scholarly journals MARCH8 targets cytoplasmic lysine residues of various viral envelope glycoproteins

2021 ◽  
Author(s):  
Yanzhao Zhang ◽  
Seiya Ozono ◽  
Takuya Tada ◽  
Minoru Tobiume ◽  
Masanori Kameoka ◽  
...  
Keyword(s):  
Rabies Virus ◽  
Chikungunya Virus ◽  
Transmembrane Protein ◽  
Ring Finger ◽  
Lymphocytic Choriomeningitis ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Lysine Residues ◽  
Viral Envelopes ◽  
Hiv 1

The host transmembrane protein MARCH8 is a RING finger E3 ubiquitin ligase that downregulates various host transmembrane proteins, such as MHC-II. We have recently reported that MARCH8 expression in virus-producing cells impairs viral infectivity by reducing virion incorporation of not only HIV-1 envelope glycoproteins but also vesicular stomatitis virus G-glycoprotein through two different pathways. However, the MARCH8 inhibition spectrum remains largely unknown. Here, we investigate the antiviral spectrum of MARCH8 using HIV-1 pseudotyped with a variety of viral envelope glycoproteins. Pseudotyping experiments revealed that viral envelopes derived from the rhabdovirus, arenavirus, coronavirus, and togavirus (alphavirus) families were sensitive to MARCH8-mediated inhibition. Lysine mutations at the cytoplasmic tails of rabies virus-G, lymphocytic choriomeningitis virus glycoproteins, SARS-CoV and SARS-CoV-2 spike proteins, and Chikungunya virus and Ross River virus E2 proteins conferred resistance to MARCH8. Immunofluorescence showed impaired downregulation of the mutants of these viral envelopes by MARCH8, followed by lysosomal degradation, suggesting that MARCH8-mediated ubiquitination leads to intracellular degradation of these envelopes. Indeed, rabies virus-G and Chikungunya virus E2 proteins proved to be clearly ubiquitinated. We conclude that MARCH8 has inhibitory activity on a variety of viral envelope glycoproteins whose cytoplasmic lysine residues are targeted by this antiviral factor.

scholarly journals Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes

2017 ◽  
Vol 28 (26) ◽  
pp. 3801-3814 ◽  
Author(s):  
Sunandini Chandra ◽  
Raju Kalaivani ◽  
Manoj Kumar ◽  
Narayanaswamy Srinivasan ◽  
Debi P. Sarkar
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Bioactive Molecules ◽  
Host Cells ◽  
Actin Dynamics ◽  
Envelope Glycoproteins ◽  
Animal Cells ◽  
Viral Envelopes

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

scholarly journals MARCH8 inhibits viral infection by two different mechanisms

2020 ◽  
Author(s):  
Yanzhao Zhang ◽  
Takuya Tada ◽  
Seiya Ozono ◽  
Satoshi Kishigami ◽  
Hideaki Fujita ◽  
...  
Keyword(s):  
Viral Infection ◽  
Molecular Mechanisms ◽  
Inhibitory Effect ◽  
Envelope Glycoproteins ◽  
Intracellular Protein ◽  
Virion Incorporation ◽  
Lysine Residues ◽  
Vesicular Stomatitis ◽  
Dependent Pathway ◽  
Hiv 1

ABSTRACTMembrane-associated RING-CH 8 (MARCH8) inhibits infection with both HIV-1 and vesicular stomatitis virus G-glycoprotein (VSV-G)-pseudotyped viruses by reducing virion incorporation of envelope glycoproteins. The molecular mechanisms by which MARCH8 targets envelope glycoproteins remain unknown. Here, we show two different mechanisms by which MARCH8 inhibits viral infection. Viruses pseudotyped with the VSV-G mutant, in which cytoplasmic lysine residues were mutated, were insensitive to the inhibitory effect of MARCH8, whereas those with a similar lysine mutant of HIV-1 Env remained sensitive to it. Indeed, the wild-type VSV-G, but not its lysine mutant, was ubiquitinated by MARCH8. Furthermore, the MARCH8 mutant, which had a disrupted cytoplasmic tyrosine motif that is critical for intracellular protein sorting, did not inhibit HIV-1 Env-mediated infection, while it still impaired infection by VSV-G-pseudotyped viruses. Overall, we conclude that MARCH8 reduces viral infectivity by downregulating envelope glycoproteins through two different mechanisms mediated by a ubiquitination-dependent or tyrosine motif-dependent pathway.

