Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers.

Inhibition of HIV type 1 infectivity by constrained α-helical peptides: Implications for the viral fusion mechanism

Linear peptides derived from the membrane proximal region of the gp41 ectodomain are effective inhibitors of HIV type 1 (HIV-1)-mediated fusion events. These inhibitory peptides lack structure in solution, rendering mechanistic interpretation of their activity difficult. Using structurally constrained analogs of these molecules, we demonstrate that the peptides inhibit infectivity by adopting a helical conformation. Moreover, we show that a specific face of the helix must be exposed to block viral infectivity. Recent crystal structures show that the region of gp41 corresponding to the inhibitory peptides is helical and uses the analogous face to pack against a groove formed by an N-terminal coiled-coil trimer. Our results provide a direct link between the inhibition of HIV-1 infectivity by these peptides and the x-ray structures, and suggest that the conformation of gp41 observed by crystallography represents the fusogenic state. Other agents that block HIV-1 infectivity by binding to this groove may hold promise for the treatment of AIDS.

Modeling Breastmilk Infectivity in HIV-1 Infected Mothers

Estimation of breastmilk infectivity in HIV-1 infected mothers is difficult because transmission can occur while the fetus is in-utero, during delivery, or through breastfeeding. Since transmission can only be detected through periodic testing, however, it may be impossible to determine the actual mode of transmission in any individual child. In this paper we develop a model to estimate breastmilk infectivity as well as the probabilities of in-utero and intrapartum transmission. In addition, the model allows separate estimation of early and late breastmilk infectivity and individual variation in maternal infectivity. Methods for hypothesis testing of binary risk factors and a method for assessing goodness of fit are also described. Data from a randomized trial of breastfeeding versus formula feeding among HIV-1 infected mothers in Nairobi, Kenya are used to illustrate the methods.

Quantification of HIV-1 viral RNA in the blood in needles used for venous puncture in HIV-infected individuals

INTRODUCTION: Occupational HIV infection among healthcare workers is an important issue in exposures involving blood and body fluids. There are few data in the literature regarding the potential and the duration of infectivity of HIV type 1 (HIV-1) in contaminated material under adverse conditions. METHODS: We quantified HIV-1 viral RNA in 25×8mm calibre hollow-bore needles, after punctures, in 25 HIV-1-infected patients selected during the sample collection. All of the patients selected were between the ages of 18 and 55. Five samples were collected from 16 patients: one sample for the immediate quantification of HIV-1 RNA in the plasma and blood samples from the interior of 4 needles to be analyzed at 0h, 6h, 24h, and 72h after collection. In nine patients, another test was carried out in the blood from one additional needle, in which HIV-1 RNA was assessed 168h after blood collection. The method used to assess HIV-1 RNA was nucleic acid sequence-based amplification. RESULTS: Up to 7 days after collection, HIV-1 RNA was detected in all of the needles. The viral RNA remained stable up to 168h, and there were no statistically significant differences among the needle samples. CONCLUSIONS: Although the infectivity of the viral material in the needles is unknown, the data indicate the need to re-evaluate the practices in cases of occupational accidents in which the source is not identified.

Counteraction of HLA-C-mediated immune control of HIV-1 by Nef.

A host genetic variant (-35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface, whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However, some individuals with the protective -35CC genotype exhibit high viral loads. Here, we investigated whether the ability of HIV-1 to replicate efficiently in the "protective" high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However, in individuals with the protective -35CC genotype we found a significant association between sVLs and the efficiency of Nef-mediated enhancement of virion infectivity and modulation of CD4, CD28, and the major histocompatibility complex class II (MHC-II)-associated invariant chain (Ii), while this was not observed in subjects with the -35TT genotype. Since the latter Nef functions all influence the stimulation of CD4(+) T helper cells by antigen-presenting cells, they may cooperate to affect both the activation status of infected T cells and the generation of an antiviral cytotoxic T-lymphocyte (CTL) response. In comparison, different levels of viremia in individuals with the common -35TT genotype were not associated with differences in Nef function but with differences in HLA-C mRNA expression levels. Thus, while high HLA-C expression may generally facilitate control of HIV-1, Nef may counteract HLA-C-mediated immune control in some individuals indirectly, by manipulating T-cell function and MHC-II antigen presentation.

