scholarly journals Important Role for the CA-NC Spacer Region in the Assembly of Bovine Immunodeficiency Virus Gag Protein

2004 ◽  
Vol 78 (2) ◽  
pp. 551-560 ◽  
Author(s):  
Xiaofeng Guo ◽  
Jing Hu ◽  
James B. Whitney ◽  
Rodney S. Russell ◽  
Chen Liang
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Virus Assembly ◽  
Virus Production ◽  
Bovine Immunodeficiency Virus ◽  
Gag Protein ◽  
Gag Proteins ◽  
Visna Virus ◽  
Immunodeficiency Virus ◽  
Anemia Virus ◽  
Hiv 1

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.

scholarly journals Conserved Interaction of Lentiviral Vif Molecules with HIV-1 Gag and Differential Effects of Species-Specific Vif on Virus Production

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Wenwen Zheng ◽  
Limian Ling ◽  
Zhaolong Li ◽  
Hong Wang ◽  
Yajuan Rui ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Virus Assembly ◽  
Virus Production ◽  
Bovine Immunodeficiency Virus ◽  
Reading Frame ◽  
Virion Infectivity Factor ◽  
Immunodeficiency Virus ◽  
New Strategy ◽  
Species Specific ◽  
Hiv 1

ABSTRACT The virion infectivity factor (Vif) open reading frame is conserved among most lentiviruses. Vif molecules contribute to viral replication by inactivating host antiviral factors, the APOBEC3 cytidine deaminases. However, various species of lentiviral Vif proteins have evolved different strategies for overcoming host APOBEC3. Whether different species of lentiviral Vif proteins still preserve certain common features has not been reported. Here, we show for the first time that diverse lentiviral Vif molecules maintain the ability to interact with the human immunodeficiency virus type 1 (HIV-1) Gag precursor (Pr55Gag) polyprotein. Surprisingly, bovine immunodeficiency virus (BIV) Vif, but not HIV-1 Vif, interfered with HIV-1 production and viral infectivity even in the absence of APOBEC3. Further analysis revealed that BIV Vif demonstrated an enhanced interaction with Pr55Gag compared to that of HIV-1 Vif, and BIV Vif defective for the Pr55Gag interaction lost its ability to inhibit HIV-1. The C-terminal region of capsid (CA) and the p2 region of Pr55Gag, which are important for virus assembly and maturation, were involved in the interaction. Transduction of CD4+ T cells with BIV Vif blocked HIV-1 replication. Thus, the conserved Vif-Pr55Gag interaction provides a potential target for the future development of antiviral strategies. IMPORTANCE The conserved Vif accessory proteins of primate lentiviruses HIV-1, simian immunodeficiency virus (SIV), and BIV all form ubiquitin ligase complexes to target host antiviral APOBEC3 proteins for degradation, with different cellular requirements and using different molecular mechanisms. Here, we demonstrate that BIV Vif can interfere with HIV-1 Gag maturation and suppress HIV-1 replication through interaction with the precursor of the Gag (Pr55Gag) of HIV-1 in virus-producing cells. Moreover, the HIV-1 and SIV Vif proteins are conserved in terms of their interactions with HIV-1 Pr55Gag although HIV-1 Vif proteins bind Pr55Gag less efficiently than those of BIV Vif. Our research not only sheds new light on this feature of these conserved lentiviral Vif proteins but also provides a formerly unrecognized target for the development of antiviral strategies. Since increasing the Vif-Pr55Gag interaction could potentially suppress virus proliferation, this approach could offer a new strategy for the development of HIV inhibitors.

scholarly journals Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins

1999 ◽  
Vol 73 (10) ◽  
pp. 8138-8144 ◽  
Author(s):  
Martin J. Vincent ◽  
Lawrence R. Melsen ◽  
Annelet S. Martin ◽  
Richard W. Compans
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Virus Assembly ◽  
Cytoplasmic Tail ◽  
Gag Protein ◽  
Gag Proteins ◽  
Basolateral Side ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1 ◽  
In Cells

ABSTRACT In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.

scholarly journals Identification of Retroviral Late Domains as Determinants of Particle Size

1999 ◽  
Vol 73 (3) ◽  
pp. 2309-2320 ◽  
Author(s):  
Laurence Garnier ◽  
Leslie J. Parent ◽  
Benjamin Rovinski ◽  
Shi-Xian Cao ◽  
John W. Wills
Keyword(s):  
Particle Size ◽  
Equine Infectious Anemia Virus ◽  
Gag Protein ◽  
Gag Proteins ◽  
Late Domain ◽  
Rous Sarcoma ◽  
Large Particles ◽  
Anemia Virus ◽  
Late Domains ◽  
Hiv 1

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.

