Rab11-FIP1C Is Dispensable for HIV-1 Replication in Primary CD4+ T Cells, but Its Role Is Cell Type Dependent in Immortalized Human T-Cell Lines.

The HIV-1 envelope glycoprotein (Env) contains a long cytoplasmic tail harboring highly conserved motifs that direct Env trafficking and incorporation into virions and promote efficient virus spread. The cellular trafficking factor Rab11a family interacting protein 1C (FIP1C) has been implicated in the directed trafficking of Env to sites of viral assembly. In this study, we confirm that small interfering RNA (siRNA)-mediated depletion of FIP1C in HeLa cells modestly reduces Env incorporation into virions. To determine whether FIP1C is required for Env incorporation and HIV-1 replication in physiologically relevant cells, CRISPR-Cas9 technology was used to knock out the expression of this protein in several human T-cell lines-Jurkat E6.1, SupT1, and H9-and in primary human CD4+ T cells. FIP1C knockout caused modest reductions in Env incorporation in SupT1 cells but did not inhibit virus replication in SupT1 or Jurkat E6.1 T cells. In H9 cells, FIP1C knockout caused a cell density-dependent defect in virus replication. In primary CD4+ T cells, FIP1C knockout had no effect on HIV-1 replication. Furthermore, human T-cell leukemia virus type 1 (HTLV-1)-transformed cell lines that are permissive for HIV-1 replication do not express FIP1C. Mutation of an aromatic motif in the Env cytoplasmic tail (Y795W) implicated in FIP1C-mediated Env incorporation impaired virus replication independently of FIP1C expression in SupT1, Jurkat E6.1, H9, and primary T cells. Together, these results indicate that while FIP1C may contribute to HIV-1 Env incorporation in some contexts, additional and potentially redundant host factors are likely required for Env incorporation and virus dissemination in T cells. IMPORTANCE The incorporation of the HIV-1 envelope (Env) glycoproteins, gp120 and gp41, into virus particles is critical for virus infectivity. gp41 contains a long cytoplasmic tail that has been proposed to interact with host cell factors, including the trafficking factor Rab11a family interacting protein 1C (FIP1C). To investigate the role of FIP1C in relevant cell types-human T-cell lines and primary CD4+ T cells-we used CRISPR-Cas9 to knock out FIP1C expression and examined the effect on HIV-1 Env incorporation and virus replication. We observed that in two of the T-cell lines examined (Jurkat E6.1 and SupT1) and in primary CD4+ T cells, FIP1C knockout did not disrupt HIV-1 replication, whereas FIP1C knockout reduced Env expression and delayed replication in H9 cells. The results indicate that while FIP1C may contribute to Env incorporation in some cell lines, it is not an essential factor for efficient HIV-1 replication in primary CD4+ T cells.

Biochemical evidence of a role for matrix trimerization in HIV-1 envelope glycoprotein incorporation.

The matrix (MA) domain of HIV Gag has important functions in directing the trafficking of Gag to sites of assembly and mediating the incorporation of the envelope glycoprotein (Env) into assembling particles. HIV-1 MA has been shown to form trimers in vitro; however, neither the presence nor the role of MA trimers has been documented in HIV-1 virions. We developed a cross-linking strategy to reveal MA trimers in virions of replication-competent HIV-1. By mutagenesis of trimer interface residues, we demonstrated a correlation between loss of MA trimerization and loss of Env incorporation. Additionally, we found that truncating the long cytoplasmic tail of Env restores incorporation of Env into MA trimer-defective particles, thus rescuing infectivity. We therefore propose a model whereby MA trimerization is required to form a lattice capable of accommodating the long cytoplasmic tail of HIV-1 Env; in the absence of MA trimerization, Env is sterically excluded from the assembling particle. These findings establish MA trimerization as an obligatory step in the assembly of infectious HIV-1 virions. As such, the MA trimer interface may represent a novel drug target for the development of antiretrovirals.

Identification of an HIV-1 Mutation in Spacer Peptide 1 That Stabilizes the Immature CA-SP1 Lattice.

