Human immunodeficiency virus type 1 Rev activation can be achieved without Rev-responsive element RNA if Rev is directed to the target as a Rev/MS2 fusion protein which tethers the MS2 operator RNA

Venkatesan, Sundararajan; Gerstberger, Susan; Park, H.; Holland, Steven M.; Nam, Yong Suk

doi:10.1128/jvi.66.12.7469-7480.1992

Cited by 33 publications

(15 citation statements)

References 48 publications

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“…There is also a lower-affinity Rev binding site in domain I (12). Numerous observations indicate that, although single Rev molecules can bind to an unoccupied RRE (3,17,18), the interaction of multiple Rev molecules with a single RRE is required for productive Rev-RRE interactions (14,25,26).…”

Section: Resultsmentioning

confidence: 99%

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

O’Carroll

Thappeta

et al. 2017

J Virol

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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 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. 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.

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Section: Resultsmentioning

confidence: 99%

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

O’Carroll

Thappeta

et al. 2017

J Virol

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“…This assumption is supported by the observation that the activation induced by a Rev-MS2 fusion protein tethered to the MS2 operator sequence was markedly increased if the MS2 RNA target was inserted in the context of RRE sequences. This enhancement was mediated rather by the structure than by the sequence of the RRE (Venkatesan et al, 1992). The negative effects induced by deletion of RexRE structures different from SW therefore can be explained by perturbation of an optimal context resulting in decreased stability of the RNA-binding site.…”

Section: Discussionmentioning

confidence: 96%

“…An alternative explanation could be that the monomer does not form a correct secondary structure in the context of the test systems used. The context of an RNA target was demonstrated to have strong influence on its capacity to mediate the regulatory effect of an RNA binding protein (Venkatesan et al, 1992).…”

Section: Discussionmentioning

confidence: 99%

A Single Stem-Loop Structure within the HTLV-1 Rex Response Element Is Sufficient to Mediate Rex Activity in Vivo

Gröne

Hoffmann²,

Berchtold³

et al. 1994

Virology

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“…Interestingly, YL2 or p32 (YL2/p32) interacts with the basic domain of Rev, which is important not only for its binding to the RRE but also for its effects on RNA splicing in vitro (32,33). Indeed, even when Rev is targeted to viral RNAs via a heterologous RNA-binding protein (MS2), a basic sequence is still required for the optimal function of Rev (45,50). Thus, it is not surprising that YL2 also potentiated the activities of Rex on the XRE and the hybrid Tat-Rev and Rev-MS2 proteins on their respective RNA targets (Fig.…”

Section: Discussionmentioning

confidence: 99%

Cellular protein modulates effects of human immunodeficiency virus type 1 Rev

Luo

Peterlin

1994

J Virol

137

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Replication of human immunodeficiency virus type 1 requires expression of the viral trans activator Rev. Rev binds to a highly structured RNA, the Rev response element, which is present in singly spliced and unspliced genomic viral RNAs. Although Rev helps to transport these transcripts from the nucleus to the cytoplasm, the mechanism(s) involved is not fully understood. Using the yeast two-hybrid system, we isolated a murine protein (YL2) that interacts with the basic domain of Rev, which is essential for the function of Rev in vivo and for the inhibitory splicing activity of Rev in vitro. YL2 has 92% identity to a human 32-kDa protein (p32), which copurifies with alternative splicing factor SF2/ASF. Furthermore, we found that whereas expression of YL2 greatly potentiated the activity of Rev, antisense YL2 transcripts blocked the effects of Rev in mammalian cells. YL2 also increased the activities of Rex on the Rex response element and of hybrid Rev proteins fused to Tat and the coat protein of bacteriophage MS2 on their respective RNAs. Thus, YL2 or p32 is a cellular protein that modulates the function of human immunodeficiency virus type 1 Rev.Rev is one of the earliest proteins expressed by human immunodeficiency virus type 1 (HIV-1) and is involved in the posttranscriptional regulation of viral RNAs (6,11,27,28,46). It is a 19-kDa nuclear protein that contains four functional domains (38). Of these, the basic domain binds to the Rev response element (RRE) (22,30,31,39,49,52), which forms highly structured RNA stem-loops (41). Since this basic domain also inhibits RNA splicing in vitro (32, 33) and a basic sequence is required for the optimal function of Rev even when it is tethered to RNA via a heterologous RNA-binding protein such as the coat protein of bacteriophage MS2 (45, 50), the basic region of Rev performs functions in addition to RNA binding. Furthermore, the two regions flanking this basic domain are required for multimerization of Rev on the RRE (38,42). Finally, five leucines near the C terminus form the activation domain of this protein (38). Splicing and transport of primary HIV-1 transcripts are complex. In the absence of Rev, only multiply spliced viral transcripts that direct the synthesis of Tat, Rev, and Nef are exported from the nucleus (29,48). These proteins perform important regulatory functions but are not themselves packaged into virions. In the presence of Rev, singly spliced and unspliced genomic viral transcripts also appear in the cytoplasm (7,10,19,29). Whereas singly spliced RNA codes for envelope glycoproteins gp120 and gp41, the unspliced RNA codes for Gag proteins, integrase, reverse transcriptase, and protease, which together with this RNA are packaged into the budding virion. Thus, Rev plays an essential role in the life cycle of HIV-1 (7).However, the mechanism of trans activation by Rev is not well understood. Since singly spliced and unspliced viral transcripts are detected in the nucleus but not in the cytoplasm in the absence of Rev, Rev might itself transport viral...

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Human immunodeficiency virus type 1 Rev activation can be achieved without Rev-responsive element RNA if Rev is directed to the target as a Rev/MS2 fusion protein which tethers the MS2 operator RNA

Cited by 33 publications

References 48 publications

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

Contributions of Individual Domains to Function of the HIV-1 Rev Response Element

A Single Stem-Loop Structure within the HTLV-1 Rex Response Element Is Sufficient to Mediate Rex Activity in Vivo

Cellular protein modulates effects of human immunodeficiency virus type 1 Rev

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