scholarly journals Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization

2006 ◽  
Vol 80 (24) ◽  
pp. 12070-12078 ◽  
Author(s):  
Michael J. Ciancanelli ◽  
Christopher F. Basler
Keyword(s):  
Matrix Protein ◽  
Ebola Virus ◽  
Equine Infectious Anemia Virus ◽  
Nipah Virus ◽  
Gag Protein ◽  
Late Domain ◽  
M Proteins ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus

ABSTRACT Matrix (M) proteins reportedly direct the budding of paramyxoviruses from infected cells. In order to begin to characterize the assembly process for the highly lethal, emerging paramyxovirus Nipah virus (NiV), we have examined the budding of NiV M. We demonstrated that expression of the NiV M protein is sufficient to produce budding virus-like particles (VLPs) that are physically and morphologically similar to NiV. We identified in NiV M a sequence, YMYL, with similarity to the YPDL late domain found in the equine infectious anemia virus Gag protein. When the YMYL within NiV M was mutated, VLP release was abolished and M was relocalized to the nucleus, but the mutant M proteins retained oligomerization activity. When YMYL was fused to a late-domain mutant of the Ebola virus VP40 matrix protein, VP40 budding was restored. These results suggest that the YMYL sequence may act as a trafficking signal and a late domain for NiV M.

scholarly journals Functional Replacement and Positional Dependence of Homologous and Heterologous L Domains in Equine Infectious Anemia Virus Replication

2002 ◽  
Vol 76 (4) ◽  
pp. 1569-1577 ◽  
Author(s):  
Feng Li ◽  
Chaoping Chen ◽  
Bridget A. Puffer ◽  
Ronald C. Montelaro
Keyword(s):  
Life Cycle ◽  
Virus Particle ◽  
Matrix Protein ◽  
Particle Production ◽  
Equine Infectious Anemia Virus ◽  
Virus Infectivity ◽  
Gag Protein ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Hiv 1

ABSTRACT We have previously demonstrated by Gag polyprotein budding assays that the Gag p9 protein of equine infectious anemia virus (EIAV) utilizes a unique YPDL motif as a late assembly domain (L domain) to facilitate release of the budding virus particle from the host cell plasma membrane (B. A. Puffer, L. J. Parent, J. W. Wills, and R. C. Montelaro, J. Virol. 71:6541-6546, 1997). To characterize in more detail the role of the YPDL L domain in the EIAV life cycle, we have examined the replication properties of a series of EIAV proviral mutants in which the parental YPDL L domain was replaced by a human immunodeficiency virus type 1 (HIV-1) PTAP or Rous sarcoma virus (RSV) PPPY L domain in the p9 protein or by proviruses in which the parental YPDL or HIV-1 PTAP L domain was inserted in the viral matrix protein. The replication properties of these L-domain variants were examined with respect to Gag protein expression and processing, virus particle production, and virus infectivity. The data from these experiments indicate that (i) the YPDL L domain of p9 is required for replication competence (assembly and infectivity) in equine cell cultures, including the natural target equine macrophages; (ii) all of the functions of the YPDL L domain in the EIAV life cycle can be replaced by replacement of the parental YPDL sequence in p9 with the PTAP L-domain segment of HIV-1 p6 or the PPPY L domain of RSV p2b; and (iii) the assembly, but not infectivity, functions of the EIAV proviral YPDL substitution mutants can be partially rescued by inclusions of YPDL and PTAP L-domain sequences in the C-terminal region of the EIAV MA protein. Taken together, these data demonstrate that the EIAV YPDL L domain mediates distinct functions in viral budding and infectivity and that the HIV-1 PTAP and RSV PPPY L domains can effectively facilitate these dual replication functions in the context of the p9 protein. In light of the fact that YPDL, PTAP, and PPPY domains evidently have distinct characteristic binding specificities, these observations may indicate different portals into common cellular processes that mediate EIAV budding and infectivity, respectively.

scholarly journals Equine Infectious Anemia Virus and the Ubiquitin-Proteasome System

2002 ◽  
Vol 76 (6) ◽  
pp. 3038-3044 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Raymond C. Sowder ◽  
Julian Adams ◽  
Kunio Nagashima ◽  
...  
Keyword(s):  
Time Course ◽  
Equine Infectious Anemia Virus ◽  
Proteasome Inhibitors ◽  
Ubiquitin Proteasome System ◽  
Gag Protein ◽  
Different Types ◽  
Ubiquitin Proteasome ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus

ABSTRACT Some retroviruses contain monoubiquitinated Gag and do not bud efficiently from cells treated with proteasome inhibitors, suggesting an interaction between the ubiquitin-proteasome system and retrovirus assembly. We examined equine infectious anemia virus (EIAV) particles and found that approximately 2% of the p9Gag proteins are monoubiquitinated, demonstrating that this Gag protein interacts with an ubiquitinating activity. Different types of proteasome inhibitors were used to determine if proteasome inactivation affects EIAV release from chronically infected cells. Pulse-chase immunoprecipitation and time course immunoblot analyses showed that proteasome inactivation slightly decreased virus release (at most a twofold effect), while it did not affect Gag processing. These results contrast with those obtained with other viruses which are sensitive to these inhibitors. This suggests that, although its Gag is monoubiquitinated, the requirements for EIAV release are somewhat different from those for retroviruses that are sensitive to proteasome inhibitors.

