Enhanced Virus Release Research Articles

Tetherin antagonism by SARS-CoV-2 ORF3a and spike protein enhances virus release.

The antiviral restriction factor, tetherin, blocks the release of several different families of enveloped viruses, including the Coronaviridae. Tetherin is an interferon-induced protein that forms parallel homodimers between the host cell and viral particles, linking viruses to the surface of infected cells and inhibiting their release. We demonstrate that SARS-CoV-2 infection causes tetherin downregulation and that tetherin depletion from cells enhances SARS-CoV-2 viral titres. We investigate the potential viral proteins involved in abrogating tetherin function and find that SARS-CoV-2 ORF3a reduces tetherin localisation within biosynthetic organelles where Coronaviruses bud, and increases tetherin localisation to late endocytic organelles via reduced retrograde recycling. We also find that expression of Spike protein causes a reduction in cellular tetherin levels. Our results confirm that tetherin acts as a host restriction factor for SARS-CoV-2 and highlight the multiple distinct mechanisms by which SARS-CoV-2 subverts tetherin function.

The envelope proteins from SARS-CoV-2 and SARS-CoV potently reduce the infectivity of human immunodeficiency virus type 1 (HIV-1)

BackgroundViroporins are virally encoded ion channels involved in virus assembly and release. Human immunodeficiency virus type 1 (HIV-1) and influenza A virus encode for viroporins. The human coronavirus SARS-CoV-2 encodes for at least two viroporins, a small 75 amino acid transmembrane protein known as the envelope (E) protein and a larger 275 amino acid protein known as Orf3a. Here, we compared the replication of HIV-1 in the presence of four different β-coronavirus E proteins.ResultsWe observed that the SARS-CoV-2 and SARS-CoV E proteins reduced the release of infectious HIV-1 yields by approximately 100-fold while MERS-CoV or HCoV-OC43 E proteins restricted HIV-1 infectivity to a lesser extent. Mechanistically, neither reverse transcription nor mRNA synthesis was involved in the restriction. We also show that all four E proteins caused phosphorylation of eIF2-α at similar levels and that lipidation of LC3-I could not account for the differences in restriction. However, the level of caspase 3 activity in transfected cells correlated with HIV-1 restriction in cells. Finally, we show that unlike the Vpu protein of HIV-1, the four E proteins did not significantly down-regulate bone marrow stromal cell antigen 2 (BST-2).ConclusionsThe results of this study indicate that while viroporins from homologous viruses can enhance virus release, we show that a viroporin from a heterologous virus can suppress HIV-1 protein synthesis and release of infectious virus.

Crosstalk Between Pyroptosis and Apoptosis in Hepatitis C Virus-induced Cell Death.

Extensive inflammation in the liver is known to contribute to the pathogenesis of hepatitis C virus (HCV) infection. Apoptosis has, for a long time, been known to act as a mechanism of hepatocyte death, but our previous research also identified inflammasome-mediated pyroptosis in infected and uninfected bystander cells as an additional mechanism of HCV-induced cytopathicity. The purpose of this study was to investigate the mechanism of HCV-induced cell death and to determine the timing and relative contributions of apoptosis and pyroptosis during HCV infection. In a model employing a cell culture-adapted strain of JFH-1 HCV and Huh-7.5 hepatocyte-like cells, we found that pyroptosis occurred earlier than did apoptosis during infection. CRISPR knockout of NLRP3 resulted in decreased caspase-1 activation, but not complete elimination, indicating multiple sensors are likely involved in HCV-induced pyroptosis. Knockout of gasdermin-D resulted in increased activation of apoptosis-related caspase-3, suggesting potential crosstalk between the two cell death pathways. An unexpected decrease in activated caspase-1 levels was observed when caspase-3 was knocked out, implying that caspase-3 may have a role in the initiation of pyroptosis, at least in the context of HCV infection. Lower viral titres in culture fluids and increased ratios of intracellular to extracellular levels of infectious virus were observed in knockout versus wild-type Huh-7.5 cells, suggesting that HCV may induce programmed cell death in order to enhance virus release from infected cells. These results contribute to the understanding of HCV pathogenesis and add to the increasing volume of literature suggesting various programmed cell death pathways are not mutually exclusive.

