scholarly journals Phenotypic analysis of human immunodeficiency virus type 1 Rev trimerization-interface mutants in human cells

2005 ◽  
Vol 86 (5) ◽  
pp. 1509-1513 ◽  
Author(s):  
Roochi Trikha ◽  
David W. Brighty
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Dominant Negative ◽  
Phenotypic Analysis ◽  
Rev Response Element ◽  
Immunodeficiency Virus

Nuclear export of unspliced and incompletely spliced human immunodeficiency virus type 1 mRNA is mediated by the viral Rev protein. Rev binds to a structured RNA motif known as the Rev-response element (RRE), which is present in all Rev-dependent transcripts, and thereby promotes entry of the ribonucleoprotein complex into the nuclear-export pathway. Recent evidence indicates that a dimerization interface and a genetically separable ‘trimerization’ interface are required for multimeric assembly of Rev on the RRE. In this report, the effect of mutations within the trimerization interface on Rev function was examined in mammalian cells. All trimerization-defective Rev molecules had profoundly compromised Rev function and a range of localization defects was observed. However, despite the potential for formation of heterodimers between functional and non-functional Rev proteins, trimerization-defective Rev mutants were unable to inhibit wild-type Rev function in a trans-dominant-negative manner.

scholarly journals A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein

2003 ◽  
Vol 77 (13) ◽  
pp. 7236-7243 ◽  
Author(s):  
L. K. Venkatesh ◽  
T. Gettemeier ◽  
G. Chinnadurai
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Cell Leukemia Virus Type ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Rev Protein

ABSTRACT The Rev protein of human immunodeficiency virus type 1 (HIV-1) is essential for the nucleocytoplasmic transport of unspliced and partially spliced HIV mRNAs containing the Rev response element (RRE). In a yeast two-hybrid screen of a HeLa cell-derived cDNA expression library for human factors interacting with the Rev leucine-rich nuclear export sequence (NES), we identified a kinesin-like protein, REBP (Rev/Rex effector binding protein), highly homologous to Kid, the carboxy-terminal 75-residue region of which interacts specifically with the NESs of HIV-1 Rev, human T-cell leukemia virus type 1 Rex, and equine infectious anemia virus Rev but not with functionally inactive mutants thereof. REBP is a nuclear protein that colocalizes with Rev in the nucleoplasm and nuclear periphery of transfected cells. Specific, albeit weak, interaction between REBP and Rev could be demonstrated in coimmunoprecipitation assays in BSC-40 cells. REBP can modestly enhance Rev-dependent RRE-linked reporter gene expression both independently and in cooperation with the nucleoporin cofactor Rab/hRIP. Thus, REBP displays the characteristics expected of an authentic mediator of Rev NES function and may play a role in RRE RNA transport during HIV infection.

scholarly journals Matrix Mediates the Functional Link between Human Immunodeficiency Virus Type 1 RNA Nuclear Export Elements and the Assembly Competency of Gag in Murine Cells

2009 ◽  
Vol 83 (17) ◽  
pp. 8525-8535 ◽  
Author(s):  
Nathan M. Sherer ◽  
Chad M. Swanson ◽  
Stelios Papaioannou ◽  
Michael H. Malim
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Particle Production ◽  
Functional Link ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) assembles poorly in murine cells, reflecting inefficient targeting of the Gag structural polyprotein to the plasma membrane. Virus particle production can be restored by replacing the cis-acting Rev response element (RRE) in Gag-Pol mRNAs with multiple copies of the CTE (4×CTE), suggesting a mechanistic link between HIV-1 RNA trafficking and productive Gag assembly. In this report, we demonstrate that Gag molecules generated from RRE-dependent transcripts are intrinsically defective for assembly in murine 3T3 cells. When controlled for the intracellular Gag level, modulations of the Gag matrix (MA) domain that enhance Gag membrane association (e.g., deletion of the MA globular head) substantially improve assembly for Gag derived from RRE- but not 4×CTE-dependent transcripts. Gag mutants carrying a leucine zipper replacement of the nucleocapsid (NC) domain remain largely assembly defective when derived from RRE-dependent transcripts, indicating that the defect does not reflect aberrant NC/RNA-driven Gag multimerization. We further demonstrate that single changes in uncharged amino acids implicated in Gag/MA myristoyl switch regulation, most notably replacing the leucine at position 21 with serine, improve assembly for Gag derived from RRE-dependent transcripts. In sum, we provide genetic evidence to suggest that HIV-1 RNA metabolism specifically modulates the activation of MA-dependent membrane targeting.

