Real-Time Visualization of HIV-1 GAG Trafficking in Infected Macrophages

HIV-1 particle production is driven by the Gag precursor protein Pr55Gag. 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

The Role of WWP1-Gag Interaction and Gag Ubiquitination in Assembly and Release of Human T-Cell Leukemia Virus Type 1▿

The PPPY motif in the matrix (MA) domain of human T-cell leukemia virus type 1 (HTLV-1) Gag associates with WWP1, a member of the HECT domain containing family of E3 ubiquitin ligases. Mutation of the PPPY motif arrests particle assembly at an early stage and abolishes ubiquitination of MA. Similar effects are seen when Gag is expressed in the presence of a truncated form of WWP1 that lacks the catalytically active HECT domain (C2WW). To understand the role of ubiquitination in budding, we mutated the four lysines in MA to arginines and identified lysine 74 as the unique site of ubiquitination. Virus-like particles produced by the K74R mutant did not contain ubiquitinated MA and showed a fourfold reduction in the release of infectious particles. Furthermore, the K74R mutation rendered assembly hypersensitive to C2WW inhibition; K74R Gag budding was inhibited at significantly lower levels of expression of C2WW compared with wild-type Gag. This finding indicates that the interaction between Gag and WWP1 is required for functions other than Gag ubiquitination. Additionally, we show that the PPPY− mutant Gag exerts a strong dominant-negative effect on the budding of wild-type Gag, further supporting the importance of recruitment of WWP1 to achieve particle assembly

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

Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity

Abstract Background Human endogenous retroviruses (HERVs), the remnants of ancient retroviral infections, constitute approximately 8% of human genomic DNA. Since HERV-K Gag expression is induced by HIV-1 Tat in T cells, induced HERV-K proteins could affect HIV-1 replication. Indeed, previously we showed that HERV-K Gag and HIV-1 Gag coassemble and that this appears to correlate with the effect of HERV-K Gag expression on HIV-1 particle release and its infectivity. We further showed that coassembly requires both MA and NC domains, which presumably serve as scaffolding for Gag via their abilities to bind membrane and RNA, respectively. Notably, however, despite possessing these abilities, MLV Gag failed to coassemble with HIV-1 Gag and did not affect assembly and infectivity of HIV-1 particles. It is unclear how the specificity of coassembly is determined. Results Here, we showed that coexpression of HERV-K Gag with HIV-1 Gag changed size and morphology of progeny HIV-1 particles and severely diminished infectivity of such progeny viruses. We further compared HERV-K-MLV chimeric constructs to identify molecular determinants for coassembly specificity and for inhibition of HIV-1 release efficiency and infectivity. We found that the CA N-terminal domain (NTD) of HERV-K Gag is important for the reduction of the HIV-1 release efficiency, whereas both CA-NTD and major homology region of HERV-K Gag contribute to colocalization with HIV-1 Gag. Interestingly, these regions of HERV-K Gag were not required for reduction of progeny HIV-1 infectivity. Conclusions Our results showed that HERV-K Gag CA is important for reduction of HIV-1 release and infectivity but the different regions within CA are involved in the effects on the HIV-1 release and infectivity. Altogether, these findings revealed that HERV-K Gag interferes the HIV-1 replication by two distinct molecular mechanisms.https://deepblue.lib.umich.edu/bitstream/2027.42/136631/1/12977_2017_Article_351.pd

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

Structural and Functional Insights into the HIV-1 Maturation Inhibitor Binding Pocket

