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

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.

2002 ◽  
Vol 76 (4) ◽  
pp. 1569-1577 ◽  
Author(s):  
Feng Li ◽  
Chaoping Chen ◽  
Bridget A. Puffer ◽  
Ronald C. Montelaro

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.


2006 ◽  
Vol 80 (24) ◽  
pp. 12070-12078 ◽  
Author(s):  
Michael J. Ciancanelli ◽  
Christopher F. Basler

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.


2004 ◽  
Vol 78 (2) ◽  
pp. 724-732 ◽  
Author(s):  
Miranda Shehu-Xhilaga ◽  
Sherimay Ablan ◽  
Dimiter G. Demirov ◽  
Chaoping Chen ◽  
Ronald C. Montelaro ◽  
...  

ABSTRACT The Gag proteins of a number of different retroviruses contain late or L domains that promote the release of virions from the plasma membrane. Three types of L domains have been identified to date: Pro-Thr-Ala-Pro (PTAP), Pro-Pro-X-Tyr, and Tyr-Pro-Asp-Leu. It has previously been demonstrated that overexpression of the N-terminal, E2-like domain of the endosomal sorting factor TSG101 (TSG-5′) inhibits human immunodeficiency virus type 1 (HIV-1) release but does not affect the release of the PPPY-containing retrovirus murine leukemia virus (MLV), whereas overexpression of the C-terminal portion of TSG101 (TSG-3′) potently disrupts both HIV-1 and MLV budding. In addition, it has been reported that, while the release of a number of retroviruses is disrupted by proteasome inhibitors, equine infectious anemia virus (EIAV) budding is not affected by these agents. In this study, we tested the ability of TSG-5′, TSG-3′, and full-length TSG101 (TSG-F) overexpression, a dominant negative form of the AAA ATPase Vps4, and proteasome inhibitors to disrupt the budding of EIAV particles bearing each of the three types of L domain. The results indicate that (i) inhibition by TSG-5′ correlates with dependence on PTAP; (ii) the release of wild-type EIAV (EIAV/WT) is insensitive to TSG-3′, whereas this C-terminal TSG101 fragment potently impairs the budding of EIAV when it is rendered PTAP or PPPY dependent; (iii) budding of all EIAV clones is blocked by dominant negative Vps4; and (iv) EIAV/WT release is not impaired by proteasome inhibitors, while EIAV/PTAP and EIAV/PPPY release is strongly disrupted by these compounds. These findings highlight intriguing similarities and differences in host factor utilization by retroviral L domains and suggest that the insensitivity of EIAV to proteasome inhibitors is conferred by the L domain itself and not by determinants in Gag outside the L domain.


1999 ◽  
Vol 73 (3) ◽  
pp. 2309-2320 ◽  
Author(s):  
Laurence Garnier ◽  
Leslie J. Parent ◽  
Benjamin Rovinski ◽  
Shi-Xian Cao ◽  
John W. Wills

ABSTRACT Retroviral Gag proteins, in the absence of any other viral products, induce budding and release of spherical, virus-like particles from the plasma membrane. Gag-produced particles, like those of authentic retrovirions, are not uniform in diameter but nevertheless fall within a fairly narrow distribution of sizes. For the human immunodeficiency virus type 1 (HIV-1) Gag protein, we recently reported that elements important for controlling particle size are contained within the C-terminal region of Gag, especially within the p6 sequence (L. Garnier, L. Ratner, B. Rovinski, S.-X. Cao, and J. W. Wills, J. Virol. 72:4667–4677, 1998). Deletions and substitutions throughout this sequence result in the release of very large particles. Because the size determinant could not be mapped to any one of the previously defined functions within p6, it seemed likely that its activity requires the overall proper folding of this region of Gag. This left open the possibility of the size determinant residing in a subdomain of p6, and in this study, we examined whether the late domain (the region of Gag that is critical for the virus-cell separation step) is involved in controlling particle size. We found that particles of normal size are produced when p6 is replaced with the totally unrelated late domain sequences from Rous sarcoma virus (contained in its p2b sequence) or equine infectious anemia virus (contained in p9). In addition, we found that the large particles released in the absence of p6 require the entire CA and adjacent spacer peptide sequences, whereas these internal sequences of HIV-1 Gag are not needed for budding (or proper size) when a late domain is present. Thus, it appears the requirements for budding are very different in the presence and absence of p6.


Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Alžběta Dostálková ◽  
Barbora Vokatá ◽  
Filip Kaufman ◽  
Pavel Ulbrich ◽  
Tomáš Ruml ◽  
...  

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.


2009 ◽  
Vol 83 (7) ◽  
pp. 3365-3373 ◽  
Author(s):  
Sarah Sebastian ◽  
Christian Grütter ◽  
Caterina Strambio de Castillia ◽  
Thomas Pertel ◽  
Silvia Olivari ◽  
...  

