scholarly journals Mapping the RNA binding sites for human immunodeficiency virus type-1 Gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions

1998 ◽  
Vol 26 (16) ◽  
pp. 3667-3676 ◽  
Author(s):  
C. K. Damgaard ◽  
H. Dyhr-Mikkelsen ◽  
J. Kjems
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Sites ◽  
Rna Binding ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Rna Binding Sites

scholarly journals Naturally Occurring Human Immunodeficiency Virus Type 1 Long Terminal Repeats Have a Frequently Observed Duplication That Binds RBF-2 and Represses Transcription

1998 ◽  
Vol 72 (8) ◽  
pp. 6465-6474 ◽  
Author(s):  
Mario Clemente Estable ◽  
Brendan Bell ◽  
Martin Hirst ◽  
Ivan Sadowski
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Sites ◽  
Naturally Occurring ◽  
Immunodeficiency Virus ◽  
Aids Study ◽  
Hiv 1 ◽  
In Cells

ABSTRACT Approximately 38% of human immunodeficiency virus type 1 (HIV-1)-infected patients within the Vancouver Lymphadenopathy-AIDS Study have proviruses bearing partial 15- to 34-nucleotide duplications upstream of the NF-κB binding sites within the 5′ long terminal repeat (LTR). This most frequent naturally occurring length polymorphism (MFNLP) of the HIV-1 5′ LTR encompasses potential binding sites for several candidate transcription factors, including TCF-1α/hLEF, c-Ets, AP-4, and Ras-responsive binding factor 2 (RBF-2) (M. C. Estable et al., J. Virol. 70:4053–4062, 1996). RBF-2 and an apparently related factor, RBF-1, bind to at least fourcis elements within the LTR which are required for full transcriptional responsiveness to protein-tyrosine kinases and v-Ras (B. Bell and I. Sadowski, Oncogene 13:2687–2697, 1996). Here we demonstrate that representative MFNLPs from two patients specifically bind RBF-2. In both cases, deletion of the MFNLP caused elevated LTR-directed transcription in cells expressing RBF-2 but not in cells with undetectable RBF-2. RBF-1, but not RBF-2, appears to contain the Ets transcription factor family member GABPα/GABPβ1. Taken together with the fact that every MFNLP from a comparative study of over 500 LTR sequences from 42 patients contains a predicted binding site for RBF-2, our data suggest that the MFNLP is selected in vivo because it provides a duplicated RBF-2 cis element, which may limit transcription in monocytes and activated T cells.

scholarly journals Redundant Roles for Nucleocapsid and Matrix RNA-Binding Sequences in Human Immunodeficiency Virus Type 1 Assembly

2005 ◽  
Vol 79 (22) ◽  
pp. 13839-13847 ◽  
Author(s):  
David E. Ott ◽  
Lori V. Coren ◽  
Tracy D. Gagliardi
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Matrix Protein ◽  
Particle Production ◽  
Rna Binding ◽  
Immunodeficiency Virus ◽  
The Matrix ◽  
Hiv 1

ABSTRACT RNA appears to be required for the assembly of retroviruses. This is likely due to binding of RNA by multiple Gags, which in turn organizes and stabilizes the Gag-Gag interactions that form the virion. While the nucleocapsid (NC) domain is the most conspicuous RNA-binding region of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein, we have previously shown that NC is not strictly required for efficient particle production. To determine if an RNA requirement for HIV-1 assembly exists, we analyzed virions produced by an NC deletion mutant for the presence of RNA. The results revealed that virions without NC still contained significant amounts of RNA. Since these packaged RNAs are probably incorporated by other RNA-binding sequences in Gag, an RNA-binding site in the matrix protein (MA) of Gag was mutated. While this mutation did not interfere with HIV-1 replication, a construct with both MA and NC mutations (MX/NX) failed to produce particles. The MX/NX mutant was rescued in trans by coassembly with several forms of Gag: wild-type Gag, either of the single-mutant Gags, or Gag truncations that contain MA or NC sequences. Addition of basic sequences to the MX/NX mutant partially restored particle production, consistent with a requirement for Gag-RNA binding in addition to Gag-Gag interactions. Together, these results support an RNA-binding requirement for Gag assembly, which relies on binding of RNA by MA or NC sequences to condense, organize, and stabilize the HIV-1 Gag-Gag interactions that form the virion.

scholarly journals Stoichiometry of Monoclonal Antibody Neutralization of T-Cell Line-Adapted Human Immunodeficiency Virus Type 1

1999 ◽  
Vol 73 (10) ◽  
pp. 8364-8370 ◽  
Author(s):  
Kristian Schønning ◽  
Ole Lund ◽  
Ole Søgaard Lund ◽  
John-Erik Stig Hansen
Keyword(s):  
Monoclonal Antibody ◽  
Human Immunodeficiency Virus ◽  
T Cell ◽  
Cell Line ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Sites ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4− HEK293 cells. MAb neutralization of chimeric virions could be described as a third-order function of the proportion of Env antigen refractory to MAb binding. This scenario is consistent with the Env oligomer constituting the minimal functional unit and neutralization occurring incrementally as each Env oligomer binds MAb. Alternatively, the data could be fit to a sigmoid function. Thus, these data could not exclude the existence of a threshold for neutralization. However, results from MAb neutralization of chimeric virus containing wild-type Env and Env defective in CD4 binding was readily explained by a model of incremental MAb neutralization. In summary, the data indicate that MAb neutralization of T-cell line-adapted HIV-1 is incremental rather than all or none and that each MAb binding an Env oligomer reduces the likelihood of infection.

