In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone^{1, 2}. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form³ in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells⁴. This indicates that the stability of the capsid is critical for HIV-1 infection.HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability^{4, 5, 6}. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.Download video file.^{(65M, mov)}     

Candida albicans Delays HIV-1 Replication in Macrophages

Macrophages are one of the most important HIV-1 target cells. Unlike CD4⁺ T cells, macrophages are resistant to the cytophatic effect of HIV-1. They are able to produce and harbor the virus for long periods acting as a viral reservoir. Candida albicans (CA) is a commensal fungus that colonizes the portals of HIV-1 entry, such as the vagina and the rectum, and becomes an aggressive pathogen in AIDS patients. In this study, we analyzed the ability of CA to modulate the course of HIV-1 infection in human monocyte-derived macrophages. We found that CA abrogated HIV-1 replication in macrophages when it was evaluated 7 days after virus inoculation. A similar inhibitory effect was observed in monocyte-derived dendritic cells. The analysis of the mechanisms responsible for the inhibition of HIV-1 production in macrophages revealed that CA efficiently sequesters HIV-1 particles avoiding its infectivity. Moreover, by acting on macrophages themselves, CA diminishes their permissibility to HIV-1 infection by reducing the expression of CD4, enhancing the production of the CCR5-interacting chemokines CCL3/MIP-1α, CCL4/MIP-1β, and CCL5/RANTES, and stimulating the production of interferon-α and the restriction factors APOBEC3G, APOBEC3F, and tetherin. Interestingly, abrogation of HIV-1 replication was overcome when the infection of macrophages was evaluated 2-3 weeks after virus inoculation. However, this reactivation of HIV-1 infection could be silenced by CA when added periodically to HIV-1-challenged macrophages. The induction of a silent HIV-1 infection in macrophages at the periphery, where cells are continuously confronted with CA, might help HIV-1 to evade the immune response and to promote resistance to antiretroviral therapy.     

Oligomerization of the Macrophage Mannose Receptor Enhances gp120-mediated Binding of HIV-1