Functional interactions between herpes simplex virus pUL51, pUL7 and gE reveal cell-specific mechanisms for epithelial cell-to-cell spread

Herpes simplex virus spread between epithelial cells is mediated by virus tegument and envelope protein complexes including gE/gI and pUL51/pUL7. pUL51 interacts with both pUL7 and gE/gI in infected cells. We show that amino acids 30–90 of pUL51 mediate interaction with pUL7. We also show that deletion of amino acids 167–244 of pUL51, or ablation of pUL7 expression both result in failure of gE to concentrate at junctional surfaces of Vero cells. We also tested the hypothesis that gE and pUL51 function on the same pathway for cell-to-cell spread by analyzing the phenotype of a double gE/UL51 mutant. In HaCaT cells, pUL51 and gE function on the same spread pathway, whereas in Vero cells they function on different pathways. Deletion of the gE gene strongly enhanced virus release to the medium in Vero cells, suggesting that the gE-dependent spread pathway may compete with virion release to the medium.

MG-132 reduces virus release in Bovine herpesvirus-1 infection

Bovine herpesvirus 1 (BoHV-1) can provoke conjunctivitis, abortions and shipping fever. BoHV-1 infection can also cause immunosuppression and increased susceptibility to secondary bacterial infections, leading to pneumonia and occasionally to death. Herein, we investigated the influence of MG-132, a proteasome inhibitor, on BoHV-1 infection in bovine kidney (MDBK) cells. Infection of MDBK cells with BoHV-1 induces apoptotic cell death that enhances virus release. Whereas, MG-132 inhibited virus-induced apoptosis and stimulated autophagy. Protein expression of viral infected cell protein 0 (bICP0), which is constitutively expressed during infection and is able to stimulate Nuclear factor kappa B (NF-κB), was completely inhibited by MG-132. These results were accompanied by a significant delay in the NF-κB activation. Interestingly, the efficient virus release provoked by BoHV-1-induced apoptosis was significantly reduced by MG-132. Overall, this study suggests that MG-132, through the activation of autophagy, may limit BoHV-1 replication during productive infection, by providing an antiviral defense mechanism.

Functional conservation and coherence of HIV-1 subtype A Vpu alleles

Functional studies of HIV-1 proteins are normally conducted using lab adapted strains of HIV-1. The extent of those functions in clinical strains is sometimes unknown. In this study, we amplified and sequenced HIV-1 Vpu from 10 Iranian patients infected with HIV-1. Phylogenetic analysis indicated that the Vpu alleles were closely related to the CRF35_AD from Iran and subtype A Vpu. We addressed some of the well-established functions of the HIV-1 Vpu, as well as some of its recently reported functions. Ability of the clinical strains of subtype A Vpu alleles for downregulation of CD4 was similar to that of the lab adapted NL4.3 Vpu. Majority of the subtype A Vpu alleles performed stronger than NL4.3 Vpu for downregulation of SNAT1. The Vpu alleles differentially induced downregulation of HLA-C, ranging from no effect to 88% downregulation of surface HLA-C. Downregulation of tetherin and enhancement of virus release was similar for the subtype A Vpu alleles and NL4.3. Subtype A Vpu alleles were more potent when compared with NL4.3 for inhibition of NF-κB activation. Our study shows that subtype A Vpu alleles exert the classical functions of HIV-1 Vpu.

Functional conservation and coherence of HIV-1 subtype A Vpu alleles

Functional studies of HIV-1 proteins are normally conducted using lab adapted strains of HIV-1. The extent of those functions in clinical strains is sometimes unknown. In this study, we amplified and sequenced HIV-1 Vpu from 10 Iranian patients infected with HIV-1. Phylogenetic analysis indicated that the Vpu alleles were closely related to the CRF35_AD from Iran and subtype A Vpu. We addressed some of the well-established functions of the HIV-1 Vpu, as well as some of its recently reported functions. Ability of the clinical strains of subtype A Vpu alleles for downregulation of CD4 was similar to that of the lab adapted NL4.3 Vpu. Majority of the subtype A Vpu alleles performed stronger than NL4.3 Vpu for downregulation of SNAT1. The Vpu alleles differentially induced downregulation of HLA-C, ranging from no effect to 88% downregulation of surface HLA-C. Downregulation of tetherin and enhancement of virus release was similar for the subtype A Vpu alleles and NL4.3. Subtype A Vpu alleles were more potent when compared with NL4.3 for inhibition of NF-κB activation. Our study shows that subtype A Vpu alleles exert the classical functions of HIV-1 Vpu.