Human TOP1 residues implicated in species specificity of HIV-1 infection are required for interaction with BTBD2, and RNAi of BTBD2 in old world monkey and human cells increases permissiveness to HIV-1 infection

Host determinants of HIV-1 viral tropism include factors from producer cells that affect the efficiency of productive infection and factors in target cells that block infection after viral entry. TRIM5 restricts HIV-1 infection at an early post-entry step through a mechanism associated with rapid disassembly of the retroviral capsid. Topoisomerase I (TOP1) appears to play a role in HIV-1 viral tropism by incorporating into or otherwise modulating virions affecting the efficiency of a post-entry step, as the expression of human TOP1 in African Green Monkey (AGM) virion-producing cells increased the infectivity of progeny virions by five-fold. This infectivity enhancement required human TOP1 residues 236 and 237 as their replacement with the AGM counterpart residues abolished the infectivity enhancement. Our previous studies showed that TOP1 interacts with BTBD1 and BTBD2, two proteins which co-localize with the TRIM5 splice variant TRIM5 in cytoplasmic bodies. Because BTBD1 and BTBD2 interact with one HIV-1 viral tropism factor, TOP1, and co-localize with a splice variant of another, we investigated the potential involvement of BTBD1 and BTBD2 in HIV-1 restriction.

Inhibition of the Nef regulatory protein of HIV-1 by a single-domain antibody

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (K(d) = 2 × 10(-9)M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4(+) T-cell maturation defects and peripheral CD4(+) T-cell activation in the CD4C/HIV-1(Nef) transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

Identification of a highly conserved valine-glycine-phenylalanine amino acid triplet required for HIV-1 Nef function

Background The Nef protein of HIV facilitates virus replication and disease progression in infected patients. This role as pathogenesis factor depends on several genetically separable Nef functions that are mediated by interactions of highly conserved protein-protein interaction motifs with different host cell proteins. By studying the functionality of a series of nef alleles from clinical isolates, we identified a dysfunctional HIV group O Nef in which a highly conserved valine-glycine-phenylalanine (VGF) region, which links a preceding acidic cluster with the following proline-rich motif into an amphipathic surface was deleted. In this study, we aimed to study the functional importance of this VGF region. Results The dysfunctional HIV group O8 nef allele was restored to the consensus sequence, and mutants of canonical (NL4.3, NA-7, SF2) and non-canonical (B2 and C1422) HIV-1 group M nef alleles were generated in which the amino acids of the VGF region were changed into alanines (VGF→AAA) and tested for their capacity to interfere with surface receptor trafficking, signal transduction and enhancement of viral replication and infectivity. We found the VGF motif, and each individual amino acid of this motif, to be critical for downregulation of MHC-I and CXCR4. Moreover, Nef’s association with the cellular p21-activated kinase 2 (PAK2), the resulting deregulation of cofilin and inhibition of host cell actin remodeling, and targeting of Lck kinase to the trans-golgi-network (TGN) were affected as well. Of particular interest, VGF integrity was essential for Nef-mediated enhancement of HIV virion infectivity and HIV replication in peripheral blood lymphocytes. For targeting of Lck kinase to the TGN and viral infectivity, especially the phenylalanine of the triplet was essential. At the molecular level, the VGF motif was required for the physical interaction of the adjacent proline-rich motif with Hck. Conclusion Based on these findings, we propose that this highly conserved three amino acid VGF motif together with the acidic cluster and the proline-rich motif form a previously unrecognized amphipathic surface on Nef. This surface appears to be essential for the majority of Nef functions and thus represents a prime target for the pharmacological inhibition of Nef.