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 Analysis of Human Immunodeficiency Virus Type 1 Gag Ubiquitination

2005 ◽  
Vol 79 (14) ◽  
pp. 9134-9144 ◽  
Author(s):  
Eva Gottwein ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Gag Protein ◽  
Gag Proteins ◽  
Immunodeficiency Virus ◽  
Lysine Residues ◽  
The Matrix ◽  
Hiv 1

ABSTRACT Ubiquitin is important for the release of human immunodeficiency virus type 1 (HIV-1) and several other retroviruses, but the functional significance of Gag ubiquitination is unknown. To address this problem, we decided to analyze Gag ubiquitination in detail. A low percentage of the HIV-1 p6 protein has previously been shown to be ubiquitinated, and published mutagenesis data suggested that Gag ubiquitination is largely lost upon mutation of the two lysine residues in p6. In this study, we show that Gag proteins lacking the p6 domain or the two lysine residues within p6 are ubiquitinated at levels comparable to those of the wild-type Gag protein. We detected monoubiquitinated forms of the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins in mature virus preparations. Protease digestion of Gag polyproteins extracted from immature virions indicated that ubiquitinated MA, CA, and possibly NC are as abundant as ubiquitinated p6. The HIV-1 late-domain motifs PTAP and LRSLF were not required for Gag ubiquitination, and mutation of the PTAP motif even resulted in an increase in the amount of Gag-Ub conjugates detected. Finally, at steady state, ubiquitinated Gag proteins were not enriched in either membrane-associated or virus-derived Gag fractions. In summary, these results indicate that HIV-1 Gag can be monoubiquitinated in all domains and that ubiquitination of lysine residues outside p6 may thus contribute to viral release and/or infectivity.

scholarly journals Ubiquitination of Human Immunodeficiency Virus Type 1 Gag Is Highly Dependent on Gag Membrane Association

2007 ◽  
Vol 81 (17) ◽  
pp. 9193-9201 ◽  
Author(s):  
Stefanie Jäger ◽  
Eva Gottwein ◽  
Hans-Georg Kräusslich
Keyword(s):  
Human Immunodeficiency Virus ◽  
Membrane Association ◽  
Gag Protein ◽  
Gag Proteins ◽  
Immunodeficiency Virus ◽  
Membrane Bound ◽  
Ptap Motif ◽  
Ubiquitination Machinery ◽  
Hiv 1

ABSTRACT Ubiquitin is important for the release of human immunodeficiency virus 1 (HIV-1) and several other retroviruses. All major domains of the HIV-1 Gag protein are monoubiquitinated, but the modifying machinery and the function of HIV-1 Gag ubiquitination remain unclear. Here, we show that the induction of a late budding arrest by mutation of the HIV-1 PTAP motif or by specific inhibition of selected ESCRT components leads to an increase of Gag-ubiquitin conjugates in cells, which coincides with an accumulation of detergent-insoluble, multimerized Gag at the plasma membrane. Membrane flotation experiments revealed that ubiquitinated Gag is highly enriched in membrane-bound fractions. Based on these findings, we propose that a blocking of virus release results in increased Gag ubiquitination as a consequence of its prolonged membrane association. Consistent with this, ubiquitination of a membrane-binding-defective (G2A)Gag mutant was dramatically reduced and the ubiquitination levels of truncated Gag proteins correlated with their abilities to bind to membranes. We therefore propose that membrane association and multimerization of HIV-1 Gag proteins, rather than a specific motif within Gag, trigger recognition by the cellular ubiquitination machinery.

scholarly journals Lentiviral Vif Degrades the APOBEC3Z3/APOBEC3H Protein of Its Mammalian Host and Is Capable of Cross-Species Activity

2010 ◽  
Vol 84 (16) ◽  
pp. 8193-8201 ◽  
Author(s):  
Rebecca S. LaRue ◽  
Joy Lengyel ◽  
Stefán R. Jónsson ◽  
Valgerdur Andrésdóttir ◽  
Reuben S. Harris
Keyword(s):  
Host Species ◽  
Equine Infectious Anemia Virus ◽  
Bovine Immunodeficiency Virus ◽  
Mammalian Host ◽  
Neutralization Capacity ◽  
Type Protein ◽  
Immunodeficiency Virus ◽  
Species Activity ◽  
Species Specific ◽  
Anemia Virus

