José Alcamí

Membrane raft microdomains mediate lateral assemblies required for HIV-1 infection

HIV‐1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120–CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV‐1 co‐receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV‐1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV‐1 envelope glycoprotein‐mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV‐1 infection.

Absolute dependence on kappa B responsive elements for initiation and Tat-mediated amplification of HIV transcription in blood CD4 T lymphocytes.

The role of NF‐kappa B‐dependent signals in activating the transcriptional activity of the HIV regulatory region (LTR) was analyzed by systematic comparison of HIV LTR activity in human CD4 T cells purified from peripheral blood and a transformed lymphoblastoid T cell line. In normal CD4 T cells we also analyzed the role played by the viral kappa B responsive elements in HIV replication. Analysis of nuclear extracts of resting, normal T lymphocytes revealed the presence of the p50, but not the p65, NF‐kappa B subunit and the induction by phorbol esters of bona fide (p50‐p65) NF‐kappa B complexes. In parallel, we observed clear enhancer‐dependent HIV LTR transactivation comparable in intensity with that observed in lymphoblastoid cells. We show that unstimulated CD4 T lymphocytes offer a cellular environment of very low permissivity to HIV LTR functioning. This was in sharp contrast to the high spontaneous LTR activity observed in lymphoblastoid T cells, where LTR activity was essentially independent of kappa B‐responsive elements. Due to the low basal LTR activity in resting T lymphocytes, NF‐kappa B‐dependent transactivation was a sine qua non event for induction of the HIV LTR. Surprisingly, even the function of HIV Tat in resting CD4 T lymphocytes was found to be absolutely dependent on LTR kappa B responsive elements. The relevance of these observations obtained in transient transfections was confirmed by the incapacity of blood CD4 T lymphocytes infected with an HIV infectious provirus carrying critical point mutations in the kappa B responsive elements to show any detectable transcriptional activity upon cell activation and prolonged culture in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Understanding HIV-1 latency provides clues for the eradication of long-term reservoirs

HIV-1 can infect both activated and resting, non-dividing cells, following which the viral genome can be permanently integrated into a host cell chromosome. Latent HIV-1 reservoirs are established early during primary infection and constitute a major barrier to eradication, even in the presence of highly active antiretroviral therapy. This Review analyses the molecular mechanisms that are necessary for the establishment of HIV-1 latency and their relationships with different cellular and anatomical reservoirs, and discusses the current treatment strategies for targeting viral persistence in reservoirs, their main limitations and future perspectives.

Induction of Apoptosis by Double-Stranded-RNA-Dependent Protein Kinase (PKR) Involves the α Subunit of Eukaryotic Translation Initiation Factor 2 and NF-κB

ABSTRACT The double-stranded (ds) RNA-dependent protein kinase (PKR) is a key mediator of antiviral effects of interferon (IFN) and an active player in apoptosis induced by different stimuli. The translation initiation factor eIF-2α (α subunit of eukaryotic translation initiation factor 2) and IκBα, the inhibitor of the transcription factor NF-κB, have been proposed as downstream mediators of PKR effects. To evaluate the involvement of NF-κB and eIF-2α in the induction of apoptosis by PKR, we have used vaccinia virus (VV) recombinants that inducibly express PKR concomitantly with a dominant negative mutant of eIF-2α or a repressor form of IκBα. We found that while expression of PKR by a VV vector resulted in extensive inhibition of protein synthesis and induction of apoptosis, coexpression of PKR with a dominant negative mutant of eIF-2α (Ser-51→Ala) reversed both the PKR-mediated translational block and PKR-induced apoptosis. Coexpression of PKR with a repressor form of IκBα (Ser-32,36-Ala) also leads to the inhibition of apoptosis by abolishing NF-κB induction, while translation remains blocked. Treating cells with two different proteasome inhibitors which block IκBα degradation, prevented PKR-induced apoptosis, supporting results from coexpression studies. Biochemical analysis and transient assays revealed that PKR expression by a VV vector induced NF-κB binding and transactivation. In addition, upregulation of Fas mRNA transcription occurred during PKR activation. Our findings provide direct evidence for the involvement of eIF-2α and NF-κB in the induction of apoptosis by PKR.