Crystal structure of chemically synthesized [N33A] stromal cell-derived factor 1α, a potent ligand for the HIV-1 “fusin” coreceptor

Stromal cell-derived factor-1α (SDF-1α ) is a member of the chemokine superfamily and functions as a growth factor and chemoattractant through activation of CXCR4/LESTR/Fusin, a G protein-coupled receptor. This receptor also functions as a coreceptor for T-tropic syncytium-inducing strains of HIV-1. SDF-1α antagonizes infectivity of these strains by competing with gp120 for binding to the receptor. The crystal structure of a variant SDF-1α ([N33A]SDF-1α ) prepared by total chemical synthesis has been refined to 2.2-Å resolution. Although SDF-1α adopts a typical chemokine β-β-β-α topology, the packing of the α-helix against the β-sheet is strikingly different. Comparison of SDF-1α with other chemokine structures confirms the hypothesis that SDF-1α may be either an ancestral protein from which all other chemokines evolved or the chemokine that is the least divergent from a primordial chemokine. The structure of SDF-1α reveals a positively charged surface ideal for binding to the negatively charged extracellular loops of the CXCR4 HIV-1 coreceptor. This ionic complementarity is likely to promote the interaction of the mobile N-terminal segment of SDF-1α with interhelical sites of the receptor, resulting in a biological response.

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21–65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21–65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41–65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21–65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21–65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31–8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.

Proteasome inhibition interferes with Gag polyprotein processing, release, and maturation of HIV-1 and HIV-2

Retrovirus assembly and maturation involve folding and transport of viral proteins to the virus assembly site followed by subsequent proteolytic cleavage of the Gag polyprotein within the nascent virion. We report that inhibiting proteasomes severely decreases the budding, maturation, and infectivity of HIV. Although processing of the Env glycoproteins is not changed, proteasome inhibitors inhibit processing of Gag polyprotein by the viral protease without affecting the activity of the HIV-1 viral protease itself, as demonstrated by in vitro processing of HIV-1 Gag polyprotein Pr55. Furthermore, this effect occurs independently of the virus release function of the HIV-1 accessory protein Vpu and is not limited to HIV-1, as proteasome inhibitors also reduce virus release and Gag processing of HIV-2. Electron microscopy analysis revealed ultrastructural changes in budding virions similar to mutants in the late assembly domain of p6gag, a C-terminal domain of Pr55 required for efficient virus maturation and release. Proteasome inhibition reduced the level of free ubiquitin in HIV-1-infected cells and prevented monoubiquitination of p6gag. Consistent with this, viruses with mutations in PR or p6gag were resistant to detrimental effects mediated by proteasome inhibitors. These results indicate the requirement for an active proteasome/ubiquitin system in release and maturation of infectious HIV particles and provide a potential pharmaceutical strategy for interfering with retrovirus replication.

Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP–neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.

Amphipathic domains in the C terminus of the transmembrane protein (gp41) permeabilize HIV-1 virions: a molecular mechanism underlying natural endogenous reverse transcription.

Reverse transcription of HIV-1, without detergent or amphipathic peptide-induced permeability of the viral envelope, has been demonstrated to occur in the intact HIV-1 virion. In this report, we demonstrate that the amphipathic domains in the C terminus of the transmembrane glycoprotein (gp41) account for the natural permeability of the HIV-1 envelope to deoxyribonucleoside triphosphates, the substrates for DNA polymerization. In addition, nonphysiological deoxyribonucleoside triphosphates, such as 3'-azido-3'-deoxythymidine 5'-triphosphate and 3'-deoxythymidine 5'-triphosphate, can also penetrate the viral envelope, incorporate into, and irreversibly terminate reverse transcripts. As a result, viral infectivity is potently inhibited. Since the lentiviral envelope with these newly demonstrated characteristics can serve as a delivery pathway for anti-reverse transcription agents, we propose a unique strategy to prevent HIV-1 interand, possibly, intrahost transmission.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.