C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.The mannose receptor (MR)² is a C-type lectin receptor that is expressed on the surface of a variety of cells, including immature monocyte-derived dendritic cells (MDDC), dermal dendritic cells, macrophages, and hepatic endothelial cells. It is a multifunctional protein, involved in antigen recognition and internalization during the early stages of the innate immune response (1) as well as physiological clearance of the endogenous pituitary hormones lutropin and thyrotropin (2, 3). Recognition of foreign antigens occurs via mannose, fucose, and GlcNAc residues (4, 5), which are generally not found as terminal residues on mammalian glycoproteins but are highly abundant on surface proteins of pathogens such as the HIV-1 envelope gp120 (6, 7). Once bound, pathogens can be internalized by endocytosis or phagocytosis, where they are targeted to lysosomes for proteolytic degradation and presentation on major histocompatibility complex class II (8). In immature DCs, soluble recombinant HIV envelope proteins are processed by this pathway, initially binding to both dendritic cell-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR but not DC-SIGN in lysosomes (9). Furthermore, in immature DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a unique vesicular compartment that co-localizes with tetraspanin proteins such as CD81 (10, 11). Recently, this compartment has been shown to be continuous with the plasma membrane (11) and does not represent a continuation of the endolysosomal network. Interestingly, this compartment can translocate virus from DCs to CD4 T cells, upon the formation of a virological synapse (10–12). Although viral uptake can occur in DCs independent of HIV env (2), the efficiency of HIV binding and uptake is greatly enhanced by the presence of C-type lectin-env interactions. At least initial binding to DC-SIGN (and most likely also MR) is required for T cell trans-infection (13).Structurally, the extracellular domain of MR consists of an N-terminal cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and eight carbohydrate recognition domains (CRD) on a single polypeptide backbone (1). Of the eight CRDs, CRD 4–8 have been shown to be required for high affinity binding of ligands containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having demonstrable monosaccharide binding in isolation (14). Binding and release of ligand within the low pH environment of the endolysosomal compartment are also Ca²⁺-dependent. Acid-induced removal of Ca²⁺ binding in CRD 4 and 5 was shown to cause a conformational rearrangement of the domain, resulting in a loss of carbohydrate binding activity (15). In contrast, binding of sulfated carbohydrates to the Cys-RD appears to be Ca²⁺-independent as no Ca²⁺-binding sites were observed in its crystal structure (2, 16).Oligomerization of CLRs such as DC-SIGN (17), Langerin (18), and mannose-binding protein (19) has been reported to be essential for binding of oligosaccharide-bearing ligands. Early studies on MR suggested that it exists solely as a monomeric molecule and that clustering of multiple CRDs within the single polypeptide backbone was necessary for high affinity binding of oligosaccharide moieties (20). However, more recent studies have shown that dimerization is possible in the presence of Ca²⁺ (21) and that an equilibrium may exist between monomeric and dimeric forms on the cell surface (22). It is currently unclear what effect dimerization has on ligand binding to the CRDs; however, there is evidence that dimerization of MR is required for high affinity binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate (GalNAc-4-SO₄) such as lutropin and thyrotropin (22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR have used solubilized protein from cell lysates (20) or purified recombinant fragments (21). Because the membrane microenvironment can influence protein associations, soluble forms of MR may not necessarily be a true model of the quaternary structure and function of the native protein. Here, we used a well established method of cross-linking (23) on MDDCs, monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to preserve lateral protein-protein interactions between MR on the cell surface prior to solubilization. Mass spectrometry analysis of affinity-purified complexes showed they were homo-oligomers, and further resolution of the complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates that they are dimers. Dimerization of MR was also found to be essential for binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral particles. Persistence of monomeric gp120 and trimeric gp140 binding to dimeric MR in the presence of EGTA and various CRD and other inhibitors, however, suggested that gp120-mediated HIV-1 binding is not Ca²⁺-dependent and that at least binding probably occurs to both Ca²⁺-dependent and -independent CRDs and also the Cys-RD.     