scholarly journals Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

2005 ◽  
Vol 16 (12) ◽  
pp. 5502-5513 ◽  
Author(s):  
Ruben M. Markosyan ◽  
Fredric S. Cohen ◽  
Grigory B. Melikyan
Keyword(s):  
Temperature Jump ◽  
Viral Envelope ◽  
Pseudotyped Virus ◽  
Target Cells ◽  
Time Resolved ◽  
Virus Content ◽  
Long Time ◽  
Viral Envelopes ◽  
Time Resolved Imaging ◽  
Hiv 1

A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge.

scholarly journals The Transmembrane Protein of HIV-1 Primary Isolates Modulates Cell Surface Expression of Their Envelope Glycoproteins

Virology ◽  
2001 ◽  
Vol 290 (1) ◽  
pp. 136-142
Author(s):  
Sarah Lebigot ◽  
Philippe Roingeard ◽  
Gilles Thibault ◽  
Franck Lemiale ◽  
Bernard Verrier ◽  
...  
Keyword(s):  
Cell Surface ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Envelope Glycoproteins ◽  
Hiv 1

scholarly journals Role of Matrix in an Early Postentry Step in the Human Immunodeficiency Virus Type 1 Life Cycle

1998 ◽  
Vol 72 (5) ◽  
pp. 4116-4126 ◽  
Author(s):  
Rosemary E. Kiernan ◽  
Akira Ono ◽  
George Englund ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Envelope Glycoprotein ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The matrix protein of human immunodeficiency virus type 1 (HIV-1) has been reported to play a crucial role in the targeting of the Gag polyprotein precursor to the plasma membrane and in the incorporation of viral envelope glycoproteins into budding virions. In this report, we present evidence that mutation of a highly conserved Leu at matrix amino acid 20 blocks or markedly delays virus replication in a range of cell types, including T-cell lines, primary human peripheral blood mononuclear cells, and monocyte-derived macrophages. These mutations do not impair virus assembly and release, RNA encapsidation, or envelope glycoprotein incorporation into virions but rather cause significant defects in an early step in the virus life cycle, as measured by single-cycle infectivity assays and the analysis of viral DNA synthesis early postinfection. This infectivity defect is independent of the type of envelope glycoprotein carried on mutant virions; similar results are obtained in pseudotyping experiments using wild-type or truncated HIV-1 envelope glycoproteins, the amphotropic murine leukemia virus envelope, or the vesicular stomatitis G protein. Intriguingly, matrix residue 20 mutations also increase the apparent binding of Gag to membrane, accelerate the kinetics of Gag processing, and induce defects in endogenous reverse transcriptase activity without affecting virion density or morphology. These results help elucidate the function of matrix in HIV-1 replication.

scholarly journals Blockade of HIV-1 Infection of New World Monkey Cells Occurs Primarily at the Stage of Virus Entry

2002 ◽  
Vol 196 (4) ◽  
pp. 431-445 ◽  
Author(s):  
Jason A. LaBonte ◽  
Gregory J. Babcock ◽  
Trushar Patel ◽  
Joseph Sodroski
Keyword(s):  
New World ◽  
Virus Entry ◽  
World Monkey ◽  
Squirrel Monkeys ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
New World Monkeys ◽  
Common Marmosets ◽  
New World Monkey ◽  
Hiv 1

HIV-1 naturally infects chimpanzees and humans, but does not infect Old World monkeys because of replication blocks that occur after virus entry into the cell. To understand the species-specific restrictions operating on HIV-1 infection, the ability of HIV-1 to infect the cells of New World monkeys was examined. Primary cells derived from common marmosets and squirrel monkeys support every phase of HIV-1 replication with the exception of virus entry. Efficient HIV-1 entry typically requires binding of the viral envelope glycoproteins and host cell receptors, CD4 and either CCR5 or CXCR4 chemokine receptors. HIV-1 did not detectably bind or utilize squirrel monkey CD4 for entry, and marmoset CD4 was also very inefficient compared with human CD4. A marmoset CD4 variant, in which residues 48 and 59 were altered to the amino acids found in human CD4, supported HIV-1 entry efficiently. The CXCR4 molecules of both marmosets and squirrel monkeys supported HIV-1 infection, but the CCR5 proteins of both species were only marginally functional. These results demonstrate that the CD4 and CCR5 proteins of New World monkeys represent the major restriction against HIV-1 replication in these primates. Directed adaptation of the HIV-1 envelope glycoproteins to common marmoset receptors might allow the development of New World monkey models of HIV-1 infection.