Insights into the activity of maturation inhibitor PF-46396 on HIV-1 clade C

HIV maturation inhibitors are an emerging class of anti-retroviral compounds that inhibit the viral protease-mediated cleavage of the Gag, CA-SP1 (capsid-spacer peptide 1) peptide to mature CA. The first-in-class maturation inhibitor bevirimat (BVM) displayed potent activity against HIV-1 clade B but was ineffective against other HIV-1 clades including clade C. Another pyridone-based maturation inhibitor, PF-46396 displayed potent activity against HIV-1 clade B. In this study, we aimed at determining the activity of PF-46396 against HIV-1 clade C. We employed various biochemical and virological assays to demonstrate that PF-46396 is effective against HIV-1 clade C. We observed a dose dependent accumulation of CA-SP1 intermediate in presence of the compound. We carried out mutagenesis in the CA- SP1 region of HIV-1 clade C Gag and observed that the mutations conferred resistance against the compound. Many mutations inhibited Gag processing thereby reducing virus release in the absence of the compound. However, presence of PF-46396 rescued these defects and enhanced virus release, replication capacity and infectivity of HIV-1 clade C. These results put together identify PF-46396 as a broadly active maturation inhibitor against HIV-1 clade B and C and help in rational designing of novel analogs with reduced toxicity and increased efficacy for its potential use in clinics.

The Tax-Inducible Actin-Bundling Protein Fascin Is Crucial for Release and Cell-to-Cell Transmission of Human T-Cell Leukemia Virus Type 1 (HTLV-1).

The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4+ B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging of chronically infected MS-9 T-cells in co-culture with Jurkat T-cells revealed that Fascin’s localization at tight cell-cell contacts is accompanied by gag polarization suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Taken together, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission.

Antagonism of BST-2/Tetherin Is a Conserved Function of the Env Glycoprotein of Primary HIV-2 Isolates.

Lentiviruses such as HIV-1 and HIV-2 encode accessory proteins whose function is to overcome host restriction mechanisms. Vpu is a well-studied HIV-1 accessory protein that enhances virus release by antagonizing the host restriction factor BST-2. HIV-2 does not encode a vpu gene. Instead, the HIV-2 Env glycoprotein was found to antagonize BST-2 in some isolates. Here, we cloned multiple Env sequences from 7 HIV-2-infected patients and found that about half were able to antagonize BST-2. Importantly, most HIV-2 patients harbored a mixed population of viruses containing or lacking the ability to antagonize BST-2. In fact, in comparing Env sequences from one patient combined with site-directed mutagenesis, we were able to restore BST-2 antagonism to an inactive Env protein by a single-amino-acid change. Our data suggest that targeting BST-2 by HIV-2 Env is a dynamic process that can be regulated by simple changes in the Env sequence.

The Vpu-interacting Protein SGTA Regulates Expression of a Non-glycosylated Tetherin Species.

The HIV-1 accessory protein Vpu enhances virus release by counteracting the host restriction factor tetherin. To further understand the role of host cell proteins in Vpu function, we carried out yeast two-hybrid screening and identified a previously reported Vpu-interacting host factor, small glutamine-rich tetratricopeptide repeat-containing protein (SGTA). While RNAi-mediated depletion of SGTA did not significantly affect levels of tetherin or virus release efficiency, we observed that overexpression of SGTA inhibited HIV-1 release in a Vpu- and tetherin-independent manner. Overexpression of SGTA in the presence of Vpu, but not in its absence, resulted in a marked stabilization and cytosolic relocalization of a 23-kDa, non-glycosylated tetherin species. Coimmunoprecipitation studies indicated that non-glycosylated tetherin is stabilized through the formation of a ternary SGTA/Vpu/tetherin complex. This accumulation of non-glycosylated tetherin is due to inhibition of its degradation, independent of the ER-associated degradation (ERAD) pathway. Because the SGTA-stabilized tetherin species is partially localized to the cytosol, we propose that overexpression of SGTA in the presence of Vpu blocks the translocation of tetherin across the ER membrane, resulting in cytosolic accumulation of a non-glycosylated tetherin species. Although our results do not provide support for a physiological function of SGTA in HIV-1 replication, they demonstrate that SGTA overexpression regulates tetherin expression and stability, thus providing insights into the function of SGTA in ER translocation and protein degradation.