Interactions of HIV-1 Inhibitory Peptide T20 with the gp41 N-HR Coiled Coil

Cellular entry of human immunodeficiency virus type 1 (HIV-1) involves fusion of viral and cellular membranes and is mediated by structural transitions in viral glycoprotein gp41. The antiviral C-peptide T20 targets the gp41 N-terminal heptad repeat region (N-HR), blocking gp41 conformational changes essential for the entry process. To probe the T20 structure-activity relationship, we engineered a molecular mimic of the entire gp41 N-HR coiled coil using the 5-Helix design strategy. T20 bound this artificial protein (denoted 5H-ex) with nanomolar affinity (K_D = 30 nm), close to its IC₅₀ concentration (∼3 nm) but much weaker than the affinity of a related inhibitory C-peptide C37 (K_D = 0.0007 nm). T20/C37 competitive binding assays confirmed that T20 interacts with the hydrophobic groove on the surface of the N-HR coiled coil outside of a deep pocket region crucial for C37 binding. We used 5H-ex to investigate how the T20 N and C termini contributed to the inhibitor binding activity. Mutating three aromatic residues at the T20 C terminus (WNWF → ANAA) had no effect on affinity, suggesting that these amino acids do not participate in T20 binding to the gp41 N-HR. The results support recent evidence pointing to a different role for these residues in T20 inhibition (Peisajovich, S. G., Gallo, S. A., Blumenthal, R., and Shai, Y. (2003) J. Biol. Chem. 278, 21012–21017; Liu, S., Jing, W., Cheung, B., Lu, H., Sun, J., Yan, X., Niu, J., Farmar, J., Wu, S., and Jiang, S. (2007) J. Biol. Chem. 282, 9612–9620). By contrast, mutations near the T20 N terminus substantially influenced inhibitor binding strength. When Ile was substituted for Thr in the second T20 position, a 40-fold increase in binding affinity was measured (K_D = 0.75 nm). The effect of this affinity enhancement on T20 inhibitory potency varied among different viral strains. The original T20 and the higher affinity T20 variant had similar potency against wild type HIV-1. However, the higher affinity T20 variant was significantly more potent against T20-resistant virus. The findings suggest that other factors in addition to binding affinity play a role in limiting T20 potency. As a mimetic of the complete gp41 N-HR coiled coil region, 5H-ex will be a useful tool to further elucidate mechanistic profiles of C-peptide inhibitors.The HIV-1² surface glycoprotein Env promotes viral entry through the fusion of viral and cellular membranes (3). Env consists of three gp120 surface subunits and three gp41 transmembrane subunits arranged as a trimer-of-heterodimers on the virion surface. In the current model of HIV-1 entry, cellular receptor binding to gp120 initiates a series of coordinated structural transformations that stimulate gp41 to extend and insert its N-terminal fusion peptide into target cell membranes (see Fig. 1A) (4, 5). This high energy extended intermediate structure ultimately collapses into a trimer-of-hairpins conformation that juxtaposes the gp41 fusion peptide and transmembrane domain. Because the fusion peptide and transmembrane domain are inserted in target cell and viral membranes, formation of the trimer-of-hairpins is proposed to bring these membranes into the close proximity required for efficient fusion.Open in a separate windowFIGURE 1.HIV-1 gp41 and its role in viral membrane fusion. A, a model of HIV-1 entry (46). In native Env prior to receptor activation, gp41 is held in a metastable conformation by a canopy of gp120 proteins (green). Receptor binding to gp120 stimulates gp41 to extend and insert its fusion peptide segment (red) into the target cell membrane. The N-HR (gray) and C-HR (blue) regions of the gp41 ectodomain are transiently exposed in this prehairpin state. Subsequently, gp41 collapses into the trimer-of-hairpins conformation that brings the gp41 fusion peptides, transmembrane regions (purple), and their associated membranes into the close proximity for membrane fusion. The actual disposition of gp120 in both the prehairpin and trimer-of-hairpins states is uncertain; for clarity, the protein is omitted in the schematic of the trimer-of-hairpins conformation. B, a diagram of HIV-1 gp41 identifying its fusion peptide (FP), N-HR, C-HR, MPER (MP), transmembrane (TM), and cytoplasmic (cyto) domains. Amino acid sequences above and below the diagram are derived from the N-HR and C-HR/MPER regions of Env_HXB2; all but the MPER sequence WNWF (magenta) were used in the design of 5H-ex. The N-HR and C-HR segments found in the original 5-Helix are boxed in gray and blue, respectively, whereas the sequences of C37 and T20 are denoted by lines. The side chains of the C-HR amino acids marked with + pack into the hydrophobic pocket at the C terminus of the N-HR coiled coil.The core of the trimer-of-hairpins is a bundle of six α-helices formed by two hydrophobic heptad repeat sequences in the N- and C-terminal regions of the gp41 ectodomain (N-HR and C-HR, respectively) (6, 7). In the trimer-of-hairpins, the N-HR segments from three gp41 ectodomains form a central trimeric coiled coil, around which the three C-HR segments pack as antiparallel helices into hydrophobic grooves (8–11). In the prehairpin extended conformation, the N-HR and C-HR segments are unassociated and transiently accessible to inhibitors of HIV-1 entry (5, 12). Several such inhibitors are formed from the peptide sequence of the C-HR and adjacent gp41 regions (4, 6, 13, 14). Denoted C-peptides, they work in a dominant negative fashion by binding to the exposed N-HR coiled coil, thereby blocking trimer-of-hairpins formation and inhibiting viral membrane fusion (4, 15–21). One C-peptide, T20 (also called enfuvirtide), has shown antiviral activity in vivo and has been approved for use in the treatment of HIV-1 infection (22, 23).T20 is a 36-amino acid peptide extending from Tyr⁶³⁸ in the middle of the C-HR to Phe⁶⁷³ in the Trp-rich membrane proximal external region (MPER) that precedes the gp41 transmembrane domain (residue numbering is according to the Env_HXB2 sequence; see Fig. 1B) (13). In T20, these C-terminal MPER-derived residues are critical for inhibitory activity, although their structure and function in the gp41-bound state are currently unknown (1, 24, 25). A second class of similarly potent C-peptides includes C34 (residues 628–661) and the slightly larger C37 (residues 625–661) (4, 6, 26, 27). These peptides are derived entirely from the C-HR sequence and thus are shifted in the N-terminal direction compared with T20 (Fig. 1B). The interactions of C34 and C37 with gp41 are greatly stabilized by residues Trp⁶²⁸, Trp⁶³¹, and Ile⁶³⁵ near the C-HR N terminus (4). Their bulky hydrophobic side chains pack into a deep hydrophobic pocket on the surface of the N-HR coiled coil. T20 lacks these pocket binding residues and their stabilizing effect. However, T20 does contain bulky hydrophobic residues (Trp⁶⁷⁰, Trp⁶⁷², and Phe⁶⁷³) at its C terminus that might pack into a similar pocket at the other end of the N-HR coiled coil.High resolution structures of the gp41 trimer-of-hairpins have aided our understanding of the mechanism of C-peptide inhibition. These structures have enabled the design of polypeptides that mimic the gp41 N-HR coiled coil and bind C34/C37, thereby providing a tool to probe the structure-activity relationships of the inhibitors (26, 28–30). No similar tool is available for investigating T20 inhibition in detail. The structures of the gp41 trimer-of-hairpins do not include the T20 C terminus (9 residues) nor the gp41 N-terminal segments that putatively interact with it. Furthermore, gp41 N-HR-derived peptides predicted to interact with T20 are poorly soluble and difficult to use in solution phase interaction assays (6). Here we describe the design of a soluble protein (denoted 5H-ex) that mimics the putative T20-binding site on the N-HR coiled coil. 5H-ex interacts with T20 with an equilibrium dissociation constant (K_D) of 30 nm, close to the T20 50% inhibitory concentration (IC₅₀) of 3 nm. Using this protein, we explored the extent to which the N and C termini of T20 contribute to its binding activity. First, we showed that the MPER-derived residues at the peptide C terminus do not stabilize the 5H-ex/T20 interaction. Second, we identified an N-terminal substitution that significantly enhanced T20 binding affinity and improved peptide inhibitory activity against T20-resistant HIV-1. The results suggest that T20 binding to the N-HR coiled coil is stabilized primarily by residues derived from the C-HR and not the MPER. 5H-ex is likely to be a useful tool in probing the structure-activity relationship of T20.     

