ubiquitin‐proteasome system is involved in the pathogenesis of cancer as well as autoimmune and several neurodegenerative diseases, and is thus a target for novel therapeutics. One disease that is related to aberrant protein degradation is multiple sclerosis, an autoimmune disorder involving the processing and presentation of myelin autoantigens that leads to the destruction of axons. Here, we show that brain‐derived proteasomes from SJL mice with experimental autoimmune encephalomyelitis (EAE) in an ubiquitin‐independent manner generate significantly increased amounts of myelin basic protein peptides that induces cytotoxic lymphocytes to target mature oligodendrocytes ex vivo. Ten times enhanced release of immunogenic peptides by cerebral proteasomes from EAE‐SJL mice is caused by a dramatic shift in the balance between constitutive and β1ihigh immunoproteasomes in the CNS of SJL mice with EAE. We found that during EAE, β1i is increased in resident CNS cells, whereas β5i is imported by infiltrating lymphocytes through the blood‐brain barrier. Peptidyl epoxyketone specifically inhibits brain‐derived β1ihigh immunoproteasomes in vitro (kobs/[I] = 240 M‐1s‐1), and at a dose of 0.5 mg/kg, it ameliorates ongoing EAE in vivo. Therefore, our findings provide novel insights into myelin metabolism in pathophysiologic conditions and reveal that the β1i subunit of the immunoproteasome is a potential target to treat autoimmune neurologic diseases.—Belogurov Jr., A., Kuzina, E., Kudriaeva, A., Kononikhin, A., Kovalchuk, S., Surina, Y., Smirnov, I., Lomakin, Y., Bacheva, A., Stepanov, A., Karpova, Y., Lyupina, Y., Kharybin, O., Melamed, D., Ponomarenko, N., Sharova, N., Nikolaev, E., Gabibov, A. Ubiquitin‐independent proteosomal degradation of myelin basic protein contributes to development of neurodegenerative autoimmunity. FASEB J. 29, 1901‐1913 (2015). www.fasebj.org