Negatively Charged Glyconanoparticles Modulate and Stabilize the Secondary Structures of a gp120 V3 Loop Peptide: Toward Fully Synthetic HIV Vaccine Candidates

The third variable region (V3 peptide) of the HIV-1 gp120 is a major immunogenic domain of HIV-1. Controlling the formation of the immunologically active conformation is a crucial step to the rational design of fully synthetic candidate vaccines. Herein, we present the modulation and stabilization of either the α-helix or β-strand conformation of the V3 peptide by conjugation to negatively charged gold glyconanoparticles (GNPs). The formation of the secondary structure can be triggered by the variation of the buffer concentration and/or pH as indicated by circular dichoism. The peptide on the GNPs shows increased stability toward peptidase degradation as compared to the free peptide. Moreover, only the V3β-GNPs bind to the anti-V3 human broadly neutralizing mAb 447-52D as demonstrated by surface plasmon resonance (SPR). The strong binding of V3β-GNPs to the 447-52D mAb was the starting point to address its study as immunogen. V3β-GNPs elicit antibodies in rabbits that recognize a recombinant gp120 and the serum displayed low but consistent neutralizing activity. These results open up the way for the design of new fully synthetic HIV vaccine candidates.

Activation of NF-κB by the dsRNA-dependent protein kinase, PKR involves the IκB kinase complex

Besides its known role as a translational controlling factor, the double stranded RNA-dependent protein kinase (PKR) is a key transcriptional regulator exerting antiviral and antitumoural activities. We have recently described that induction of NF-κB by PKR is involved in apoptosis commitment. To define how PKR mediates NF-κB activation by dsRNA, we have used two different approaches, one based on expression of PKR by a vaccinia virus (VV) recombinant and the other based on induction of endogenous PKR by poly I:C (pIC) treatment. We found that NF-κB complexes induced by PKR are composed primarily of p50-p65 heterodimers and also of c-rel-p50 heterodimers. As described for other stimuli, following pIC treatment, PKR phosphorylates the NF-κB inhibitor IκBα at serine 32 before degradation. Expression by VV recombinants of IKK1 or IKK2 dominant negative mutants together with PKR showed inhibition of PKR-induced NF-κB activation, as measured both by gel shift and luciferase reporter assays. Immunoprecipitation analysis revealed that PKR interacts with the IKK complex. Our findings demonstrate that physiological function(s) of PKR involve activation of the IκB kinase complex.

TRAF Family Proteins Link PKR with NF-κB Activation

ABSTRACT The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-κB via the IκB kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-κB activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-κB by the dsRNA-dependent protein kinase.

International Network for Comparison of HIV Neutralization Assays: The NeutNet Report

Background Neutralizing antibodies provide markers for vaccine-induced protective immunity in many viral infections. By analogy, HIV-1 neutralizing antibodies induced by immunization may well predict vaccine effectiveness. Assessment of neutralizing antibodies is therefore of primary importance, but is hampered by the fact that we do not know which assay(s) can provide measures of protective immunity. An international collaboration (NeutNet) involving 18 different laboratories previously compared different assays using monoclonal antibodies (mAbs) and soluble CD4 (Phase I study). Methods In the present study (Phase II), polyclonal reagents were evaluated by 13 laboratories. Each laboratory evaluated nine plasmas against an 8 virus panel representing different genetic subtypes and phenotypes. TriMab, a mixture of three mAbs, was used as a positive control allowing comparison of the results with Phase I in a total of nine different assays. The assays used either uncloned virus produced in peripheral blood mononuclear cells (PBMCs) (Virus Infectivity Assays, VIA), or Env (gp160)-pseudotyped viruses (pseudoviruses, PSV) produced in HEK293T cells from molecular clones or from uncloned virus. Target cells included PBMC and genetically engineered cell lines in either single- or multiple-cycle infection format. Infection was quantified by using a range of assay read-outs including extra- or intra-cellular p24 antigen detection, luciferase, beta-galactosidase or green fluorescent protein (GFP) reporter gene expression. Findings Using TriMab, results of Phase I and Phase II were generally in agreement for six of the eight viruses tested and confirmed that the PSV assay is more sensitive than PBMC (p = 0.014). Comparisons with the polyclonal reagents showed that sensitivities were dependent on both virus and plasma. Conclusions Here we further demonstrate clear differences in assay sensitivities that were dependent on both the neutralizing reagent and the virus. Consistent with the Phase I study, we recommend parallel use of PSV and VIA for vaccine evaluation.