Differential Reovirus-Specific and Herpesvirus-Specific Activator Protein 1 Activation of Secretogranin II Leads to Altered Virus Secretion.

Viruses utilize host cell machinery for propagation and manage to evade cellular host defense mechanisms in the process. Much remains unknown regarding how the host responds to viral infection. We recently performed global proteomic screens of mammalian reovirus TIL- and T3D-infected and herpesvirus (herpes simplex virus 1 [HSV-1])-infected HEK293 cells. The nonenveloped RNA reoviruses caused an upregulation, whereas the enveloped DNA HSV-1 caused a downregulation, of cellular secretogranin II (SCG2). SCG2, a member of the granin family that functions in hormonal peptide sorting into secretory vesicles, has not been linked to virus infections previously. We confirmed SCG2 upregulation and found SCG2 phosphorylation by 18 h postinfection (hpi) in reovirus-infected cells. We also found a decrease in the amount of reovirus secretion from SCG2 knockdown cells. Similar analyses of cells infected with HSV-1 showed an increase in the amount of secreted virus. Analysis of the stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) pathway indicated that each virus activates different pathways leading to activator protein 1 (AP-1) activation, which is the known SCG2 transcription activator. We conclude from these experiments that the negative correlation between SCG2 quantity and virus secretion for both viruses indicates a virus-specific role for SCG2 during infection. Mammalian reoviruses affect the gastrointestinal system or cause respiratory infections in humans. Recent work has shown that all mammalian reovirus strains (most specifically T3D) may be useful oncolytic agents. The ubiquitous herpes simplex viruses cause common sores in mucosal areas of their host and have coevolved with hosts over many years. Both of these virus species are prototypical representatives of their viral families, and investigation of these viruses can lead to further knowledge of how they and the other more pathogenic members of their respective families interact with the host. Here we show that secretogranin II (SCG2), a protein not previously studied in the context of virus infections, alters virus output in a virus-specific manner and that the quantity of SCG2 is inversely related to amounts of infectious-virus secretion. Herpesviruses may target this protein to facilitate enhanced virus release from the host.

In COS cells Vpu can both stabilize tetherin expression and counteract its antiviral activity.

The interferon-inducible cellular protein tetherin (CD317/BST-2) inhibits the release of a broad range of enveloped viruses. The HIV-1 accessory protein Vpu enhances virus particle release by counteracting this host restriction factor. While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood. Here, we show that human tetherin is expressed at low levels in African green monkey kidney (COS) cells. However, Vpu markedly increases tetherin expression in this cell line, apparently by sequestering it in an internal compartment that bears lysosomal markers. This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin. Although Vpu stabilizes human tetherin in COS cells, it still counteracts the ability of tetherin to suppress virus release. The enhancement of virus release by Vpu in COS cells is associated with a modest reduction in cell-surface tetherin expression, even though the overall expression of tetherin is higher in the presence of Vpu. This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.