In Vivo Functions of CPSF6 for HIV-1 as Revealed by HIV-1 Capsid Evolution in HLA-B27-Positive Subjects

The host protein CPSF6 possesses a domain that can interact with the HIV-1 capsid (CA) protein. CPSF6 has been implicated in regulating HIV-1 nuclear entry. However, its functional significance for HIV-1 replication has yet to be firmly established. Here we provide evidence for two divergent functions of CPSF6 for HIV-1 replication in vivo. We demonstrate that endogenous CPSF6 exerts an inhibitory effect on naturally occurring HIV-1 variants in individuals carrying the HLA-B27 allele. Conversely, we find a strong selective pressure in these individuals to preserve CPSF6 binding, while escaping from the restrictive activity by CPSF6. This active maintenance of CPSF6 binding during HIV-1 CA evolution in vivo contrasts with the in vitro viral evolution, which can reduce CPSF6 binding to evade from CPSF6-mediated restriction. Thus, these observations argue for a beneficial role of CPSF6 for HIV-1 in vivo. CPSF6-mediated restriction renders HIV-1 less dependent or independent from TNPO3, RanBP2 and Nup153, host factors implicated in HIV-1 nuclear entry. However, viral evolution that maintains CPSF6 binding in HLA-B27+ subjects invariably restores the ability to utilize these host factors, which may be the major selective pressure for CPSF6 binding in vivo. Our study uncovers two opposing CA-dependent functions of CPSF6 in HIV-1 replication in vivo; however, the benefit for binding CPSF6 appears to outweigh the cost, providing support for a vital function of CPSF6 during HIV-1 replication in vivo.     

