Perturbation of the carboxy terminus of HIV-1 Rev affects multimerization on the Rev responsive element

Daly, Thomas J.; Rennert, Paul D.; Lynch, P.M.; Barry, Jennifer K.; Dundas, Moira; Rusche, James R.; Doten, R C; Auer, Manfred; Farrington, G K

doi:10.1021/bi00085a028

Cited by 38 publications

(35 citation statements)

References 26 publications

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“…Although the kinetics presented here cannot settle this dispute, the fact that the minimal high affinity binding site (RRE/SLIIB) bound Rev at a 1:1 ratio and the slightly larger RRE/HH yields a 2:1 protein:RNA ratio suggested that the initial binding to RRE must involve a Rev monomer, followed by multimerization in situ, in agreement with previous reports (10,58,81,82). It has also been suggested that a critical threshold level of Rev on RRE is required for trans-activation (59,61,79,80) and also that the RRE acts as a molecular rheostat (59) regulating the sequential addition of Rev monomers to achieve the critical threshold.…”

Section: Discussionsupporting

confidence: 91%

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Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions

Ryk

Venkatesan

1999

Journal of Biological Chemistry

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The kinetics of interaction between the human immunodeficiency virus-1 Rev protein and its RNA target, Rev response element (RRE) RNA was determined in vitro using a biosensor technique. Our results showed that the primary Rev binding site is a core stem-loop RNA molecule of 30 nucleotides that bound Rev at a 1:1 ratio, whereas the 244-nucleotide full-length RRE bound four Rev monomers. At high Rev concentrations, additional binding of Rev to RRE was observed with ratios of more than 10:1. Because RRE mutants that lacked the core binding site and were inactive in vivo bound Rev nonspecifically at these concentrations, the real stoichiometric ratio of Rev-RRE is probably closer to 4:1. Binding affinity of Rev for RRE was approximately 10 ؊10 M, whereas the affinity for the core RNA was about 10 ؊11 M, the difference being due to the contribution of low affinity binding sites on the RRE. Mathematical analysis suggested cooperativity of Rev binding, probably mediated by the Rev oligomerization domains. C-terminal deletions of Rev had no effect on RRE binding, but truncation of the N terminus by as few as 11 residues significantly reduced binding specificity. This method was also useful to rapidly evaluate the potential of aminoglycoside antibiotics, to inhibit the Rev-RRE interaction.The genome of HIV-1 1 is transcribed as a full-length 9-kilobase mRNA that can follow two different "pathways" (1-4). Early in infection, the primary HIV transcript is fully spliced to mRNAs of approximately 2 kilobases in length and then transported from the nucleus to the cytoplasm, where the mRNAs are translated into a variety of small accessory proteins, such as Tat, Rev,. Later in the virus lifecycle, unspliced or partially spliced RNAs are exported from the nucleus to the cytosol, where the unspliced RNA is packaged into virus particles; the different partially spliced mRNAs are translated into Gag, Pol, Env, and smaller proteins, such as vif, vpR, and vpU (4, 6 -10). This temporal switch in the HIV RNA complexity and the coding potential is mediated by virus coded Rev protein (11)(12)(13)(14)(15)(16)(17)(18)(19), an approximately 16-kDa basic RNA-binding protein with two functional domains (Fig. 1A). The N-terminal domain is involved in RNA binding and oligomerization (20 -25), whereas the more C-terminal activation domain (13, 26 -30) is thought to interact with the nuclear pore-associated proteins, such as hRIP/Rab (31-35), CRM1 (36 -39), and nuclear eIF-5A (40, 41). In the absence of Rev, the RNA transcript is fully spliced; the full-length transcript is never observed in the cytoplasm (3, 8, 9, 13, 16 -19).Rev binds specifically to a highly structured 244-nucleotide RNA sequence, the Rev response element (RRE), located in the env gene of the primary transcript (16,42,43); this RNA binding (43-48) is essential for the nuclear export of unspliced and partially spliced HIV mRNAs (8, 9, 15, 16,). Lack of functional Rev or RRE completely blocks viral replication. The secondary structure of the RRE RNA is presumed to fold into fou...

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Section: Discussionsupporting

confidence: 91%

“…Because RRE/SLIIB can only bind one molecule of Rev, cooperative binding was not an issue. Furthermore, because Rev bound with high affinity to RRE/SLIIB, this showed that high affinity binding is possible in the absence of multimerization, in agreement with the suggestion of Daly et al (81,82).…”

Section: Discussionsupporting

confidence: 90%

Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions

Ryk

Venkatesan

1999

Journal of Biological Chemistry

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“…As discussed earlier, mutants of this type exhibit RRE binding and nucleolar localization characteristics that are indistinguishable from those of the wild-type protein (Fig. 2i,j;Malim et al 1989a;Olsen et al 1990;Zapp et al 1991;Daly et al 1993). Importantly, and in striking contrast to Rev, the M10 protein failed to accumulate in the cytoplasms of transfected HeLa cells following treatment with 5 p.g/ml of actinomycin D (Fig.…”

Section: R E S U L T Smentioning

confidence: 55%

“…The second essential domain contains three closely spaced leucine residues and is positioned toward the carboxyl terminus (Venkatesh and Chinnadurai 1990;. The mutation of any of these leucines generates nonfunctional proteins (Malim et al 1989a;Hope et al 1990b;Mermer et al 1990;Olsen et al 1990;Venkatesh and Chinnadurai 1990) whose predominant accumulation in the nucleoli and ability to bind to the RRE are both indistinguishable from wild-type Rev (Malim et al 1989a;Olsen et al 1990;Zapp et al 1991;Tiley et al 1992;Daly et al 1993). It has therefore been proposed that the defect in Rev mutants with this phenotype lies in their inability to interact with the nuclear factors, presumably proteins, required for the transport of unspliced HIV-1 mRNA to the cytoplasm {Malim et al 1991).…”

Section: Receivedmentioning

confidence: 99%

The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm.