scholarly journals Conformational Differences between Functional Human Immunodeficiency Virus Envelope Glycoprotein Trimers and Stabilized Soluble Trimers

2018 ◽  
Vol 93 (3) ◽  
Author(s):  
Luis R. Castillo-Menendez ◽  
Hanh T. Nguyen ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Envelope Glycoprotein ◽  
Coiled Coil ◽  
Viral Envelope ◽  
Host Cells ◽  
Heptad Repeat ◽  
Envelope Glycoproteins ◽  
Immunodeficiency Virus ◽  
Functional Membrane ◽  
Hiv 1

ABSTRACTBinding to the receptor CD4 triggers entry-related conformational changes in the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer, (gp120/gp41)3. Soluble versions of HIV-1 Env trimers (sgp140 SOSIP.664) stabilized by a gp120-gp41 disulfide bond and a change (I559P) in gp41 have been structurally characterized. Here, we use cross-linking/mass spectrometry to evaluate the conformations of functional membrane Env and sgp140 SOSIP.664. Differences were detected in the gp120 trimer association domain and C terminus and in the gp41 heptad repeat 1 (HR1) region. Whereas the membrane Env trimer exposes the gp41 HR1 coiled coil only after CD4 binding, the sgp140 SOSIP.664 HR1 coiled coil was accessible to the gp41 HR2 peptide even in the absence of CD4. Our results delineate differences in both gp120 and gp41 subunits between functional membrane Env and the sgp140 SOSIP.664 trimer and provide distance constraints that can assist validation of candidate structural models of the native HIV-1 Env trimer.IMPORTANCEHIV-1 envelope glycoprotein spikes mediate the entry of the virus into host cells and are a major target for vaccine-induced antibodies. Soluble forms of the envelope glycoproteins that are stable and easily produced have been characterized extensively and are being considered as vaccines. Here, we present evidence that these stabilized soluble envelope glycoproteins differ in multiple respects from the natural HIV-1 envelope glycoproteins. By pinpointing these differences, our results can guide the improvement of envelope glycoprotein preparations to achieve greater similarity to the viral envelope glycoprotein spike, potentially increasing their effectiveness as a vaccine.

scholarly journals Antibody Binding Is a Dominant Determinant of the Efficiency of Human Immunodeficiency Virus Type 1 Neutralization

2006 ◽  
Vol 80 (22) ◽  
pp. 11404-11408 ◽  
Author(s):  
Xinzhen Yang ◽  
Inna Lipchina ◽  
Simon Cocklin ◽  
Irwin Chaiken ◽  
Joseph Sodroski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Antibody Binding ◽  
Viral Envelope ◽  
Envelope Glycoproteins ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Primary and laboratory-adapted variants of human immunodeficiency virus type 1 (HIV-1) exhibit a wide range of sensitivities to neutralization by antibodies directed against the viral envelope glycoproteins. An antibody directed against an artificial FLAG epitope inserted into the envelope glycoproteins of three HIV-1 isolates with vastly different neutralization sensitivities inhibited all three viruses equivalently. Thus, naturally occurring HIV-1 isolates that are neutralization resistant are not necessarily more impervious to the inhibitory consequences of bound antibody. Moreover, the binding affinity of the anti-FLAG antibody correlated with neutralizing potency, underscoring the dominant impact on neutralization of antibody binding to the envelope glycoproteins.

scholarly journals The HIV-1 Antisense Protein ASP Is a Transmembrane Protein of the Cell Surface and an Integral Protein of the Viral Envelope

2019 ◽  
Vol 93 (21) ◽  
Author(s):  
Yvonne Affram ◽  
Juan C. Zapata ◽  
Zahra Gholizadeh ◽  
William D. Tolbert ◽  
Wei Zhou ◽  
...  
Keyword(s):  
Cell Surface ◽  
Viral Replication ◽  
Plasma Membranes ◽  
Transmembrane Protein ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Integral Protein ◽  
Viral Particles ◽  
Infected Cells ◽  
Hiv 1