<div><p>Processing of the Gag precursor protein by the viral protease during particle release triggers virion maturation, an essential step in the virus replication cycle. The first-in-class HIV-1 maturation inhibitor dimethylsuccinyl betulinic acid [PA-457 or bevirimat (BVM)] blocks HIV-1 maturation by inhibiting the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA. A structurally distinct molecule, PF-46396, was recently reported to have a similar mode of action to that of BVM. Because of the structural dissimilarity between BVM and PF-46396, we hypothesized that the two compounds might interact differentially with the putative maturation inhibitor-binding pocket in Gag. To test this hypothesis, PF-46396 resistance was selected for <em>in vitro</em>. Resistance mutations were identified in three regions of Gag: around the CA-SP1 cleavage site where BVM resistance maps, at CA amino acid 201, and in the CA major homology region (MHR). The MHR mutants are profoundly PF-46396-dependent in Gag assembly and release and virus replication. The severe defect exhibited by the inhibitor-dependent MHR mutants in the absence of the compound is also corrected by a second-site compensatory change far downstream in SP1, suggesting structural and functional cross-talk between the HIV-1 CA MHR and SP1. When PF-46396 and BVM were both present in infected cells they exhibited mutually antagonistic behavior. Together, these results identify Gag residues that line the maturation inhibitor-binding pocket and suggest that BVM and PF-46396 interact differentially with this putative pocket. These findings provide novel insights into the structure-function relationship between the CA MHR and SP1, two domains of Gag that are critical to both assembly and maturation. The highly conserved nature of the MHR across all orthoretroviridae suggests that these findings will be broadly relevant to retroviral assembly. Finally, the results presented here provide a framework for increased structural understanding of HIV-1 maturation inhibitor activity.</p> </div

PF-46396-dependent mutants are not rescued by BVM.

<p>[A] Radioimmunoprecipitation analysis of cell- and virion-associated proteins in the absence (−) and presence (+) of 2 µM BVM. Positions of Pr55^Gag (PrGag), Pr41^Gag (p41), CA-SP1 and CA are indicated. Lower panels show phosphorimager-based quantification of relative virus release efficiency (VRE) and % virion CA-SP1, calculated as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat-1002997-g006" target="_blank">Fig. 6</a> legend. Error bars denote SD; N = 4. [B] Virus replication kinetics in the absence and presence of BVM. The Jurkat T-cell line was transfected with WT or mutant pNL4-3 and propagated in the presence of 0, 0.1, or 1.0 µM BVM. Virus replication was monitored by RT activity, shown in cpm/µl.</p

Schematic molecular model of PF-46396 and BVM binding sites.

<p>The ribbon diagrams illustrate two adjacent CA-CTD/SP1 monomers in the hexagonal complex of the immature capsid. The atomic coordinates of the CA-CTD and SP1 domains were obtained from PDB 3H4E <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Pornillos1" target="_blank">[66]</a> and PDB 1U57 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Morellet1" target="_blank">[27]</a>, respectively. Residues 220–223 that link the CA-CTD and SP1 domains of each monomer were not modeled due to a lack of experimental data. The relative orientations of the adjacent CA-CTD domains were taken from the models of Bharat et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Bharat1" target="_blank">[61]</a>. The sites of resistance mutations are labeled and color-coded for location: Green, blue and red for the MHR, CA-CTD/SP1 boundary, and individual I201 residue, respectively. Yellow indicates the sites of secondary substitutions that rescue the G156E and P157S mutants. Also shown are stick figures of the PF-46396 and BVM compounds at the heights of their respective binding sites predicted from the locations of the resistance mutations (color-code: C – brown, N - blue, O - red, H – white, and Fl – green). The rotational orientation of PF-46396 is arbitrary, but that of BVM is taken from the photoafinity data of Nguyen et al. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Nguyen1" target="_blank">[45]</a>. The proximity of the MHR of the left monomer to the CA-CTD/SP1 region of the right one suggests that the binding sites of the two compounds straddle adjacent monomers. This would explain the observed necessity of Gag assembly into the immature capsid structure for BVM cleavage inhibition (see text).</p

Summary of PF-46396-resistance mutations selected.

#<p>From ref. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002997#ppat.1002997-Adamson5" target="_blank">[51]</a>.</p>*<p>In parentheses is the number of times the indicated mutation was selected.</p