scholarly journals Interaction of YB-1 with human immunodeficiency virus type 1 Tat and TAR RNA modulates viral promoter activity

1999 ◽  
Vol 80 (10) ◽  
pp. 2629-2638 ◽  
Author(s):  
Sameer A. Ansari ◽  
Mahmut Safak ◽  
Gary L. Gallia ◽  
Bassel E. Sawaya ◽  
Shohreh Amini ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Binding ◽  
Regulatory Element ◽  
Binding Activity ◽  
Full Length ◽  
Immunodeficiency Virus ◽  
Hiv 1

Transcriptional regulation of the human immunodeficiency virus type 1 (HIV-1) genome is mediated by viral and cellular factors. TAR, an unusual RNA regulatory element with a stem–bulge–loop structure at the 5′ ends of all nascent viral transcripts is critical for HIV-1 transcription. TAR is the target for Tat, a viral transcription factor encoded early in the HIV-1 life-cycle and essential for gene expression. Evidence demonstrating the interaction of a cellular ssDNA/RNA binding protein, YB-1, with TAR through a region which is important for Tat interaction is presented. Interestingly, results from protein–protein interaction studies revealed that YB-1 can also form a complex with Tat. Results from mapping experiments suggest that while the region spanning aa 125–203 within YB-1 is essential for its association with TAR, a truncated YB-1 spanning aa 1–125 can weakly bind to Tat. Functionally, overexpression of full-length YB-1 enhanced Tat-induced activation of the HIV-1 minimal promoter containing TAR sequences, whereas mutant YB-1 with no ability to bind to Tat and TAR failed to affect Tat-mediated activation. Expression of mutant YB-1(1–125), which binds to Tat but not RNA, decreased Tat- mediated enhancement of virus transcription. These observations suggest that while full-length YB-1 may function as a facilitator and, by interaction with both Tat and TAR, increase the level of Tat:TAR association, mutant YB-1 with no TAR binding activity, by complexing with Tat, may prevent Tat interaction with TAR. The importance of these findings in light of the proposed mechanism of Tat function is discussed.

scholarly journals Low TRBP Levels Support an Innate Human Immunodeficiency Virus Type 1 Resistance in Astrocytes by Enhancing the PKR Antiviral Response

2005 ◽  
Vol 79 (20) ◽  
pp. 12763-12772 ◽  
Author(s):  
Chi L. Ong ◽  
Janine C. Thorpe ◽  
Paul R. Gorry ◽  
Sylvie Bannwarth ◽  
Anthony Jaworowski ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Antiviral Response ◽  
Immunodeficiency Virus ◽  
Low Levels ◽  
Hiv 1

ABSTRACT Acute human immunodeficiency virus type 1 (HIV-1) replication in astrocytes produces minimal new virus particles due, in part, to inefficient translation of viral structural proteins despite high levels of cytoplasmic viral mRNA. We found that a highly reactive double-stranded (ds) RNA-binding protein kinase (PKR) response in astrocytes underlies this inefficient translation of HIV-1 mRNA. The dsRNA elements made during acute replication of HIV-1 in astrocytes triggers PKR activation and the specific inhibition of HIV-1 protein translation. The heightened PKR response results from relatively low levels of the cellular antagonist of PKR, the TAR RNA binding protein (TRBP). Efficient HIV-1 production was restored in astrocytes by inhibiting the innate PKR response to HIV-1 dsRNA with dominant negative PKR mutants, or PKR knockdown by siRNA gene silencing. Increasing the expression of TRBP in astrocytes restored acute virus production to levels comparable to those observed in permissive cells. Therefore, the robust innate PKR antiviral response in astrocytes results from relatively low levels of TRBP expression and contributes to their restricted infection. Our findings highlight TRBP as a novel cellular target for therapeutic interventions to block productive HIV-1 replication in cells that are fully permissive for HIV-1 infection.

scholarly journals Single Point Mutations in the Zinc Finger Motifs of the Human Immunodeficiency Virus Type 1 Nucleocapsid Alter RNA Binding Specificities of the Gag Protein and Enhance Packaging and Infectivity