Counteraction of Tetherin Antiviral Activity by Two Closely Related SIVs Differing by the Presence of a Vpu Gene

In different primate lentiviruses, three proteins (Vpu, Env and Nef) have been shown to have anti-tetherin activities. SIVden is a primate lentivirus harbored by a Cercopithecus denti (C. denti) whose genome code for a Vpu gene. We have compared the activity of HIV-1 Vpu and of SIVden Vpu on tetherin proteins from humans, from C. denti and from Cercopithecus neglectus (C. neglectus), a monkey species that is naturally infected by SIVdeb, a virus closely related to SIVden but which does not encode a Vpu protein. Here, we demonstrate that SIVden Vpu, is active against C. denti tetherin, but not against human tetherin. Interestingly, C. neglectus tetherin was more sensitive to SIVden Vpu than to HIV-1 Vpu. We also identify residues in the tetherin transmembrane domains that are responsible for the species-specific Vpu effect. Simultaneous mutation (P40L and T45I) of human tetherin conferred sensitivity to SIVden Vpu, while abolishing its sensitivity to HIV-1 Vpu. We next analyzed the anti-tetherin activity of the Nef proteins from HIV-1, SIVden and SIVdeb. All three Nef proteins were unable to rescue virus release in the presence of human or C. denti tetherin. Conversely, SIVdeb Nef enhanced virus release in the presence of C. neglectus tetherin, suggesting that SIVdeb relies on Nef in its natural host. Finally, while HIV-1 Vpu not only removed human tetherin from the cell surface but also directed it for degradation, SIVden Vpu only induced the redistribution of both C. denti and C. neglectus tetherins, resulting in a predominantly perinuclear localization.

Generation and Biological Characterization of a Neutralization-Resistant Mutant of Newcastle Disease Virus

A neutralization-resistant mutant of Newcastle disease virus (NDV) Kr005 strain belonging to class II genotype VII was generated using a neutralizing monoclonal antibody and its biological effects were assessed. The mutant showed single amino acid substitution (E to K) at position 347 of the hemagglutinin-neuraminidase (HN) protein (E347K mutant). The E347K mutant exhibited marked rounding of the cells and few syncytia in infected chicken embryofibroblast (CEF) cells. The hemadsorption and neuraminidase activities of the E347K mutant of the wild-type virus were 118% and 166%, respectively. The mutant produced a rapid elution pattern whereas the wild type had a slow elution pattern. Growth kinetics studies showed that the E347K mutant produced an 80-times higher yield of extracellular virus in CEF cells compared with the wild-type virus. The time-course virus titer showed a marked increase in mutant-infected cells from 6 h to 12 h post infection (pi), which was consistent with the titer pattern time-course for NA activity. The E347K mutant virus showed a slight decrease in virulence compared to the wild-type virus, but there was no change in pathotype when measured by in vivo pathogenicity testing. These results suggest that an E347K mutation in HN protein might be associated with increased NA activity and subsequent enhancement of virus release from infected cells without change in viral pathotype.

The role of murine leukemia virus glycosated Gag protein in virus replication

Murine leukemia viruses (MuLVs) encode an alternate form of Gag protein in addition to the standard Gag precursor polyprotein, glycosylated Gag (glyco-gag or gPr80gag). gPr80gag contains additional amino-terminal peptides, and it is glycosylated. Until recently the function of gPr80gag was unclear, but we have recently shown that gPr80gag facilitates viral budding and release through lipid rafts. Glyco-gag mutant virus is less efficiently released from fibroblasts, mutant virus has lower cholesterol content (and higher buoyant density), and less Gag precursor polyprotein (Pr65gag) is associated with detergent-resistant membranes in infected cells. The mechanism by which gPr80gag facilitates virus release is under investigation. The N-terminal 88 amino acids of gPr80gag are sufficient to enhance virus release, indicating that the Gag sequences on this protein are not required. Cellular La protein is involved in gPr80gag function, since over-expression of La phenocopies gPr80gag in facilitating virus release, and knockdown of La abrogates gPr80gag-enhancement of virus release. MuLV glyco-gag can also facilitate release of HIV-1 particles from transfected cells, which suggests that these two viruses may share mechanisms for directing virus release through lipid rafts. gPr80gagalso counteracts the murine APOBEC3 restriction factor (mA3) since gPr80gagmutant virus shows a defect for establishment of infection in wild-type mice, but not in mA3 knockout animals.

