Expression of Concern: Evidence that Vpu Modulates HIV-1 Gag-Envelope Interaction towards Envelope Incorporation and Infectivity in a Cell Type Dependent Manner.
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SUBMITTER: PLOS ONE Editors
PROVIDER: S-EPMC10965049 | biostudies-literature | 2024
REPOSITORIES: biostudies-literature
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Expression of Concern: Evidence that Vpu Modulates HIV-1 Gag-Envelope Interaction towards Envelope Incorporation and Infectivity in a Cell Type Dependent Manner.
PloS one 20240326 3
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Project description:The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env) incorporation defect caused by a Gag matrix (MA) mutation (L30E) was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.
| S-EPMC3628852 | biostudies-literature
Project description:BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication. RESULTS: A proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages. CONCLUSION: Our current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.
| S-EPMC3905668 | biostudies-literature
Project description:The HIV-1 envelope glycoprotein (Env) is sparsely incorporated onto assembling virus particles on the host cell plasma membrane in order for the virus to balance infectivity and evade the immune response. Env becomes trapped in a nascent particle on encounter with the polymeric viral protein Gag, which forms a dense protein lattice on the inner leaflet of the plasma membrane. While Env incorporation efficiency is readily measured biochemically from released particles, very little is known about the spatiotemporal dynamics of Env trapping events. Herein, we demonstrate, via high-resolution single-molecule tracking, that retention of Env trimers within single virus assembly sites requires the Env cytoplasmic tail (CT) and the L12 residue in the matrix (MA) domain of Gag but does not require curvature of the viral lattice. We further demonstrate that Env trimers are confined to subviral regions of a budding Gag lattice, supporting a model where direct interactions and/or steric corralling between the Env-CT and a lattice of MA trimers promote Env trapping and infectious HIV-1 assembly.
| S-EPMC6911212 | biostudies-literature
Project description:Abnormal deposition of aggregated wild-type (WT) human transthyretin (TTR) and its pathogenic variants is responsible for cardiomyopathy and neuropathy related to TTR amyloidosis. The tryptophan (Trp) fluorescence measurements typically used to study structural changes of TTR do not yield site-specific information on the two Trp residues per TTR protomer. To obtain such information, tryptophan labeled with fluorine at the 5 and 6 positions (5FW and 6FW) was incorporated into TTR. Fluorescence of 5FW and 6FW-labeled WT-TTR (WT-5FW and WT-6FW) and a single-Trp mutant W41Y showed that the photophysics of incorporated fluoro-Trp is consistent with site-specific solvation of the indole ring of W41 and W79. 19F-NMR showed that solvent accessibility depends on both the location of the Trp and the position of the fluorine substituent in the indole ring. Unexpectedly, differences were observed in the rates of aggregation, with WT-6FW aggregating more rapidly than WT-5FW or WT-TTR. Real-time 19F-NMR urea unfolding experiments revealed that WT-5FW is kinetically more stable than WT-6FW, consistent with the aggregation assay. In addition, structural perturbations of residues distant from either Trp site are more extensive in WT-6FW. Notably, residues in the dimer interfaces are perturbed by 6FW at residue 79; pathogenic mutations in these regions are associated with reduced tetramer stability and amyloidogenesis. The differences in behavior that arise from the replacement of a fluorine at the 5-position of a tryptophan with one at the adjacent 6-position emphasize the delicate balance of stability in the TTR tetramer.
| S-EPMC5645263 | biostudies-literature
Project description:Viral protein U (Vpu) is a protein encoded by human immunodeficiency virus type 1 (HIV-1) that promotes the degradation of the virus receptor, CD4, and enhances the release of virus particles from cells. We isolated a cDNA that encodes a novel cellular protein that interacts with Vpu in vitro, in vivo, and in yeast cells. This Vpu-binding protein (UBP) has a molecular mass of 41 kDa and is expressed ubiquitously in human tissues at the RNA level. UBP is a novel member of the tetratricopeptide repeat (TPR) protein family containing four copies of the 34-amino-acid TPR motif. Other proteins that contain TPR motifs include members of the immunophilin superfamily, organelle-targeting proteins, and a protein phosphatase. UBP also interacts directly with HIV-1 Gag protein, the principal structural component of the viral capsid. However, when Vpu and Gag are coexpressed, stable interaction between UBP and Gag is diminished. Furthermore, overexpression of UBP in virus-producing cells resulted in a significant reduction in HIV-1 virion release. Taken together, these data indicate that UBP plays a role in Vpu-mediated enhancement of particle release.
| S-EPMC110095 | biostudies-literature
Project description:The matrix (MA) domain of the human immunodeficiency virus (HIV) 1 Gag is responsible for Gag targeting to the plasma membrane where virions assemble. MA also plays a role in the incorporation of the viral envelope (Env) glycoproteins and can influence particle infectivity post-maturation and post-entry. A highly basic region of MA targets Gag to the plasma membrane via specific binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. This binding also triggers exposure of an amino-terminal myristate moiety, which anchors Gag to the membrane. An MA mutant deficient for PI(4,5)P2 binding, 29KE/31KE, has been shown to mislocalize within the cell, leading to particle assembly in a multivesicular body compartment and defective release of cell-free particles in HeLa and 293T cells. Despite the defect in virus production in these cells, release of the 29KE/31KE mutant is not significantly reduced in primary T cells, macrophages and Jurkat T cells. 29KE/31KE virions also display an infectivity defect associated with impaired Env incorporation, irrespective of the producer cell line. Here we examine the properties of 29KE/31KE by analyzing compensatory mutations obtained by a viral adaptation strategy. The MA mutant 16EK restores virus release through enhanced membrane binding. 16EK also influences the infectivity defect, in combination with an additional MA mutant, 62QR. Additionally, the 29KE/31KE MA mutant displays a defect in proteolytic cleavage of the murine leukemia virus Env cytoplasmic tail in pseudotyped virions. Our findings elucidate the mechanism whereby an MA mutant defective in PI(4,5)P2 binding can be rescued and highlight the ability of MA to influence Env glycoprotein function.
| S-EPMC4844178 | biostudies-literature
Project description:Our examination of RNA helicases for effects on HIV-1 protein production and particle assembly identified Rocaglamide (RocA), a known modulator of eIF4A1 function, as an inhibitor of HIV-1 replication in primary CD4+ T cells and three cell systems. HIV-1 attenuation by low-nM RocA doses was associated with reduced viral particle formation without a marked decrease in Gag production. Rather, the co-localization of Gag and HIV-1 genomic RNA (gRNA) assemblies was impaired by RocA treatment in a reversible fashion. Ribonucleoprotein (RNP) immunoprecipitation studies recapitulated the loss of Gag-gRNA assemblies upon RocA treatment. Parallel biophysical studies determined that neither RocA nor eIF4A1 independently affected the ability of Gag to interact with viral RNA, but together, they distorted the structure of the HIV-1 RNP visualized by electron microscopy. Taken together, several lines of evidence indicate that RocA induces stable binding of eIF4A1 onto the viral RNA genome in a manner that interferes with the ordered assembly of Gag along Gag-gRNA assemblies required to generate infectious virions.
| S-EPMC11437399 | biostudies-literature
Project description:BackgroundHuman 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.ResultsHere, 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.ConclusionsOur 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.
| S-EPMC5406883 | biostudies-literature
Project description:The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a "snorkeling" model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.
| S-EPMC2772686 | biostudies-literature
Project description:Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called "activating adaptors" that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein's motility in a cargo-specific manner.
| S-EPMC7655957 | biostudies-literature