Multivalent manno-glyconanoparticles inhibit DC-SIGN-mediated HIV-1 trans-infection of human T cells.

Viral entry is a critical step in human immunodeficiency virus (HIV) infection that takes place through a series of sequential interactions between the envelope glycoprotein gp120, cellular receptor CD4, and coreceptors CCR5 or CXCR4 on T cells. Through a mechanism named trans-infection, dendritic cells (DCs) efficiently transfer the virus to T lymphocytes where viral replication occurs. Interference in HIV transmission in the DC–lymphocyte synapse is a preferential but complex target in the development of new compounds displaying anti-HIV activity. HIV–DC interaction is mediated by the glycans of gp120 and the C-type lectin DC-SIGN (dendritic cell-specific ICAM 3grabbing nonintegrin) expressed on DCs. DC-SIGN is tetrameric and specifically recognizes N-linked high-mannose oligosaccharides (Man9GlcNAc2) through multivalent and Ca -dependent protein–carbohydrate interactions. Mimicking the cluster presentation of the oligomannosides on the surface of the virus is a strategy for designing carbohydrate-based antiviral agents. Mannose multivalent systems based on proteins, peptides, liposomes, and dendrimers as scaffolds have recently been prepared for targeting DCs and tested as inhibitors of DC-SIGN binding to gp120 in ELISA and SPR experiments. Nevertheless, none of these systems has so far been tested in cell-based trans-infection models. Glyconanoparticles (GNPs) are polyvalent, biocompatible sugar-functionalized gold nanoclusters. 10] Glyconanoparticle platforms offer the unique potential for simultaneous incorporation of different ligands in variable densities on a single gold cluster. We have prepared a small library of GNPs (mannoGNPs) coated with sets of different structural motifs of the Nlinked high-mannose undecasaccharide Man9ACHTUNGTRENNUNG(GlcNAc)2 of gp120 (or with an unnatural heptasaccharide) and have observed that GNPs that display multiple copies of the disacchaACHTUNGTRENNUNGride Mana1-2Mana block DC-SIGN/gp120 binding at 120 nm in surface plasmon resonance (SPR) experiments. The mannoGNPs were designed to target DC-SIGN receptors present on DCs by mimicking the clustered carbohydrate display of gp120. In this work we present the results obtained with manno-GNPs (Scheme 1) as inhibitors of DC-SIGN-mediated HIV trans-infection of human activated peripheral blood mononuclear cells (PBMCs). We show that manno-GNPs coated with the linear tetrasaccharide Mana1-2Mana1-2Mana1-3Mana inhibit HIV trans-infection of human T cells similarly to GNPs coated with more complex branched pentaand heptaoligomannosides. Hybrid gold manno-GNPs displaying different densities of linear and branched mannose oligosaccharides were prepared (Scheme 1). The manno-conjugates 6–10 were synthesized by conjugation of ethylamino mannosides 1–5, obtained by the protocol of Wong et al. , with isothiocyanate linker 11 and subsequent removal of the acetyl group (Scheme 1 A). A shorter five-carbon atom aliphatic linker was used for the preparation of 5’-mercaptopentyl b-d-glucopyranoside (GlcC5S), and was hidden internally in order to allow correct presentation of the oligomannosides. Manno-GNPs with 100, 50, or 10 % densities of dimannoside (D-100, D-50, and D-10, respectively), trimannoside (T-50 and T-10), tetramannoside (Te-50 and Te-10), pentamannoside (P-50 and P-10), and