ABSTRACT TRIM5α is a retrovirus restriction factor in the host cell cytoplasm that blocks infection before provirus establishment. Restriction activity requires capsid (CA)-specific recognition by the PRYSPRY domain of TRIM5α. To better understand the restriction mechanism, nine charge-cluster-to-triple-alanine mutants in the TRIM5α PRYSPRY domain were assessed for CA-specific restriction activity. Five mutants distributed along the TRIM5α PRYSPRY primary sequence disrupted restriction activity against N-tropic murine leukemia virus and equine infectious anemia virus. Modeling of the TRIM5α PRYSPRY domain based on the crystal structures of PRYSPRY-19q13.4.1, GUSTAVUS, and TRIM21 identified a surface patch where disruptive mutants clustered. All mutants in this patch retained CA-binding activity, a reticular distribution in the cytoplasm, and steady-state protein levels comparable to those of the wild type. Residues in the essential patch are conserved in TRIM5α orthologues and in closely related paralogues. The same surface patch in the TRIM18 and TRIM20 PRYSPRY domains is the site of mutants causing Opitz syndrome and familial Mediterranean fever. These results indicate that, in addition to CA-specific binding, the PRYSPRY domain possesses a second function, possibly binding of a cofactor, that is essential for retroviral restriction activity by TRIM5α.


2016 ◽  
Author(s):  
Ajit Chande ◽  
Cristiana Cuccurullo ◽  
Annachiara Rosa ◽  
Serena Ziglio ◽  
Susan Carpenter ◽  
...  

ABSTRACTThe lentivirus equine infectious anemia virus (EIAV) encodes S2, a pathogenic determinant important for virus replication and disease progression in horses. No molecular function has yet been linked to this accessory protein. We now report that S2 can replace the activity of Nef on HIV-1 infectivity, being required to antagonize the inhibitory activity of SERINC proteins on Nef-defective HIV-1. Similar to Nef, S2 excludes SERINC5 from virus particles and requires an ExxxLL motif predicted to recruit the clathrin adaptor AP2. Accordingly, a functional endocytic machinery is essential for S2-mediated infectivity enhancement, which is impaired by inhibitors of clathrin-mediated endocytosis. In addition to retargeting SERINC5 to a late endosomal compartment, S2 promotes the host factor degradation. Emphasizing the similarity with Nef, we show that S2 is myristoylated and, compatible with a crucial role of the post-translational modification, its N-terminal glycine is required for the anti-SERINC5 activity.EIAV-derived vectors devoid of S2 are less susceptible than HIV-1 to the inhibitory effect of both human and equine SERINC5. We then identified the envelope glycoprotein of EIAV as a determinant which also modulates retrovirus susceptibility to SERINC5, indicating a bi-modular ability of the equine lentivirus to counteract the host factor.S2 shares no sequence homology with other retroviral factors known to counteract SERINC5. Adding to primate lentivirus Nef and gammaretrovirus glycoGag, the accessory protein from EIAV makes another example of a retroviral virulence determinant which independently evolved SERINC5-antagonizing activity. SERINC5 therefore plays a critical role for the interaction of the host with diverse retrovirus pathogens.Significance StatementSERINC5 and SERINC3 are recently discovered cellular inhibitors of retroviruses, which are incorporated into virus particles and impair their ability to propagate the infection to target cells. Only two groups of viruses (represented by HIV-1 and MLV) have so far been identified to have evolved the ability of counteracting SERINC inhibition. We now discovered that Equine infectious anemia virus, which causes a debilitating disease in horses, also acquired the ability to protect the virus particle from inhibition by SERINC5 and SERINC3, using its small protein S2. The evidence that three different retroviruses have independently evolved the ability to elude inhibition bySERINC5 and SERINC3 indicates that these cellular factors play a fundamental role against various retrovirus pathogens.


2002 ◽  
Vol 76 (6) ◽  
pp. 3038-3044 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Raymond C. Sowder ◽  
Julian Adams ◽  
Kunio Nagashima ◽  
...  

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.


2004 ◽  
Vol 78 (2) ◽  
pp. 551-560 ◽  
Author(s):  
Xiaofeng Guo ◽  
Jing Hu ◽  
James B. Whitney ◽  
Rodney S. Russell ◽  
Chen Liang

ABSTRACT Lentiviral Gag proteins contain a short spacer sequence that separates the capsid (CA) from the downstream nucleocapsid (NC) domain. This short spacer has been shown to play an important role in the assembly of human immunodeficiency virus type 1 (HIV-1). We have now extended this finding to the CA-NC spacer motif within the Gag protein of bovine immunodeficiency virus (BIV). Mutation of this latter spacer sequence led to dramatic reductions in virus production, which was mainly attributed to the severely disrupted association of the mutated Gag with the plasma membrane, as shown by the results of membrane flotation assays and confocal microscopy. Detailed mutagenesis analysis of the BIV CA-NC spacer region for virus assembly determinants led to the identification of two key residues, L368 and M372, which are separated by three amino acids, 369-VAA-371. Incidentally, the same two residues are present within the HIV-1 CA-NC spacer region at positions 364 and 368 and have also been shown to be crucial for HIV-1 assembly. Regardless of this conservation between these two viruses, the BIV CA-NC spacer could not be replaced by its HIV-1 counterpart without decreasing virus production, as opposed to its successful replacement by the CA-NC spacer sequences from the nonprimate lentiviruses such as feline immunodeficiency virus (FIV), equine infectious anemia virus and visna virus, with the sequence from FIV showing the highest effectiveness in this regard. Taken together, these data suggest a pivotal role for the CA-NC spacer region in the assembly of BIV Gag; however, the mechanism involved therein may differ from that for the HIV-1 CA-NC spacer.


1998 ◽  
Vol 72 (12) ◽  
pp. 10218-10221 ◽  
Author(s):  
Bridget A. Puffer ◽  
S. C. Watkins ◽  
Ronald C. Montelaro

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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