In Silico Prediction of Mutant HIV-1 Proteases Cleaving a Target Sequence

HIV-1 protease represents an appealing system for directed enzyme re-design, since it has various different endogenous targets, a relatively simple structure and it is well studied. Recently Chaudhury and Gray (Structure (2009) 17: 1636–1648) published a computational algorithm to discern the specificity determining residues of HIV-1 protease. In this paper we present two computational tools aimed at re-designing HIV-1 protease, derived from the algorithm of Chaudhuri and Gray. First, we present an energy-only based methodology to discriminate cleavable and non cleavable peptides for HIV-1 proteases, both wild type and mutant. Secondly, we show an algorithm we developed to predict mutant HIV-1 proteases capable of cleaving a new target substrate peptide, different from the natural targets of HIV-1 protease. The obtained in silico mutant enzymes were analyzed in terms of cleavability and specificity towards the target peptide using the energy-only methodology. We found two mutant proteases as best candidates for specificity and cleavability towards the target sequence.     

The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.     

HIV-1 Tat protein directly induces mitochondrial membrane permeabilization and inactivates cytochrome c oxidase

The Trans-activator protein (Tat) of human immunodeficiency virus (HIV) is a pleiotropic protein involved in different aspects of AIDS pathogenesis. As a number of viral proteins Tat is suspected to disturb mitochondrial function. We prepared pure synthetic full-length Tat by native chemical ligation (NCL), and Tat peptides, to evaluate their direct effects on isolated mitochondria. Submicromolar doses of synthetic Tat cause a rapid dissipation of the mitochondrial transmembrane potential (ΔΨ_m) as well as cytochrome c release in mitochondria isolated from mouse liver, heart, and brain. Accordingly, Tat decreases substrate oxidation by mitochondria isolated from these tissues, with oxygen uptake being initially restored by adding cytochrome c. The anion-channel inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) protects isolated mitochondria against Tat-induced mitochondrial membrane permeabilization (MMP), whereas ruthenium red, a ryanodine receptor blocker, does not. Pharmacologic inhibitors of the permeability transition pore, Bax/Bak inhibitors, and recombinant Bcl-2 and Bcl-XL proteins do not reduce Tat-induced MMP. We finally observed that Tat inhibits cytochrome c oxidase (COX) activity in disrupted mitochondria isolated from liver, heart, and brain of both mouse and human samples, making it the first described viral protein to be a potential COX inhibitor.     

Efficacy and Safety of Three Antiretroviral Regimens for Initial Treatment of HIV-1: A Randomized Clinical Trial in Diverse Multinational Settings

Background

Antiretroviral regimens with simplified dosing and better safety are needed to maximize the efficiency of antiretroviral delivery in resource-limited settings. We investigated the efficacy and safety of antiretroviral regimens with once-daily compared to twice-daily dosing in diverse areas of the world.