UNLABELLED: Upon release of HIV-1 particles from the infected cell, the viral protease cleaves the Gag polyprotein at specific sites, triggering maturation. During this process, which is essential for infectivity, the capsid protein (CA) reassembles into a conical core. Maturation inhibitors (MIs) block HIV-1 maturation by interfering with protease-mediated CA-spacer peptide 1 (CA-SP1) processing, concomitantly stabilizing the immature CA-SP1 lattice; virions from MI-treated cells retain an immature-like CA-SP1 lattice, whereas mutational abolition of cleavage at the CA-SP1 site results in virions in which the CA-SP1 lattice converts to a mature-like form. We previously reported that propagation of HIV-1 in the presence of MI PF-46396 selected for assembly-defective, compound-dependent mutants with amino acid substitutions in the major homology region (MHR) of CA. Propagation of these mutants in the absence of PF-46396 resulted in the acquisition of second-site compensatory mutations. These included a Thr-to-Ile substitution at SP1 residue 8 (T8I), which results in impaired CA-SP1 processing. Thus, the T8I mutation phenocopies PF-46396 treatment in terms of its ability to rescue the replication defect imposed by the MHR mutations and to impede CA-SP1 processing. Here, we use cryo-electron tomography to show that, like MIs, the T8I mutation stabilizes the immature-like CA-SP1 lattice. These results have important implications for the mechanism of action of HIV-1 MIs; they also suggest that T8I may provide a valuable tool for structural definition of the CA-SP1 boundary region, which has thus far been refractory to high-resolution analysis, apparently because of conformational flexibility in this region of Gag. IMPORTANCE: HIV-1 maturation involves dissection of the Gag polyprotein by the viral protease and assembly of a conical capsid enclosing the viral ribonucleoprotein. Maturation inhibitors (MIs) prevent the final cleavage step at the site between the capsid protein (CA) and spacer peptide 1 (SP1), apparently by binding at this site and denying the protease access. Additionally, MIs stabilize the immature-like CA-SP1 lattice, preventing release of CA into the soluble pool. We previously found that T8I, a mutation in SP1, rescues a PF-46396-dependent CA mutant and blocks CA-SP1 cleavage. In this study, we imaged T8I virions by cryo-electron tomography and showed that T8I mutants, like MI-treated virions, contain an immature CA-SP1 lattice. These results lay the groundwork needed to understand the structure of the CA-SP1 interface region and further illuminate the mechanism of action of MIs.

Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain.

UNLABELLED: HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. IMPORTANCE: Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients.

TIM-family proteins inhibit HIV-1 release.

Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors.

MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production.

MicroRNAs (miRNAs) are small, 18-22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA-protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA-Gag complexes interfere with viral-RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA's target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts.

Alkyl Amine Bevirimat Derivatives Are Potent and Broadly Active HIV-1 Maturation Inhibitors.

Concomitant with the release of human immunodeficiency virus type 1 (HIV-1) particles from the infected cell, the viral protease cleaves the Gag polyprotein precursor at a number of sites to trigger virus maturation. We previously reported that a betulinic acid-derived compound, bevirimat (BVM), blocks HIV-1 maturation by disrupting a late step in protease-mediated Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. BVM was shown in multiple clinical trials to be safe and effective in reducing viral loads in HIV-1-infected patients. However, naturally occurring polymorphisms in the SP1 region of Gag (e.g., SP1-V7A) led to a variable response in some BVM-treated patients. The reduced susceptibility of SP1-polymorphic HIV-1 to BVM resulted in the discontinuation of its clinical development. To overcome the loss of BVM activity induced by polymorphisms in SP1, we carried out an extensive medicinal chemistry campaign to develop novel maturation inhibitors. In this study, we focused on alkyl amine derivatives modified at the C-28 position of the BVM scaffold. We identified a set of derivatives that are markedly more potent than BVM against an HIV-1 clade B clone (NL4-3) and show robust antiviral activity against a variant of NL4-3 containing the V7A polymorphism in SP1. One of the most potent of these compounds also strongly inhibited a multiclade panel of primary HIV-1 isolates. These data demonstrate that C-28 alkyl amine derivatives of BVM can, to a large extent, overcome the loss of susceptibility imposed by polymorphisms in SP1.

Global rescue of defects in HIV-1 envelope glycoprotein incorporation: implications for matrix structure.