Meyer¹,

Malim²

1994

Genes Dev.

291

239

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The HIV-1 Rev protein is a nuclear trans-activator essential for the transport of unspliced viral transcripts to the cytoplasm. In this paper we demonstrate that Rev, rather than being confined to the nucleus, is constantly shuttling between the nucleus and the cytoplasm. We also show that inactivation of Rev's leucine-rich activation domain generates mutant proteins that not only fail to induce the nuclear export of viral transcripts but are also unable to enter the cytoplasm. On the basis of this correlation, we propose that Rev activates viral mRNA transport by directly binding to these RNAs and translocating, with them, to the cytoplasm. In addition, these results also identify, for the first time, a peptide sequence that is important for nuclear export.

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“…Oligomerization of Rev requires the Rev multimerization domain that has also been shown to be necessary for Rev function (12,34,39,42,54). Additionally, the molecular details of the interaction of the Rev monomer and its primary binding site have been well characterized through the use of an isolated Rev peptide containing only the arginine-rich binding motif (ARM) and the stem IIB RNA hairpin (3,4,48,49).…”

Section: Cells Infected With Human Immunodeficiency Virus (Hiv)mentioning

confidence: 99%

Single-Nucleotide Changes in the HIV Rev-Response Element Mediate Resistance to Compounds That Inhibit Rev Function

Shuck-Lee¹,

Chang

Sloan³

et al. 2011

J Virol

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Previously we described the identification of two compounds (3-amino-5-ethyl-4,6-dimethylthieno[2,3-b] pyridine-2-carboxamide [103833] and 4-amino-6-methoxy-2-(trifluoromethyl)-3-quinolinecarbonitrile[104366]) that interfered with HIV replication through the inhibition of Rev function. We now describe resistant viral variants that arose after drug selection, using virus derived from two different HIV proviral clones, NL4-3 and R7/3. With HIV NL4-3 , each compound selected a different single point mutation in the Rev response element (RRE) at the bottom of stem-loop IIC. Either mutation led to the lengthening of the stem-loop IIC stem by an additional base pair, creating an RRE that was more responsive to lower concentrations of Rev than the wild type. Surprisingly, wild-type HIV R7/3 was also found to be inhibited when tested with these compounds, in spite of the fact this virus already has an RNA stem-loop IIC similar to the one in the resistant NL4-3 variant. When drug resistance was selected in HIV R7/3 , a virus arose with two nucleotide changes that mapped to the envelope region outside the RRE. One of these nucleotide changes was synonymous with respect to env, and one was not. The combination of both nucleotide changes appeared to be necessary for the resistance phenotype as the individual point mutations by themselves did not convey resistance. Thus, although drug-resistant variants can be generated with both viral strains, the underlying mechanism is clearly different. These results highlight that minor nucleotide changes in HIV RNA, outside the primary Rev binding site, can significantly alter the efficiency of the Rev/RRE pathway. Cells infected with human immunodeficiency virus (HIV)produce stable species of unspliced and incompletely spliced mRNAs that require the Rev protein for their nuclear-cytoplasmic export and expression (for a review, see reference 29). Rev is a small basic protein of about 116 amino acids that is encoded in the HIV genome (for a review, see reference 44). One well-defined function of Rev in HIV infection is to bind (7,10,11,30,31,41,53) to the Rev response element (RRE) (15,26,45) that is found in these unspliced and incompletely spliced mRNAs and promote their nucleo-cytoplasmic export (21,28,40) by interacting with the Crm1 cellular export receptor and Ran-GTP (1).Many previous studies identified stem-loop IIB of the RRE as the primary binding site for a Rev protein monomer and suggested that 6 to 10 subunits of Rev can subsequently bind to the RRE (2, 9, 32, 33, 36, 50). Oligomerization of Rev requires the Rev multimerization domain that has also been shown to be necessary for Rev function (12,34,39,42,54). Additionally, the molecular details of the interaction of the Rev monomer and its primary binding site have been well characterized through the use of an isolated Rev peptide containing only the arginine-rich binding motif (ARM) and the stem IIB RNA hairpin (3,4,48,49).Recently, Rev has been shown to form a cooperative highaffinity oligomeric complex with the RRE (13)....

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Perturbation of the carboxy terminus of HIV-1 Rev affects multimerization on the Rev responsive element

Cited by 38 publications

References 26 publications

Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions

Real-time Kinetics of HIV-1 Rev-Rev Response Element Interactions

The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm.

Single-Nucleotide Changes in the HIV Rev-Response Element Mediate Resistance to Compounds That Inhibit Rev Function

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