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Sam68 is absolutely required for Rev function and HIV-1 production.

ABSTRACT: Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68DeltaC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to create stable Sam68 knockdown HeLa (SSKH) cells. In SSKH cells, Rev failed to activate both RRE-mediated reporter gene [chloramphenicol acetyltransferase (CAT) and/or gag] expressions. Importantly, reduction of Sam68 expression led to a dramatic inhibition of HIV-1 production. Inhibition of the reporter gene expression and HIV production correlated with the failure to export RRE-containing CAT mRNA and unspliced viral mRNAs to the cytoplasm, confirming that SSKH cells are defective for Rev-mediated RNA export. Taken together, these results suggest that Sam68 is involved in Rev-mediated RNA export and is absolutely required for HIV production.

SUBMITTER: Modem S

PROVIDER: S-EPMC549398 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Sam68 is absolutely required for Rev function and HIV-1 production.

Modem Suhasini S Badri Kameswara R KR Holland Thomas C TC Reddy Thipparthi R TR

Nucleic acids research 20050208 3

Sam68 functionally complements for, as well as synergizes with, HIV-1 Rev in Rev response element (RRE)-mediated gene expression and virus production. Furthermore, C-terminal deletion/point mutants of Sam68 (Sam68DeltaC/Sam68-P21) exert a transdominant negative phenotype for Rev function and HIV-1 production. However, the relevance of Sam68 in Rev/RRE function is not well defined. To gain more insight into the mechanism of Sam68 in Rev function, we used an RNAi (RNA interference) strategy to cre ...[more]

PMID: 15701759

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Project description:Combination antiretroviral therapy fails in complete suppression of HIV-1 due to drug resistance and persistent latency. Novel therapeutic intervention requires knowledge of intracellular pathways responsible for viral replication, specifically those untargeted by antiretroviral drugs. An understudied phenomenon is the nucleolar localization of Rev phosphoprotein, which completes nucleocytoplasmic transport of unspliced/partially spliced HIV mRNA through multimerization with intronic cis-acting targets-the Rev-response element (RRE). Rev contains a nucleolar localization signal (NoLS) comprising the COOH terminus of the arginine-rich motif for accumulation within nucleoli-speculated as the interaction ground for Rev with cellular proteins mediating mRNA-independent nuclear export and splicing. Functionality of Rev nucleolar access during HIV-1 production and infection was investigated in the context of deletion and single-point mutations within Rev-NoLS. Mutations induced upon Rev-NoLS are hypothesized to inactivate the HIV-1 infectious cycle. HIV-1HXB2 replication ceased with Rev mutations lacking nucleolar access due to loss or replacement of multiple arginine residues. Rev mutations missing single arginine residues remained strictly nucleolar in pattern and participated in proviral production, however, with reduced efficiency. Viral RNA packaging also decreased in efficiency after expression of nucleolar-localizing mutations. These results were observed during propagation of variant HIV-1NL4-3 containing nucleolar-localizing mutations within the viral backbone (M4, M5, and M6). Lentiviral particles produced with Rev single-point mutations were transducible at extremely low frequency. Similarly, HIV-1NL4-3 Rev-NoLS variants lost infectivity, unlike virulent WT (wild type) HIV-1NL4-3. HIV-1NL4-3 variants were capable of CD4+ host entry and reverse transcription as WT HIV-1NL4-3, but lacked ability to complete a full infectious cycle. We currently reveal that viral integration is deregulated in the presence of Rev-NoLS mutations.

Project description:The HIV-1 Rev response element (RRE) is a 351-base element in unspliced and partially spliced viral RNA; binding of the RRE by the viral Rev protein induces nuclear export of RRE-containing RNAs, as required for virus replication. It contains one long, imperfect double helix (domain I), one branched domain (domain II) containing a high-affinity Rev-binding site, and two or three additional domains. We previously reported that the RRE assumes an "A" shape in solution and suggested that the location of the Rev binding sites in domains I and II, opposite each other on the two legs of the A, is optimal for Rev binding and explains Rev's specificity for RRE-containing RNAs. Using small-angle X-ray scattering (SAXS) and a quantitative functional assay, we have now analyzed a panel of RRE mutants. All the results support the essential role of the A shape for RRE function. Moreover, they suggest that the distal portion of domain I and the three crowning domains all contribute to the maintenance of the A shape. Domains I and II are necessary and sufficient for substantial RRE function, provided they are joined by a flexible linker that allows the two domains to face each other.IMPORTANCE Retroviral replication requires that some of the viral RNAs transcribed in the cell nucleus be exported to the cytoplasm without being spliced. To achieve this, HIV-1 encodes a protein, Rev, which binds to a complex, highly structured element within viral RNA, the Rev response element (RRE), and escorts RRE-containing RNAs from the nucleus. We previously reported that the RRE is "A" shaped and suggested that this architecture, with the 2 legs opposite one another, can explain the specificity of Rev for the RRE. We have analyzed the functional contributions of individual RRE domains and now report that several domains contribute, with some redundancy, to maintenance of the overall RRE shape. The data strongly support the hypothesis that the opposed placement of the 2 legs is essential for RRE function.

Project description:A cis-acting RNA regulatory element, the Rev-responsive element (RRE), has essential roles in replication of lentiviruses, including human immunodeficiency virus (HIV-1) and equine infection anemia virus (EIAV). The RRE binds the viral trans-acting regulatory protein, Rev, to mediate nucleocytoplasmic transport of incompletely spliced mRNAs encoding viral structural genes and genomic RNA. Because of its potential as a clinical target, RRE-Rev interactions have been well studied in HIV-1; however, detailed molecular structures of Rev-RRE complexes in other lentiviruses are still lacking. In this study, we investigate the secondary structure of the EIAV RRE and interrogate regulatory protein-RNA interactions in EIAV Rev-RRE complexes. Computational prediction and detailed chemical probing and footprinting experiments were used to determine the RNA secondary structure of EIAV RRE-1, a 555 nt region that provides RRE function in vivo. Chemical probing experiments confirmed the presence of several predicted loop and stem-loop structures, which are conserved among 140 EIAV sequence variants. Footprinting experiments revealed that Rev binding induces significant structural rearrangement in two conserved domains characterized by stable stem-loop structures. Rev binding region-1 (RBR-1) corresponds to a genetically-defined Rev binding region that overlaps exon 1 of the EIAV rev gene and contains an exonic splicing enhancer (ESE). RBR-2, characterized for the first time in this study, is required for high affinity binding of EIAV Rev to the RRE. RBR-2 contains an RNA structural motif that is also found within the high affinity Rev binding site in HIV-1 (stem-loop IIB), and within or near mapped RRE regions of four additional lentiviruses. The powerful integration of computational and experimental approaches in this study has generated a validated RNA secondary structure for the EIAV RRE and provided provocative evidence that high affinity Rev binding sites of HIV-1 and EIAV share a conserved RNA structural motif. The presence of this motif in phylogenetically divergent lentiviruses suggests that it may play a role in highly conserved interactions that could be targeted in novel anti-lentiviral therapies.

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Sam68 is absolutely required for Rev function and HIV-1 production.

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Sam68 is absolutely required for Rev function and HIV-1 production.

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