scholarly journals A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.

1996 ◽  
Vol 16 (12) ◽  
pp. 7144-7150 ◽  
Author(s):  
F Stutz ◽  
E Izaurralde ◽  
I W Mattaj ◽  
M Rosbash
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Pore ◽  
Immunodeficiency Virus ◽  
Rna Export ◽  
Rev Protein

The human immunodeficiency virus type 1 Rev protein contains a nuclear export signal (NES) that is required for Rev-mediated RNA export in mammals as well as in the yeast Saccharomyces cerevisiae. The Rev NES has been shown to specifically interact with a human (hRIP/RAB1) and a yeast (yRip1p) protein in the two-hybrid assay. Both of these interacting proteins are related to FG nucleoporins on the basis of the presence of typical repeat motifs. This paper shows that Rev is able to interact with multiple FG repeat-containing nucleoporins from both S. cerevisiae and mammals; moreover, the ability of Rev NES mutants to interact with these FG nucleoporins parallels the ability of the mutants to promote RNA export in yeast and mammalian cells. The data also show that, after Xenopus oocyte nuclear injection, several FG nucleoporin repeat domains inhibit the export of both Rev protein and U small nuclear RNAs, suggesting that these nucleoporins participate in Rev-mediated and cellular RNA export. Interestingly, not all FG nucleoporin repeat domains produced the same pattern of RNA export inhibition. The results suggest that Rev and cellular mediators of RNA export can interact with multiple components of the nuclear pore complex during transport, analogous to the proposed mode of action of the nuclear protein import receptor.

scholarly journals Human Immunodeficiency Virus Type 1 Nucleocapsid p1 Confers ESCRT Pathway Dependence

2010 ◽  
Vol 84 (13) ◽  
pp. 6590-6597 ◽  
Author(s):  
Elena Popova ◽  
Sergei Popov ◽  
Heinrich G. Göttlinger
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Leucine Zipper ◽  
Particle Production ◽  
Dominant Negative ◽  
Dimerization Domain ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT To facilitate the release of infectious progeny virions, human immunodeficiency virus type 1 (HIV-1) exploits the Endosomal Sorting Complex Required for Transport (ESCRT) pathway by engaging Tsg101 and ALIX through late assembly (L) domains in the C-terminal p6 domain of Gag. However, the L domains in p6 are known to be dispensable for efficient particle production by certain HIV-1 Gag constructs that have the nucleocapsid (NC) domain replaced by a foreign dimerization domain to substitute for the assembly function of NC. We now show that one such L domain-independent HIV-1 Gag construct (termed ZWT) that has NC-p1-p6 replaced by a leucine zipper domain is resistant to dominant-negative inhibitors of the ESCRT pathway that block HIV-1 particle production. However, ZWT became dependent on the presence of an L domain when NC-p1-p6 was restored to its C terminus. Furthermore, when the NC domain was replaced by a leucine zipper, the p1-p6 region, but not p6 alone, conferred sensitivity to inhibition of the ESCRT pathway. In an authentic HIV-1 Gag context, the effect of an inhibitor of the ESCRT pathway on particle production could be alleviated by deleting a portion of the NC domain together with p1. Together, these results indicate that the ESCRT pathway dependence of HIV-1 budding is determined, at least in part, by the NC-p1 region of Gag.