scholarly journals Equine Infectious Anemia Virus Utilizes Host Vesicular Protein Sorting Machinery during Particle Release

2003 ◽  
Vol 77 (15) ◽  
pp. 8440-8447 ◽  
Author(s):  
Giancarlo O. Tanzi ◽  
Andrew J. Piefer ◽  
Paul Bates
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Leukemia Virus ◽  
Protein Sorting ◽  
Gag Protein ◽  
Particle Release ◽  
Late Domain ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Vacuolar Protein ◽  
Hiv 1

ABSTRACT A final step in retrovirus assembly, particle release from the cell, is modulated by a small motif in the Gag protein known as a late domain. Recently, human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (M-MuLV) were shown to require components of the cellular vacuolar protein sorting (VPS) machinery for efficient viral release. HIV-1 interacts with the VPS pathway via an association of HIV-1 Gag with TSG101, a component of the cellular complexes involved in VPS. Equine infectious anemia virus (EIAV) is unique among enveloped viruses studied to date because it utilizes a novel motif, YPDL in Gag, as a late domain. Our analysis of EIAV assembly demonstrates that EIAV Gag release is blocked by inhibition of the VPS pathway. However, in contrast to HIV-1, EIAV Gag release is insensitive to TSG101 depletion and EIAV particles do not contain significant levels of TSG101. Finally, we demonstrate that fusing EIAV Gag directly with another cellular component of the VPS machinery, VPS28, can restore efficient release of an EIAV Gag late-domain mutant. These results provide evidence that retroviruses can interact with the cellular VPS machinery in several different ways to accomplish particle release.

scholarly journals An Equine Infectious Anemia Virus Variant Superinfects Cells through Novel Receptor Interactions

2008 ◽  
Vol 82 (19) ◽  
pp. 9425-9432 ◽  
Author(s):  
Melinda A. Brindley ◽  
Baoshan Zhang ◽  
Ronald C. Montelaro ◽  
Wendy Maury
Keyword(s):  
Endothelial Cells ◽  
Equine Infectious Anemia Virus ◽  
Cell Monolayer ◽  
Wild Type ◽  
Virus Variant ◽  
Receptor Interactions ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus ◽  
Type Strains

ABSTRACT Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAVvMA-1c, can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAVvMA-1c superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAVvMA-1c overcomes this restriction. We found that the cellular receptor used by EIAV, equine lentivirus receptor 1 (ELR1), remains on the surface of cells chronically infected with EIAV, suggesting that wild-type EIAV interferes with superinfection by masking ELR1. The addition of soluble wild-type SU protein to the medium during infection blocked infection by wild-type strains of virus, implicating SU as the viral protein responsible for interfering with virion entry into previously infected cells. Additionally, interference of wild-type EIAV binding to ELR1 by the addition of either anti-ELR1 antibodies or the ELR1 ectodomain prevented entry of the wild-type strains of EIAV into two permissive cell populations. Many of these same interference treatments prevented EIAVvMA-1c infection of endothelial cells but only modestly affected the ability of EIAVvMA-1c to enter and kill previously infected ED cells. These findings indicate that EIAVvMA-1c retains the ability to use ELR1 for entry and suggest that this virus can interact with an additional, unidentified receptor to superinfect ED cells.

Replication of a Nipah Virus Encoding a Nuclear-Retained Matrix Protein

2019 ◽  
Vol 221 (Supplement_4) ◽  
pp. S389-S394 ◽  
Author(s):  
Marc Ringel ◽  
Laura Behner ◽  
Anja Heiner ◽  
Lucie Sauerhering ◽  
Andrea Maisner
Keyword(s):  
Plasma Membrane ◽  
Nuclear Export ◽  
Matrix Protein ◽  
Virus Assembly ◽  
Nipah Virus ◽  
Nuclear Export Signal ◽  
Cell Types ◽  
Surface Transport ◽  
M Proteins ◽  
Infected Cells

Abstract Nipah virus (NiV) matrix protein (NiV M) plays a major role in virus assembly. It undergoes nuclear transit before accumulating at the plasma membrane and recruiting nucleocapsids to the budding sites. Because nuclear NiV M cannot be detected in all cell types, we wondered whether it can reach the cell surface by bypassing the nucleus. Using an M mutant with a defective nuclear export signal (MNESmut), however, we revealed that the nuclear import of M is ubiquitous, because MNESmut was retained in the nuclei of all cell types tested. Because a functional nuclear transit is a general prerequisite for M surface transport, we wanted to characterize the effect of nuclear-retained M protein in a full viral context and generated a recombinant NiV-MNESmut. Mutant NiV-MNESmut caused increased cell-cell fusion and produced lower virus titers. As expected for an assembly defective NiV, perinuclear inclusions (IBperi) were formed, but inclusions at the plasma membrane (IBPM), which probably represent the viral assembly platforms, were not found. It is interesting to note that the transport-defective MNESmut was recruited to IBperi. This probably prevents overaccumulation of nonfunctional M proteins in the cytoplasm and nuclei of NiV-infected cells and thus provides first evidence that IBperi are functionally relevant aggresome-like compartments.