The matrix (MA) domain of HIV-1 Gag plays key roles in membrane targeting of Gag, and envelope (Env) glycoprotein incorporation into virions. Although a trimeric MA structure has been available since 1996, evidence for functional MA trimers has been elusive. The mechanism of HIV-1 Env recruitment into virions likewise remains unclear. Here, we identify a point mutation in MA that rescues the Env incorporation defects imposed by an extensive panel of MA and Env mutations. Mapping the mutations onto the putative MA trimer reveals that the incorporation-defective mutations cluster at the tips of the trimer, around the perimeter of a putative gap in the MA lattice into which the cytoplasmic tail of gp41 could insert. By contrast, the rescue mutation is located at the trimer interface, suggesting that it may confer rescue of Env incorporation via modification of MA trimer interactions, a hypothesis consistent with additional mutational analysis. These data strongly support the existence of MA trimers in the immature Gag lattice and demonstrate that rescue of Env incorporation defects is mediated by modified interactions at the MA trimer interface. The data support the hypothesis that mutations in MA that block Env incorporation do so by imposing a steric clash with the gp41 cytoplasmic tail, rather than by disrupting a specific MA-gp41 interaction. The importance of the trimer interface in rescuing Env incorporation suggests that the trimeric arrangement of MA may be a critical factor in permitting incorporation of Env into the Gag lattice.

The Effect of Inositol Hexakisphosphate on HIV-1 Particle Production and Infectivity can be Modulated by Mutations that Affect the Stability of the Immature Gag Lattice.

The assembly of an HIV-1 particle begins with the construction of a spherical lattice composed of hexamer subunits of the Gag polyprotein. The cellular metabolite inositol hexakisphosphate (IP6) binds and stabilizes the immature Gag lattice via an interaction with the six-helix bundle (6HB), a crucial structural feature of Gag hexamers that modulates both virus assembly and infectivity. The 6HB must be stable enough to promote immature Gag lattice formation, but also flexible enough to be accessible to the viral protease, which cleaves the 6HB during particle maturation. 6HB cleavage liberates the capsid (CA) domain of Gag from the adjacent spacer peptide 1 (SP1) and IP6 from its binding site. This pool of IP6 molecules then promotes the assembly of CA into the mature conical capsid that is required for infection. Depletion of IP6 in virus-producer cells results in severe defects in assembly and infectivity of wild-type (WT) virions. Here we show that in an SP1 double mutant (M4L/T8I) with a hyperstable 6HB, IP6 can block virion infectivity by preventing CA-SP1 processing. Thus, depletion of IP6 in virus-producer cells markedly increases M4L/T8I CA-SP1 processing and infectivity. We also show that the introduction of the M4L/T8I mutations partially rescues the assembly and infectivity defects induced by IP6 depletion on WT virions, likely by increasing the affinity of the immature lattice for limiting IP6. These findings reinforce the importance of the 6HB in virus assembly, maturation, and infection and highlight the ability of IP6 to modulate 6HB stability.

Evidence of a role for soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery in HIV-1 assembly and release.

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.

Enhanced Transmissibility and Decreased Virulence of HIV-1 CRF07_BC May Explain Its Rapid Expansion in China.

HIV-1 CRF07_BC is one of the most common circulating recombinant forms (CRFs) in China and is becoming increasingly prevalent especially in HIV-infected men who have sex with men (MSM). The reason why this strain expanded so quickly in China remains to be defined. We previously observed that individuals infected with HIV-1 CRF07_BC showed slower disease progression than those infected with HIV-1 subtype B or CRF01_AE. CRF07_BC viruses carry two unique mutations in the p6Gag protein: insertion of PTAPPE sequences downstream of the original Tsg101 binding domain, and deletion of a seven-amino-acid sequence (30PIDKELY36) that partially overlaps with the Alix binding domain. In this study, we confirmed the enhanced transmission capability of CRF07_BC over HIV-1 subtype B or CRF01_AE by constructing HIV-1 transmission networks to quantitatively evaluate the growth rate of transmission clusters of different HIV-1 genotypes. We further determined lower virus infectivity and slower replication of CRF07_BC with aforementioned PTAPPE insertion (insPTAP) and/or PIDKELY deletion (Δ7) in the p6Gag protein, which in turn may increase the pool of people infected with CRF07_BC and the risk of HIV-1 transmission. These new features of CRF07_BC may explain its quick spread and will help adjust prevention strategy of HIV-1 epidemic. IMPORTANCE HIV-1 CRF07_BC is one of the most common circulating recombinant forms (CRFs) in China. The question is why and how CRF07_BC expanded so rapidly remains unknown. To address the question, we explored the transmission capability of CRF07_BC by constructing HIV-1 transmission networks to quantitatively evaluate the growth rate of transmission clusters of different HIV-1 genotypes. We further characterized the role of two unique mutations in CRF07_BC, PTAPPE insertion (insPTAP) and/or PIDKELY deletion (Δ7) in the p6Gag in virus replication. Our results help define the molecular mechanism regarding the association between the unique mutations and the slower disease progression of CRF07_BC as well as the quick spread of CRF07_BC in China.

Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail.

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.

Functional replacement of a retroviral late domain by ubiquitin fusion.

Retroviral Gag polyprotein precursors are both necessary and sufficient for the assembly and release of virus-like particles (VLPs) from infected cells. It is well established that small Gag-encoded motifs, known as late domains, promote particle release by interacting with components of the cellular endosomal sorting and ubiquitination machinery. The Gag proteins of a number of different retroviruses are ubiquitinated; however, the role of Gag ubiquitination in particle egress remains undefined. In this study, we investigated this question by using a panel of equine infectious anemia virus (EIAV) Gag derivatives bearing the wild-type EIAV late domain, heterologous retroviral late domains or no late domain. Ubiquitin was fused in cis to the C-termini of these Gag polyproteins, and the effects on VLP budding were measured. Remarkably, fusion of ubiquitin to EIAV Gag lacking a late domain (EIAV/DeltaYPDL-Ub) largely rescued VLP release. We also determined the effects of ubiquitin fusion on the sensitivity of particle release to budding inhibitors and to depletion of key endosomal sorting factors. Ubiquitin fusion rendered EIAV/DeltaYPDL-Ub sensitive to depletion of cellular endosomal sorting factors Tsg101 and Alix and to overexpression of dominant-negative fragments of Tsg101 and Alix. These findings demonstrate that ubiquitin can functionally compensate for the absence of a retroviral late domain and provide insights into the host-cell machinery engaged by ubiquitin during particle egress.

Evidence that productive human immunodeficiency virus type 1 assembly can occur in an intracellular compartment.

Human immunodeficiency virus type 1 (HIV-1) assembly occurs predominantly at the plasma membrane of infected cells. The targeting of assembly to intracellular compartments such as multivesicular bodies (MVBs) generally leads to a significant reduction in virus release efficiency, suggesting that MVBs are a nonproductive site for HIV-1 assembly. In the current study, we make use of an HIV-1 Gag-matrix mutant, 29/31KE, that is MVB targeted. We previously showed that this mutant is severely defective for virus particle production in HeLa cells but more modestly affected in primary macrophages. To more broadly examine the consequences of MVB targeting for virus production, we investigated 29/31KE particle production in a range of cell types. Surprisingly, this mutant supported highly efficient assembly and release in T cells despite its striking MVB Gag localization. Manipulation of cellular endocytic pathways revealed that unlike Vpu-defective HIV-1, which demonstrated intracellular Gag localization as a result of Gag endocytosis from the plasma membrane, 29/31KE mutant Gag was targeted directly to an MVB compartment. The 29/31KE mutant was unable to support multiple-round replication; however, this defect could be reversed by truncating the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 and by the acquisition of a 16EK change in matrix. The 16EK/29/31KE matrix mutant replicated efficiently in the MT-4 T-cell line despite maintaining an MVB-targeting phenotype. These results indicate that MVB-targeted Gag can be efficiently released from T cells and primary macrophages, suggesting that under some circumstances, late endosomal compartments can serve as productive sites for HIV-1 assembly in these physiologically relevant cell types.

Impact of human immunodeficiency virus type 1 resistance to protease inhibitors on evolution of resistance to the maturation inhibitor bevirimat (PA-457).

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.

Real-time visualization of HIV-1 GAG trafficking in infected macrophages.

HIV-1 particle production is driven by the Gag precursor protein Pr55(Gag). Despite significant progress in defining both the viral and cellular determinants of HIV-1 assembly and release, the trafficking pathway used by Gag to reach its site of assembly in the infected cell remains to be elucidated. The Gag trafficking itinerary in primary monocyte-derived macrophages is especially poorly understood. To define the site of assembly and characterize the Gag trafficking pathway in this physiologically relevant cell type, we have made use of the biarsenical-tetracysteine system. A small tetracysteine tag was introduced near the C-terminus of the matrix domain of Gag. The insertion of the tag at this position did not interfere with Gag trafficking, virus assembly or release, particle infectivity, or the kinetics of virus replication. By using this in vivo detection system to visualize Gag trafficking in living macrophages, Gag was observed to accumulate both at the plasma membrane and in an apparently internal compartment that bears markers characteristic of late endosomes or multivesicular bodies. Significantly, the internal Gag rapidly translocated to the junction between the infected macrophages and uninfected T cells following macrophage/T-cell synapse formation. These data indicate that a population of Gag in infected macrophages remains sequestered internally and is presented to uninfected target cells at a virological synapse.

Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus.

All retroviruses encode a Gag polyprotein containing an N-terminal matrix domain (MA) that anchors Gag to the plasma membrane and recruits envelope glycoproteins to virus assembly sites. Membrane binding by the Gag protein of HIV-1 and most other lentiviruses is dependent on N-terminal myristoylation of MA by host N-myristoyltransferase enzymes (NMTs), which recognize a six-residue "myristoylation signal" with consensus sequence: M1GXXX[ST]. For unknown reasons, the feline immunodeficiency virus (FIV), which infects both domestic and wild cats, encodes a non-consensus myristoylation sequence not utilized by its host or by other mammals (most commonly: M1GNGQG). To explore the evolutionary basis for this sequence, we compared the structure, dynamics, and myristoylation properties of native FIV MA with a mutant protein containing a consensus feline myristoylation motif (MANOS) and examined the impact of MA mutations on virus assembly and ability to support spreading infection. Unexpectedly, myristoylation efficiency of MANOS in Escherichia coli by co-expressed mammalian NMT was reduced by ~70% compared to the wild-type protein. NMR studies revealed that residues of the N-terminal myristoylation signal are fully exposed and mobile in the native protein but partially sequestered in the MANOS chimera, suggesting that the unusual FIV sequence is conserved to promote exposure and efficient myristoylation of the MA N terminus. In contrast, virus assembly studies indicate that the MANOS mutation does not affect virus assembly, but does prevent virus spread, in feline kidney cells. Our findings indicate that residues of the FIV myristoylation sequence play roles in replication beyond NMT recognition and Gag-membrane binding.

Inhibition of human immunodeficiency virus type 1 assembly and release by the cholesterol-binding compound amphotericin B methyl ester: evidence for Vpu dependence.

We investigated the mechanism by which the cholesterol-binding compound amphotericin B methyl ester (AME) inhibits human immunodeficiency virus type 1 (HIV-1) particle production. We observed no significant effect of AME on Gag binding to the plasma membrane, Gag association with lipid rafts, or Gag multimerization, indicating that the mechanism of inhibition by AME is distinct from that by cholesterol depletion. Electron microscopy analysis indicated that AME significantly disrupts virion morphology. Interestingly, we found that AME does not inhibit the release of Vpu-defective HIV-1 or Vpu(-) retroviruses such as murine leukemia virus and simian immunodeficiency virus. We demonstrated that the ability of Vpu to counter the activity of CD317/BST-2/tetherin is markedly reduced by AME. These results indicate that AME interferes with the anti-CD317/BST-2/tetherin function of Vpu.

A Triazinone Derivative Inhibits HIV-1 Replication by Interfering with Reverse Transcriptase Activity.

A novel HIV-1 inhibitor, 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (compound 1), was identified from a compound library screened for the ability to inhibit HIV-1 replication. EC50 values of compound 1 were found to range from 107.9 to 145.4 nm against primary HIV-1 clinical isolates. In in vitro assays, HIV-1 reverse transcriptase (RT) activity was inhibited by compound 1 with an EC50 of 4.3 µm. An assay for resistance to compound 1 selected a variant of HIV-1 with a RT mutation (RTL100I ); this frequently identified mutation confers mild resistance to non-nucleoside RT inhibitors (NNRTIs). A recombinant HIV-1 bearing RTL100I exhibited a 41-fold greater resistance to compound 1 than the wild-type virus. Compound 1 was also effective against HIV-1 with RTK103N , one of the major mutations that confers substantial resistance to NNRTIs. Computer-assisted docking simulations indicated that compound 1 binds to the RT NNRTI binding pocket in a manner similar to that of efavirenz; however, the putative compound 1 binding site is located further from RTK103 than that of efavirenz. Compound 1 is a novel NNRTI with a unique drug-resistance profile.

Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.

The human immunodeficiency virus (HIV) hijacks the endosomal sorting complexes required for transport (ESCRT) to mediate virus release from infected cells. The nanoscale organization of ESCRT machinery necessary for mediating viral abscission is unclear. Here, we applied three-dimensional superresolution microscopy and correlative electron microscopy to delineate the organization of ESCRT components at HIV assembly sites. We observed ESCRT subunits localized within the head of budding virions and released particles, with head-localized levels of CHMP2A decreasing relative to Tsg101 and CHMP4B upon virus abscission. Thus, the driving force for HIV release may derive from initial scaffolding of ESCRT subunits within the viral bud interior followed by plasma membrane association and selective remodeling of ESCRT subunits.