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

ABSTRACTMurine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65gagfor Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80gag). gPr80gagis translated from the same unspliced viral RNA as Pr65gag, from an upstream in-frame CUG initiation codon. As a result, gPr80gagcontains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80gaginto 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 gPr80gagfacilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80gagis sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80gagfunction 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.IMPORTANCERetroviruses cause diseases such as leukemia and AIDS. An important aspect of viral replication is how viruses are released from infected cells. We previously found that a unique protein encoded by murine leukemia viruses (MuLVs), glyco-gag (or gPr80gag), enhances efficient virus release through cholesterol-rich membrane subdomains called lipid rafts. In this study, we found that the N-terminal domain of gPr80gagis sufficient to enhance viral release. A search for cellular proteins that participate in gPr80gagfunction led to cellular La protein. Overexpression of La phenocopied glyco-gag in enhancing M-MuLV release, and knockdown of La abolished glyco-gag function. M-MuLV glyco-gag also enhanced release of HIV-1, as did overexpression La in the absence of glyco-gag. Thus, M-MuLV and HIV-1 may share a cellular pathway for release through lipid rafts involving La. These results may also be relevant for other viruses that are released through lipid rafts.

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Functional Interplay Between Murine Leukemia Virus Glycogag, Serinc5, and Surface Glycoprotein Governs Virus Entry, with Opposite Effects on Gammaretroviral and Ebolavirus Glycoproteins

mBio ◽

10.1128/mbio.01985-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 24

Author(s):

Yadvinder S. Ahi ◽

Shu Zhang ◽

Yashna Thappeta ◽

Audrey Denman ◽

Amin Feizpour ◽

...

Keyword(s):

Viral Entry ◽

Equine Infectious Anemia Virus ◽

Virus Entry ◽

Cellular Protein ◽

Negative Effects ◽

Equine Infectious Anemia ◽

Anemia Virus ◽

Leukemia Viruses ◽

Hiv 1 ◽

Murine Leukemia

ABSTRACTGammaretroviruses, such as murine leukemia viruses (MLVs), encode, in addition to the canonical Gag, Pol, and Env proteins that will form progeny virus particles, a protein called “glycogag” (glycosylated Gag). MLV glycogag contains the entire Gag sequence plus an 88-residue N-terminal extension. It has recently been reported that glycogag, like the Nef protein of HIV-1, counteracts the antiviral effects of the cellular protein Serinc5. We have found, in agreement with prior work, that glycogag strongly enhances the infectivity of MLVs with some Env proteins but not those with others. In contrast, however, glycogag was detrimental to MLVs carrying Ebolavirus glycoprotein. Glycogag could be replaced, with respect to viral infectivity, by the unrelated S2 protein of equine infectious anemia virus. We devised an assay for viral entry in which virus particles deliver the Cre recombinase into cells, leading to the expression of a reporter. Data from this assay showed that both the positive and the negative effects of glycogag and S2 upon MLV infectivity are exerted at the level of virus entry. Moreover, transfection of the virus-producing cells with a Serinc5 expression plasmid reduced the infectivity and entry capability of MLV carrying xenotropic MLV Env, particularly in the absence of glycogag. Conversely, Serinc5 expression abrogated the negative effects of glycogag upon the infectivity and entry capability of MLV carrying Ebolavirus glycoprotein. As Serinc5 may influence cellular phospholipid metabolism, it seems possible that all of these effects on virus entry derive from changes in the lipid composition of viral membranes.IMPORTANCEMany murine leukemia viruses (MLVs) encode a protein called “glycogag.” The function of glycogag is not fully understood, but it can assist HIV-1 replication in the absence of the HIV-1 protein Nef under some circumstances. In turn, Nef counteracts the cellular protein Serinc5. Glycogag enhances the infectivity of MLVs with some but not all MLV Env proteins (which mediate viral entry into the host cell upon binding to cell surface receptors). We now report that glycogag acts by enhancing viral entry and that, like Nef, glycogag antagonizes Serinc5. Surprisingly, the effects of glycogag and Serinc5 upon the entry and infectivity of MLV particles carrying an Ebolavirus glycoprotein are the opposite of those observed with the MLV Env proteins. The unrelated S2 protein of equine infectious anemia virus (EIAV) is functionally analogous to glycogag in our experiments. Thus, three retroviruses (HIV-1, MLV, and EIAV) have independently evolved accessory proteins that counteract Serinc5.