Antibody-Mediated Enhancement of HIV-1 and HIV-2 Production from BST-2/Tetherin-Positive Cells

BST-2/CD317/HM1.24/tetherin is a B-cell antigen overexpressed on the surface of myeloma cell lines and on neoplastic plasma cells of patients with multiple myeloma. Antibodies to BST-2 are in clinical trial for the treatment of multiple myeloma and are considered for the treatment of solid tumors with high BST-2 antigen levels. Functionally, BST-2 restricts the secretion of retroviruses, including human immunodeficiency virus type 1, as well as members of the herpesvirus, filovirus, and arenavirus families, presumably by tethering nascent virions to the cell surface. Here we report that BST-2 antibody treatment facilitates virus release from BST-2(+) cells by interfering with the tethering activity of BST-2. BST-2 antibodies were unable to release already tethered virions and were most effective when added early during virus production. BST-2 antibody treatment did not affect BST-2 dimerization and did not reduce the cell surface expression of BST-2. Interestingly, BST-2 antibody treatment reduced the nonspecific shedding of BST-2 and limited the encapsidation of BST-2 into virions. Finally, flotation analyses indicate that BST-2 antibodies affect the distribution of BST-2 within membrane rafts. Our data suggest that BST-2 antibody treatment may enhance virus release by inducing a redistribution of BST-2 at the cell surface, thus preventing it from accumulating at the sites of virus budding.

Ion channel activity of HIV-1 Vpu is dispensable for counteraction of CD317

While the C-terminal domain of HIV-1 Vpu is critical for CD4 degradation, the transmembrane domain (TM) mediates ion channel activity, enhances virus release and is essential for counteracting CD317/Bst-2/Tetherin. Here we analyzed whether the ion channel activity of Vpu is required to antagonize CD317-mediated restriction of virion release. We examined TM-mutants of three conserved residues: the S23A mutation, which was previously shown to abrogate ion channel function, did not affect Vpu mediated augmentation of virus release. In contrast, the A14N and A18N mutation did not affect ion channel activity of Vpu, but substantially reduced its ability to support virus release and to down-regulate CD317 from the cell surface. Altogether, our data suggest that not the ion channel activity of Vpu, but its ability to remove CD317 from the cell surface is required to augment HIV-1 release.

The Cellular Protein La Functions in Enhancement of Virus Release through Lipid Rafts Facilitated by Murine Leukemia Virus Glycosylated Gag

Murine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65gag for Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80gag). gPr80gag is translated from the same unspliced viral RNA as Pr65gag, from an upstream in-frame CUG initiation codon. As a result, gPr80gag contains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80gag into the rough endoplasmic reticulum, where it is glycosylated, exported to the cell surface, and cleaved into two proteins of 55 and 40 kDa. The amino-terminal 55-kDa protein remains cell associated with the 88 unique amino acids exposed to the cytosol. We previously showed that gPr80gag facilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80gag is sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80gag function led to cellular La protein. Overexpression of mouse or human La enhanced M-MuLV particle release in the absence of glyco-gag, and the released virus had a reduced buoyant density characteristic of increased cholesterol content. Moreover, small interfering RNA (siRNA) knockdown of human La abolished glyco-gag enhancement of M-MuLV release. These results implicate La as a cellular protein involved in M-MuLV glyco-gag function. We also found that overexpression of mouse or human La could enhance HIV-1 release in the absence of gPr80gag. Therefore, M-MuLV and HIV-1 may share a pathway for release through lipid rafts involving La.

Membrane raft association of the Vpu protein of human immunodeficiency virus type 1 correlates with enhanced virus release

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) is known to enhance virion release from certain cell types. To accomplish this function, Vpu interacts with the restriction factor known as bone marrow stromal cell antigen 2 (BST-2)/tetherin. In this study, we analyzed whether the Vpu protein is associated with microdomains known as lipid or membrane rafts. Our results indicate that Vpu partially partitions into detergent-resistant membrane (DRM) fractions when expressed alone or in the context of simian–human immunodeficiency virus (SHIV) infection. The ability to be partitioned into rafts was observed with both subtype B and C Vpu proteins. The use of cholesterol lowering lovastatin/M-β-cyclodextrin and co-patching experiments confirmed that Vpu can be detected in cholesterol rich regions of membranes. Finally, we present data showing that raft association-defective transmembrane mutants of Vpu have impaired enhanced virus release function, but still maintain the ability to down-regulate CD4.