scholarly journals Differential Effects of Actin Cytoskeleton Dynamics on Equine Infectious Anemia Virus Particle Production

2004 ◽  
Vol 78 (2) ◽  
pp. 882-891 ◽  
Author(s):  
Chaoping Chen ◽  
Ora A. Weisz ◽  
Donna B. Stolz ◽  
Simon C. Watkins ◽  
Ronald C. Montelaro
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Equine Infectious Anemia Virus ◽  
Cytochalasin D ◽  
Full Length ◽  
Gag Proteins ◽  
Virion Production ◽  
Equine Infectious Anemia ◽  
Infected Cells ◽  
Anemia Virus

ABSTRACT Retrovirus assembly and budding involve a highly dynamic and concerted interaction of viral and cellular proteins. Previous studies have shown that retroviral Gag proteins interact with actin filaments, but the significance of these interactions remains to be defined. Using equine infectious anemia virus (EIAV), we now demonstrate differential effects of cellular actin dynamics at distinct stages of retrovirus assembly and budding. First, virion production was reduced when EIAV-infected cells were treated with phallacidin, a cell-permeable reagent that stabilizes actin filaments by slowing down their depolymerization. Confocal microscopy confirmed that the inhibition of EIAV production correlated temporally over several days with the incorporation dynamics of phallacidin into the actin cytoskeleton. Although the overall structure of the actin cytoskeleton and expression of viral protein appeared to be unaffected, phallacidin treatment dramatically reduced the amount of full-length Gag protein associated with the actin cytoskeleton. These data suggest that an association of full-length Gag proteins with de novo actin filaments might contribute to Gag assembly and budding. On the other hand, virion production was enhanced when EIAV-infected cells were incubated briefly (2 h) with the actin-depolymerizing drugs cytochalasin D and latrunculin B. Interestingly, the enhanced virion production induced by cytochalasin D required a functional late (L) domain, either the EIAV YPDL L-domain or the proline-rich L domains derived from human immunodeficiency virus type 1 or Rous sarcoma virus, respectively. Thus, depolymerization of actin filaments may be a common function mediated by retrovirus L domains during late stages of viral budding. Taken together, these observations indicate that dynamic actin polymerization and depolymerization may be associated with different stages of viral production.

Binding of equine infectious anemia virus matrix protein to membrane bilayers involves multiple interactions

2000 ◽  
Vol 296 (3) ◽  
pp. 887-898 ◽  
Author(s):  
Paxton Provitera ◽  
Fadilla Bouamr ◽  
Diana Murray ◽  
Carol Carter ◽  
Suzanne Scarlata
Keyword(s):  
Matrix Protein ◽  
Equine Infectious Anemia Virus ◽  
Membrane Bilayers ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Multiple Interactions

scholarly journals Equine Infectious Anemia Virus Gag Polyprotein Late Domain Specifically Recruits Cellular AP-2 Adapter Protein Complexes during Virion Assembly

1998 ◽  
Vol 72 (12) ◽  
pp. 10218-10221 ◽  
Author(s):  
Bridget A. Puffer ◽  
S. C. Watkins ◽  
Ronald C. Montelaro
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Protein Complexes ◽  
Adapter Protein ◽  
Virion Assembly ◽  
Late Domain ◽  
Rous Sarcoma ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Late Assembly Domain

ABSTRACT We have identified an interaction between the equine infectious anemia virus (EIAV) late assembly domain and the cellular AP-2 clathrin-associated adapter protein complex. A YXXL motif within the EIAV Gag late assembly domain was previously characterized as a sequence critical for release of assembling virions. We now show that this YXXL sequence interacts in vitro with the AP-50 subunit of the AP-2 complex, while the functionally interchangeable late assembly domains carried by the Rous sarcoma virus p2b protein and human immunodeficiency virus type 1 p6 protein, which utilize PPPY and PTAPP L domains, respectively, do not bind AP-50 in vitro. In addition, EIAV late domain mutants containing mutations that have previously been shown to abrogate budding also exhibit marked decreases in AP-50 binding efficiencies. A role for AP-2 complex in viral assembly is supported by immunofluorescence analysis of EIAV-infected equine dermal cells demonstrating specific colocalization of the α adaptin subunit of AP-2 with the EIAV p9 protein at sites of virus budding on the plasma membrane. These data provide strong evidence that EIAV utilizes the cellular AP-2 complex to accomplish virion assembly and release.

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