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HIV-1 Is Restricted prior to Integration of Viral DNA in Primary Cord-Derived Human CD34+Cells

Journal of Virology ◽

10.1128/jvi.01044-15 ◽

2015 ◽

Vol 89 (15) ◽

pp. 8096-8100 ◽

Cited By ~ 12

Author(s):

Daniel O. Griffin ◽

Stephen P. Goff

Keyword(s):

Transcriptional Silencing ◽

Viral Dna ◽

Retroviral Infection ◽

Human Cd34 ◽

Cd34 Cells ◽

Dominant Point ◽

Mouse Cells ◽

Leukemia Viruses ◽

Hiv 1 ◽

Murine Leukemia

Certain cells have the ability to block retroviral infection at specific stages of the viral cycle by the activities of well-characterized factors and transcriptional silencing machinery. Infection of murine stem cells (MSCs) by the murine leukemia viruses (MLVs) is profoundly blocked postintegration by transcriptional silencing. Here, we show that a dominant point of restriction of HIV-1 in human CD34+cells is prior to integration of viral DNA and that HIV-1 restriction by human CD34+cells is fundamentally different from MLV restriction by mouse cells.

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Murine leukemia virus glycosylated Gag (gPr80gag) facilitates interferon-sensitive virus release through lipid rafts

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908660107 ◽

2009 ◽

Vol 107 (3) ◽

pp. 1190-1195 ◽

Cited By ~ 26

Author(s):

T. Nitta ◽

Y. Kuznetsov ◽

A. McPherson ◽

H. Fan

Keyword(s):

Lipid Rafts ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Virus Release ◽

Murine Leukemia

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The Mason-Pfizer Monkey Virus PPPY and PSAP Motifs Both Contribute to Virus Release

Journal of Virology ◽

10.1128/jvi.77.17.9474-9485.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9474-9485 ◽

Cited By ~ 97

Author(s):

Eva Gottwein ◽

Jochen Bodem ◽

Barbara Müller ◽

Ariane Schmechel ◽

Hanswalter Zentgraf ◽

...

Keyword(s):

Cellular Protein ◽

Membrane Curvature ◽

Mutant Virus ◽

Dependent Manner ◽

Virus Release ◽

Immunodeficiency Virus ◽

Ptap Motif ◽

Hiv 1

ABSTRACT Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release.

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Angiomotin functions in HIV-1 assembly and budding

eLife ◽

10.7554/elife.03778 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 24

Author(s):

Gaelle Mercenne ◽

Steven L Alam ◽

Jun Arii ◽

Matthew S Lalonde ◽

Wesley I Sundquist

Keyword(s):

Adaptor Protein ◽

Cellular Protein ◽

Virus Release ◽

Electron Microscopic ◽

Gag Protein ◽

Virion Assembly ◽

Gag Proteins ◽

Immature Virion ◽

Late Assembly Domain ◽

Hiv 1

Many retroviral Gag proteins contain PPXY late assembly domain motifs that recruit proteins of the NEDD4 E3 ubiquitin ligase family to facilitate virus release. Overexpression of NEDD4L can also stimulate HIV-1 release but in this case the Gag protein lacks a PPXY motif, suggesting that NEDD4L may function through an adaptor protein. Here, we demonstrate that the cellular protein Angiomotin (AMOT) can bind both NEDD4L and HIV-1 Gag. HIV-1 release and infectivity are stimulated by AMOT overexpression and inhibited by AMOT depletion, whereas AMOT mutants that cannot bind NEDD4L cannot function in virus release. Electron microscopic analyses revealed that in the absence of AMOT assembling Gag molecules fail to form a fully spherical enveloped particle. Our experiments indicate that AMOT and other motin family members function together with NEDD4L to help complete immature virion assembly prior to ESCRT-mediated virus budding.