heptamannoside (H-50) were prepared from mixtures of manno-conjugates 6–10 and gluco-conjugate GlcC5S by using a previously described methodology (Scheme 1 B). GNPs were characterized by transmission electron microscopy (TEM), H NMR, IR, UV/Visible, and elemental analysis as previously reported. The average number of (oligo)mannosides per GNP and their average molecular weights (Table 1) were calculated from elemental analysis and gold cluster size (1–2 nm as determined by TEM; see the Supporting Information). For trans-infection studies, we used Raji DC-SIGN transfected lymphoblastoid B cells, which can capture and transmit HIV with an efficiency similar to that of monocyte-derived DCs. Manno-GNPs are nontoxic to Raji DC-SIGN and to human activated PBMCs at concentrations of 100 mg mL , as determined by the CellTiter cell viability assay (Figure S9 in the Supporting Information). The activities of manno-GNPs against R5 or X4 HIV-1 were evaluated through an original DC-SIGN transfer assay in which inhibition of HIV-1 infection by GNPs was assessed by use of recombinant viruses carrying the Renilla reporter genes in their genomes. In this assay, inhibition of viral replication is proportional to Renilla-luciferase activity in cell lysates. Briefly, Raji DC-SIGN cells were preincubated with GNPs for 1 h and were then pulsed with JR-Renilla (R5) or NL4.3-Renilla (X4) recombinant viruses for 2 h. Afterwards, the cell cultures were extensively washed and co-cultured with activated PBMCs that would be infected through transfer of the virus bound to DC-SIGN in Raji cells. Viral replication was as[a] Dr. O. Mart nezvila, Dr. M. Marradi, Dr. C. Clavel, Prof. Dr. S. Penad s Laboratory of GlycoNanotechnology, CIC biomaGUNE/CIBER-BBN P8 Miram n 182, 20009 San Sebasti n (Spain) Fax: (+ 34) 943-00-53-01 E-mail : spenades@cicbiomagune.es [b] Dr. L. M. Bedoya, Dr. J. Alcam AIDS Immunopathology Unit, National Center of Microbiology Instituto de Salud Carlos III, Ctra.Majadahonda-Pozuelo Km. 2,200 Madrid (Spain) E-mail : ppalcami@isciii.es [c] Dr. C. Clavel Current address : IBMM, UMR 5247CNRS-Universit s Montpellier 2 et 1 ENSCM, Rue de l’Ecole Normale 8, 34296 Montpellier Cedex 5 (France) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900294.

Genomic organization and promoter characterization of human CXCR4 gene

CXCR4 is the receptor for the CXC chemokine SDF1 that has essential functions on embryo organogenesis, immunological functions and T lymphocyte trafficking. Recently, CXCR4 has drawn unexpected attention as it was recently identified as a co‐factor required for entry of lymphotropic HIV isolates in CD4+ T lymphocytes. CXCR4 is the only SDF1 receptor identified so far. This suggests that CXCR4 expression is critical for the biological effects of SDF1. To investigate the mechanisms controlling both the constitutive and induced expression of CXCR4 receptors we have isolated and characterized the promoter region and determined the genomic structure of the human gene. The CXCR4 gene contains two exons separated by an intronic sequence. A 2.6 kb 5′‐flanking region located upstream the CXCR4 open reading frame contains a TATA box and the transcription start site characteristic of a functional promoter. This region also contains putative consensus binding sequences for different transcription factors, some of them associated with the hemopoiesis and lymphocyte development.