ABSTRACT All lentiviruses except equine infectious anemia virus (EIAV) use the small accessory protein Vif to counteract the restriction activity of the relevant APOBEC3 (A3) proteins of their host species. Prior studies have suggested that the Vif-A3 interaction is species specific. Here, using the APOBEC3H (Z3)-type proteins from five distinct mammals, we report that this is generally not the case: some lentiviral Vif proteins are capable of triggering the degradation of both the A3Z3-type protein of their normal host species and those of several other mammals. For instance, SIVmac Vif can mediate the degradation of the human, macaque, and cow A3Z3-type proteins but not of the sheep or cat A3Z3-type proteins. Maedi-visna virus (MVV) Vif is similarly promiscuous, degrading not only sheep A3Z3 but also the A3Z3-type proteins of humans, macaques, cows, and cats. In contrast to the neutralization capacity of these Vif proteins, human immunodeficiency virus (HIV), bovine immunodeficiency virus (BIV), and feline immunodeficiency virus (FIV) Vif appear specific to the A3Z3-type protein of their hosts. We conclude, first, that the Vif-A3Z3 interaction can be promiscuous and, second, despite this tendency, that each lentiviral Vif protein is optimized to degrade the A3Z3 protein of its mammalian host. Our results thereby suggest that the Vif-A3Z3 interaction is relevant to lentivirus biology.

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 Cellular Motor Protein KIF-4 Associates with Retroviral Gag

1999 ◽  
Vol 73 (12) ◽  
pp. 10508-10513 ◽  
Author(s):  
Yao Tang ◽  
Ulrike Winkler ◽  
Eric O. Freed ◽  
Ted A. Torrey ◽  
Wankee Kim ◽  
...  
Keyword(s):  
Protein Transport ◽  
Gradient Centrifugation ◽  
Motor Protein ◽  
Recombinant Vaccinia Virus ◽  
Size Exclusion ◽  
Gag Protein ◽  
Gag Proteins ◽  
Immunodeficiency Virus ◽  
Cellular Motor ◽  
Hiv 1

ABSTRACT Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4–Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.

scholarly journals Functional Roles of Equine Infectious Anemia Virus Gag p9 in Viral Budding and Infection

2001 ◽  
Vol 75 (20) ◽  
pp. 9762-9770 ◽  
Author(s):  
Chaoping Chen ◽  
Feng Li ◽  
Ronald C. Montelaro
Keyword(s):  
Amino Acids ◽  
Equine Infectious Anemia Virus ◽  
Virus Assembly ◽  
Virus Budding ◽  
Gag Protein ◽  
Functional Roles ◽  
Gag Polyprotein ◽  
Viral Budding ◽  
Equine Infectious Anemia ◽  
Anemia Virus

ABSTRACT Previous studies utilizing Gag polyprotein budding assays with transfected cells reveal that the equine infectious anemia virus (EIAV) Gag p9 protein provides a late assembly function mediated by a critical Y23P24D25L26 motif (L-domain) to release viral particles from the plasma membrane. To elucidate further the role of EIAV p9 in virus assembly and replication, we have examined the replication properties of a defined series of p9 truncation and site-directed mutations in the context of a reference infectious molecular proviral clone, EIAVuk. Characterization of these p9 proviral mutants revealed new functional properties of p9 in EIAV replication, not previously elucidated by Gag polyprotein budding assays. The results of these studies demonstrated that only the N-terminal 31 amino acids of a total of 51 residues in the complete p9 protein were required to maintain replication competence in transfected equine cells; proviral mutants with p9 C-terminal truncations of 20 or fewer amino acids remained replication competent, while mutants with truncations of 21 or more residues were completely replication defective. The inability of the defective p9 proviral mutations to produce infectious virus could not be attributed to defects in Gag polyprotein expression or processing, in virion RT activity, or in virus budding. While proviral replication competence appeared to be associated with the presence of a K30K31 motif and potential ubiquitination of the EIAV p9 protein, mutations of these lysine residues to methionines produced variant proviruses that replicated as well as the parental EIAVuk in transfected ED cells. Thus, these observations reveal for the first time that EIAV p9 is not absolutely required for virus budding in the context of proviral gene expression, suggesting that other EIAV proteins can at least in part mediate late budding functions previously associated with the p9 protein. In addition, the data define a function for EIAV p9 in the infectivity of virus particles, indicating a previously unrecognized role for this Gag protein in EIAV replication.

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