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The ESCRT-Associated Protein Alix Recruits the Ubiquitin Ligase Nedd4-1 To Facilitate HIV-1 Release through the LYPXnL L Domain Motif

Journal of Virology ◽

10.1128/jvi.00634-10 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8181-8192 ◽

Cited By ~ 60

Author(s):

Paola Sette ◽

Joshua A. Jadwin ◽

Vincent Dussupt ◽

Nana F. Bello ◽

Fadila Bouamr

Keyword(s):

Catalytic Activity ◽

Ubiquitin Ligase ◽

Ubiquitin Ligases ◽

Cellular Proteins ◽

Virus Release ◽

Yeast Two Hybrid ◽

And Function ◽

Two Hybrid ◽

Hiv 1 ◽

Gain Access

ABSTRACT The p6 region of HIV-1 Gag contains two late (L) domains, PTAP and LYPXnL, that bind Tsg101 and Alix, respectively. Interactions with these two cellular proteins recruit members of the host's fission machinery (ESCRT) to facilitate HIV-1 release. Other retroviruses gain access to the host ESCRT components by utilizing a PPXY-type L domain that interacts with cellular Nedd4-like ubiquitin ligases. Despite the absence of a PPXY motif in HIV-1 Gag, interaction with the ubiquitin ligase Nedd4-2 was recently shown to stimulate HIV-1 release. We show here that another Nedd4-like ubiquitin ligase, Nedd4-1, corrected release defects resulting from the disruption of PTAP (PTAP−), suggesting that HIV-1 Gag also recruits Nedd4-1 to facilitate virus release. Notably, Nedd4-1 remediation of HIV-1 PTAP− budding defects is independent of cellular Tsg101, implying that Nedd4-1's function in HIV-1 release does not involve ESCRT-I components and is therefore distinct from that of Nedd4-2. Consistent with this finding, deletion of the p6 region decreased Nedd4-1-Gag interaction, and disruption of the LYPXnL motif eliminated Nedd4-1-mediated restoration of HIV-1 PTAP−. This result indicated that both Nedd4-1 interaction with Gag and function in virus release occur through the Alix-binding LYPXnL motif. Mutations of basic residues located in the NC domain of Gag that are critical for Alix's facilitation of HIV-1 release, also disrupted release mediated by Nedd4-1, further confirming a Nedd4-1-Alix functional interdependence. In fact we found that Nedd4-1 binds Alix in both immunoprecipitation and yeast-two-hybrid assays. In addition, Nedd4-1 requires its catalytic activity to promote virus release. Remarkably, RNAi knockdown of cellular Nedd4-1 eliminated Alix ubiquitination in the cell and impeded its ability to function in HIV-1 release. Together our data support a model in which Alix recruits Nedd4-1 to facilitate HIV-1 release mediated through the LYPXnL/Alix budding pathway via a mechanism that involves Alix ubiquitination.

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Recent Studies on a Murine Leukemia Virus Grown in Tissue Culture

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100060544 ◽

1967 ◽

Vol 25 ◽

pp. 106-107

Author(s):

L. Z. de Tkaczevski ◽

E. de Harven ◽

C. Friend

Keyword(s):

Tissue Culture ◽

Solid Tumors ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Supernatant Fluid ◽

Virus Particles ◽

Friend Leukemia ◽

Type C ◽

Leukemia Viruses ◽

Murine Leukemia

Despite extensive studies, the correlation between the morphology and pathogenicity of murine leukemia viruses (MLV) has not yet been clarified. The virus particles found in the plasma of leukemic mice belong to 2 distinct groups, 1 or 2% of them being enveloped A particles and the vast majority being of type C. It is generally believed that these 2 types of particles represent different phases in the development of the same virus. Particles of type A have been thought to be an earlier form of type C particles. One of the tissue culture lines established from Friend leukemia solid tumors has provided the material for the present study. The supernatant fluid of the line designated C-1A contains an almost pure population of A particles as illustrated in Figure 1. The ratio is, therefore, the reverse of what is unvariably observed in the plasma of leukemic mice where C particles predominate.

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