ABSTRACT The negative strand of HIV-1 encodes a highly hydrophobic antisense protein (ASP) with no known homologs. The presence of humoral and cellular immune responses to ASP in HIV-1 patients indicates that ASP is expressed in vivo, but its role in HIV-1 replication remains unknown. We investigated ASP expression in multiple chronically infected myeloid and lymphoid cell lines using an anti-ASP monoclonal antibody (324.6) in combination with flow cytometry and microscopy approaches. At baseline and in the absence of stimuli, ASP shows polarized subnuclear distribution, preferentially in areas with low content of suppressive epigenetic marks. However, following treatment with phorbol 12-myristate 13-acetate (PMA), ASP translocates to the cytoplasm and is detectable on the cell surface, even in the absence of membrane permeabilization, indicating that 324.6 recognizes an ASP epitope that is exposed extracellularly. Further, surface staining with 324.6 and anti-gp120 antibodies showed that ASP and gp120 colocalize, suggesting that ASP might become incorporated in the membranes of budding virions. Indeed, fluorescence correlation spectroscopy studies showed binding of 324.6 to cell-free HIV-1 particles. Moreover, 324.6 was able to capture and retain HIV-1 virions with efficiency similar to that of the anti-gp120 antibody VRC01. Our studies indicate that ASP is an integral protein of the plasma membranes of chronically infected cells stimulated with PMA, and upon viral budding, ASP becomes a structural protein of the HIV-1 envelope. These results may provide leads to investigate the possible role of ASP in the virus replication cycle and suggest that ASP may represent a new therapeutic or vaccine target. IMPORTANCE The HIV-1 genome contains a gene expressed in the opposite, or antisense, direction to all other genes. The protein product of this antisense gene, called ASP, is poorly characterized, and its role in viral replication remains unknown. We provide evidence that the antisense protein, ASP, of HIV-1 is found within the cell nucleus in unstimulated cells. In addition, we show that after PMA treatment, ASP exits the nucleus and localizes on the cell membrane. Moreover, we demonstrate that ASP is present on the surfaces of viral particles. Altogether, our studies identify ASP as a new structural component of HIV-1 and show that ASP is an accessory protein that promotes viral replication. The presence of ASP on the surfaces of both infected cells and viral particles might be exploited therapeutically.

scholarly journals Arenavirus Z-Glycoprotein Association Requires Z Myristoylation but Not Functional RING or Late Domains

2007 ◽  
Vol 81 (17) ◽  
pp. 9451-9460 ◽  
Author(s):  
Althea A. Capul ◽  
Mar Perez ◽  
Emily Burke ◽  
Stefan Kunz ◽  
Michael J. Buchmeier ◽  
...  
Keyword(s):  
Function Analysis ◽  
Ring Finger ◽  
Hemorrhagic Fever ◽  
Lassa Fever ◽  
Lymphocytic Choriomeningitis ◽  
Viral Envelope ◽  
Surface Glycoprotein ◽  
Glycoprotein Precursor ◽  
Biochemical Interaction ◽  
Lassa Fever Virus

ABSTRACT Generation of infectious arenavirus-like particles requires the virus RING finger Z protein and surface glycoprotein precursor (GPC) and the correct processing of GPC into GP1, GP2, and a stable signal peptide (SSP). Z is the driving force of arenavirus budding, whereas the GP complex (GPc), consisting of hetero-oligomers of SSP, GP1, and GP2, forms the viral envelope spikes that mediate receptor recognition and cell entry. Based on the roles played by Z and GP in the arenavirus life cycle, we hypothesized that Z and the GPc should interact in a manner required for virion formation. Here, using confocal microscopy and coimmunoprecipitation assays, we provide evidence for subcellular colocalization and biochemical interaction, respectively, of Z and the GPc. Our results from mutation-function analysis reveal that Z myristoylation, but not the Z late (L) or RING domain, is required for Z-GPc interaction. Moreover, Z interacted directly with SSP in the absence of other components of the GPc. We obtained similar results with Z and GPC from the prototypical arenavirus lymphocytic choriomeningitis virus and the hemorrhagic fever arenavirus Lassa fever virus.

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