Methods and Findings

1,571 HIV-1-infected persons (47% women) from nine countries in four continents were assigned with equal probability to open-label antiretroviral therapy with efavirenz plus lamivudine-zidovudine (EFV+3TC-ZDV), atazanavir plus didanosine-EC plus emtricitabine (ATV+DDI+FTC), or efavirenz plus emtricitabine-tenofovir-disoproxil fumarate (DF) (EFV+FTC-TDF). ATV+DDI+FTC and EFV+FTC-TDF were hypothesized to be non-inferior to EFV+3TC-ZDV if the upper one-sided 95% confidence bound for the hazard ratio (HR) was ≤1.35 when 30% of participants had treatment failure.An independent monitoring board recommended stopping study follow-up prior to accumulation of 472 treatment failures. Comparing EFV+FTC-TDF to EFV+3TC-ZDV, during a median 184 wk of follow-up there were 95 treatment failures (18%) among 526 participants versus 98 failures among 519 participants (19%; HR 0.95, 95% CI 0.72–1.27; p = 0.74). Safety endpoints occurred in 243 (46%) participants assigned to EFV+FTC-TDF versus 313 (60%) assigned to EFV+3TC-ZDV (HR 0.64, CI 0.54–0.76; p<0.001) and there was a significant interaction between sex and regimen safety (HR 0.50, CI 0.39–0.64 for women; HR 0.79, CI 0.62–1.00 for men; p = 0.01). Comparing ATV+DDI+FTC to EFV+3TC-ZDV, during a median follow-up of 81 wk there were 108 failures (21%) among 526 participants assigned to ATV+DDI+FTC and 76 (15%) among 519 participants assigned to EFV+3TC-ZDV (HR 1.51, CI 1.12–2.04; p = 0.007).

Conclusion

EFV+FTC-TDF had similar high efficacy compared to EFV+3TC-ZDV in this trial population, recruited in diverse multinational settings. Superior safety, especially in HIV-1-infected women, and once-daily dosing of EFV+FTC-TDF are advantageous for use of this regimen for initial treatment of HIV-1 infection in resource-limited countries. ATV+DDI+FTC had inferior efficacy and is not recommended as an initial antiretroviral regimen.

Trial Registration

www.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00084136","term_id":"NCT00084136"}}NCT00084136 Please see later in the article for the Editors'' Summary.     

Inhibition of HIV-1 Integrase gene expression by 10-23 DNAzyme

HIV Integrase (IN) is an enzyme that is responsible for the integration of the proviral genome into the human genome, and this integration step is the first step of the virus hijacking the human cell machinery for its propagation and replication. 10-23 DNAzyme has the potential to suppress gene expressions through sequence-specific mRNA cleavage. We have designed three novel DNAzymes, DIN54, DIN116, and DIN152, against HIV-1 Integrase gene using Mfold software and evaluated them for target site cleavage activity on the in vitro transcribed mRNA. All DNAzymes were tested for its inhibition of expression of HIV Integrase protein in the transiently transfected cell lines. DIN116 and DIN152 inhibited IN-EGFP expression by 80 percent and 70 percent respectively.     

A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo

The CD4 binding site (CD4bs) on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs) to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC₈₀ value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.     

In Vitro HIV-1 Evolution in Response to Triple Reverse Transcriptase Inhibitors & In Silico Phenotypic Analysis

Background

Effectiveness of ART regimens strongly depends upon complex interactions between the selective pressure of drugs and the evolution of mutations that allow or restrict drug resistance.

Methods

Four clinical isolates from NRTI-exposed, NNRTI-naive subjects were passaged in increasing concentrations of NVP in combination with 1 µM 3 TC and 2 µM ADV to assess selective pressures of multi-drug treatment. A novel parameter inference procedure, based on a stochastic viral growth model, was used to estimate phenotypic resistance and fitness from in vitro combination passage experiments.

Results

Newly developed mathematical methods estimated key phenotypic parameters of mutations arising through selective pressure exerted by 3 TC and NVP. Concentrations of 1 µM 3 TC maintained the M184V mutation, which was associated with intrinsic fitness deficits. Increasing NVP concentrations selected major NNRTI resistance mutations. The evolutionary pathway of NVP resistance was highly dependent on the viral genetic background, epistasis as well as stochasticity. Parameter estimation indicated that the previously unrecognized mutation L228Q was associated with NVP resistance in some isolates.

Conclusion

Serial passage of viruses in the presence of multiple drugs may resemble the selection of mutations observed among treated individuals and populations in vivo and indicate evolutionary preferences and restrictions. Phenotypic resistance estimated here “in silico” from in vitro passage experiments agreed well with previous knowledge, suggesting that the unique combination of “wet-” and “dry-lab” experimentation may improve our understanding of HIV-1 resistance evolution in the future.