2005 ◽  
Vol 79 (12) ◽  
pp. 7756-7767 ◽  
Author(s):  
Michal Mark-Danieli ◽  
Nihay Laham ◽  
Michal Kenan-Eichler ◽  
Asher Castiel ◽  
Daniel Melamed ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Zinc Finger ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Rna Binding ◽  
Gag Protein ◽  
Encapsidation Signal ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT A specific interaction between the nucleocapsid (NC) domain of the Gag polyprotein and the RNA encapsidation signal (Ψ) is required for preferential incorporation of the retroviral genomic RNA into the assembled virion. Using the yeast three-hybrid system, we developed a genetic screen to detect human immunodeficiency virus type 1 (HIV-1) Gag mutants with altered RNA binding specificities. Specifically, we randomly mutated full-length HIV-1 Gag or its NC portion and screened the mutants for an increase in affinity for the Harvey murine sarcoma virus encapsidation signal. These screens identified several NC zinc finger mutants with altered RNA binding specificities. Furthermore, additional zinc finger mutants that also demonstrated this phenotype were made by site-directed mutagenesis. The majority of these mutants were able to produce normal virion-like particles; however, when tested in a single-cycle infection assay, some of the mutants demonstrated higher transduction efficiencies than that of wild-type Gag. In particular, the N17K mutant showed a seven- to ninefold increase in transduction, which correlated with enhanced vector RNA packaging. This mutant also packaged larger amounts of foreign RNA. Our results emphasize the importance of the NC zinc fingers, and not other Gag sequences, in achieving specificity in the genome encapsidation process. In addition, the described mutations may contribute to our understanding of HIV diversity resulting from recombination events between copackaged viral genomes and foreign RNA.

scholarly journals Functional Analysis and Structural Modeling of Human APOBEC3G Reveal the Role of Evolutionarily Conserved Elements in the Inhibition of Human Immunodeficiency Virus Type 1 Infection and Alu Transposition

2009 ◽  
Vol 83 (23) ◽  
pp. 12611-12621 ◽  
Author(s):  
Yannick Bulliard ◽  
Priscilla Turelli ◽  
Ute F. Röhrig ◽  
Vincent Zoete ◽  
Bastien Mangeat ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Large Scale ◽  
Rna Binding ◽  
Evolutionary Forces ◽  
Wide Range ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroelements are important evolutionary forces but can be deleterious if left uncontrolled. Members of the human APOBEC3 family of cytidine deaminases can inhibit a wide range of endogenous, as well as exogenous, retroelements. These enzymes are structurally organized in one or two domains comprising a zinc-coordinating motif. APOBEC3G contains two such domains, only the C terminal of which is endowed with editing activity, while its N-terminal counterpart binds RNA, promotes homo-oligomerization, and is necessary for packaging into human immunodeficiency virus type 1 (HIV-1) virions. Here, we performed a large-scale mutagenesis-based analysis of the APOBEC3G N terminus, testing mutants for (i) inhibition of vif-defective HIV-1 infection and Alu retrotransposition, (ii) RNA binding, and (iii) oligomerization. Furthermore, in the absence of structural information on this domain, we used homology modeling to examine the positions of functionally important residues and of residues found to be under positive selection by phylogenetic analyses of primate APOBEC3G genes. Our results reveal the importance of a predicted RNA binding dimerization interface both for packaging into HIV-1 virions and inhibition of both HIV-1 infection and Alu transposition. We further found that the HIV-1-blocking activity of APOBEC3G N-terminal mutants defective for packaging can be almost entirely rescued if their virion incorporation is forced by fusion with Vpr, indicating that the corresponding region of APOBEC3G plays little role in other aspects of its action against this pathogen. Interestingly, residues forming the APOBEC3G dimer interface are highly conserved, contrasting with the rapid evolution of two neighboring surface-exposed amino acid patches, one targeted by the Vif protein of primate lentiviruses and the other of yet-undefined function.

scholarly journals High-Mobility-Group Protein I Can Modulate Binding of Transcription Factors to the U5 Region of the Human Immunodeficiency Virus Type 1 Proviral Promoter

2000 ◽  
Vol 74 (22) ◽  
pp. 10523-10534 ◽  
Author(s):  
Angus Henderson ◽  
Michael Bunce ◽  
Nicole Siddon ◽  
Raymond Reeves ◽  
David John Tremethick
Keyword(s):  
Human Immunodeficiency Virus ◽  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Binding Sites ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT HMG I/Y appears to be a multifunctional protein that relies on in its ability to interact with DNA in a structure-specific manner and with DNA, binding transcriptional activators via distinct protein-protein interaction surfaces. To investigate the hypothesis that HMG I/Y may have a role in human immunodeficiency virus type 1 (HIV-1) expression, we have analyzed whether HMG I/Y interacts with the 5′ long terminal repeat and whether this interaction can modulate transcription factor binding. Using purified recombinant HMG I, we have identified several high-affinity binding sites which overlap important transcription factor binding sites. One of these HMG I binding sites coincides with an important binding site for AP-1 located downstream of the transcriptional start site, in the 5′ untranslated region at the boundary of a positioned nucleosome. HMG I binding to this composite site inhibits the binding of recombinant AP-1. Consistent with this observation, using nuclear extracts prepared from Jurkat T cells, we show that HMG I (but not HMG Y) is strongly induced upon phorbol myristate acetate stimulation and this induced HMG I appears to both selectively inhibit the binding of basal DNA-binding proteins and enhance the binding of an inducible AP-1 transcription factor to this AP-1 binding site. We also report the novel finding that a component present in this inducible AP-1 complex is ATF-3. Taken together, these results argue that HMG I may play a fundamental role in HIV-1 expression by determining the nature of transcription factor-promoter interactions.

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