scholarly journals The Human Immunodeficiency Virus Type 1 gp120 V2 Domain Mediates gp41-Independent Intersubunit Contacts

2000 ◽  
Vol 74 (10) ◽  
pp. 4448-4455 ◽  
Author(s):  
Rob J. Center ◽  
Patricia L. Earl ◽  
Jacob Lebowitz ◽  
Peter Schuck ◽  
Bernard Moss
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Mammalian Cells ◽  
Quaternary Structure ◽  
Gel Filtration ◽  
Sedimentation Equilibrium ◽  
Oligomeric Structure ◽  
Immunodeficiency Virus

ABSTRACT The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.

scholarly journals Functional Analysis of the Human Immunodeficiency Virus Type 1 Rev Protein Oligomerization Interface

1998 ◽  
Vol 72 (4) ◽  
pp. 2935-2944 ◽  
Author(s):  
Sarah L. Thomas ◽  
Martin Oft ◽  
Herbert Jaksche ◽  
Georg Casari ◽  
Peter Heger ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Regulatory Protein ◽  
Surface Patch ◽  
Target Sequence ◽  
Rev Response Element ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The expression of human immunodeficiency virus type 1 (HIV-1) structural proteins requires the action of the viraltrans-regulatory protein Rev. Rev is a nuclear shuttle protein that directly binds to its cis-acting Rev response element (RRE) RNA target sequence. Subsequent oligomerization of Rev monomers on the RRE and interaction of Rev with a cellular cofactor(s) result in the cytoplasmic accumulation of RRE-containing viral mRNAs. Moreover, Rev by itself is exported from the nucleus to the cytoplasm. Although it has been demonstrated that Rev multimerization is critically required for Rev activity and hence for HIV-1 replication, the number of Rev monomers required to form atrans-activation-competent complex on the RRE is unknown. Here we report a systematic analysis of the putative multimerization domains within the Rev trans-activator protein. We identify the amino acid residues which are part of the proposed single hydrophobic surface patch in the Rev amino terminus that mediates intermolecular interactions. Furthermore, we show that the expression of a multimerization-deficient Rev mutant blocks HIV-1 replication in a trans-dominant (dominant-negative) fashion.

scholarly journals Longitudinal Phenotypic Analysis of Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes: Correlation with Disease Progression

1999 ◽  
Vol 73 (11) ◽  
pp. 9153-9160 ◽  
Author(s):  
Graham S. Ogg ◽  
Stefan Kostense ◽  
Michel R. Klein ◽  
Suzanne Jurriaans ◽  
Dörte Hamann ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Lymphocytes ◽  
Disease Progression ◽  
Cytotoxic T Lymphocytes ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Phenotypic Analysis ◽  
Tetramer Binding ◽  
Immunodeficiency Virus

ABSTRACT Few studies have examined longitudinal changes in human immunodeficiency virus type 1 (HIV)-specific cytotoxic T lymphocytes (CTL). To more closely define the natural history of HIV-specific CTL, we used HLA-peptide tetrameric complexes to study the longitudinal CD8+ T-cell response evolution in 16 A*0201-positive untreated individuals followed clinically for up to 14 years. As early as 1 to 2 years after seroconversion, we found a significant association between high frequencies of A*0201-restricted p17Gag/Pol tetramer-binding cells and slower disease progression (P < 0.01). We observed that responses could remain stable over many months, but any longitudinal changes that occurred were typically accompanied by reciprocal changes in RNA viral load. Phenotypic analysis with markers CD45RO, CD45RA, and CD27 identified distinct subsets of antigen-specific cells and the preferential loss of CD27+ CD45RO+ cells during periods of rapid decline in the frequency of tetramer-binding cells. In addition we were unable to confirm previous studies showing a consistent selective loss of HIV-specific cells in the context of sustained Epstein-Barr virus-specific cell frequencies. Overall, these data support a role of HIV-specific CTL in the control of disease progression and suggest that the ultimate loss of such CTL may be preferentially from the CD27+ CD45RO+ subset.

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