Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants

Abstract

The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype. MATERIALS AND METHODSCell lines and plasmid constructs. The COS cell line, the transfection assays, the indirect immunofluorescence assay, and the plasmid pSVCMVrev expressing Rev wt have been described previously (39). The 293 cell line represents transformed human embryonal kidney cells obtained from the American Type Culture Collection (ATCC CRL 1573). Three different rev mutant plasmids, prev81, M10, and prev⌬18-23, collectively referred to as prev TD, all with the simian virus 40 origin and the rev gene controlled by the cytomegalovirus immediateearly promoter, were used. In prev81, the amino acid residue leucine 81 is changed to aspartic acid (80). In M10, the amino acid residues leucine 78 and glutamic acid 79 are substituted by aspartic acid and leucine, respectively (53). In prev⌬18-23, the amino acid residues 76 to 84 are deleted (56). Plasmid pcDNA1E7 expresses both the Rev and the Env proteins and was a gift of J. Sodroski and R. Wyatt. Plasmid pSV1X1⌬1 expresses the Env proteins only when Rev is supplemented in trans and was a gift of M.-L. Hammarskjöld and D. Rekosh (32).Antibodies. The anti-Rev monoclonal antibody (MAb) 1G10 binds to the C-terminal region of Rev, and the anti-Rev MAb 8E7 binds to the activation domain (39). MAb 8E7 recognizes Rev wt and Rev81 and M10 (but not Rev⌬18-23) in the indirect immunofluorescence assay. In Western immunoblot analysis, MAb 8E7 bound poorly to Rev81. In the radioimmunoprecipitation assay, only Rev wt was precipitated by 8E7, confirming that the leucine-rich activation domain is an essential part of the epitope. For detection of Env gp160/gp120 in Western blot analysis, MAb ADP327 (79), provided by C. Thiriart and C. Bruck, Smith Kline Biologicals, Rixensart, Belgium, and H. C. Holmes, Medical Research Council, London, United Kingdom, was used. Polyclonal rabbit anti-gp160/gp120 antibody was obtained from American Biotechnology. The human anti-sm serum (Chemicon) recognizes the sm-antigen common to small nuclear ribonucleoprotein particles (snRNPs) in the nuclear speckles. MAbs 4F4 and 4B10 against heterogeneous nuclear RNP (hnRNP) C1/C2 proteins and hnRNP A1, respectively, were gifts of G. Dreyfuss and S. Piñol-Roma (66). The antimitochondrial MAb 1273 was from Chemicon. on July 10, 2020 by guest http://jvi.asm.org/ Downloaded from 3316 SZILVAY ET AL. J. VIROL. on July 10, 2020 by guest http://jvi.a...

HIV-1 Rev Depolymerizes Microtubules to Form Stable Bilayered Rings

“…These values are compatible with the formation of a Rev/tubulin 1:1 complex. Rev concentrations in infected and transformed cells appear to be difficult to determine but are generally believed to be low—certainly significantly lower than an abundant protein such as tubulin—and quite variable, both between cellular compartments and between cells (Kalland et al 1994; Szilvay et al 1995; Bøe et al 1998) and over time. Consequently, it might seem unlikely that Rev would disrupt the MT cytoskeleton on a large scale, as is observed during HIV infection (Delezay et al 1997).…”

Analysis of Intracellular Trafficking and Interactions of Cytoplasmic HIV-1 Rev Mutants in Living Cells

“…Mutations in the Rev effector domain prevent Rev's export and cause a dominant nega-tive phenotype. This appears to be mediated by the formation of heteromultimers between Rev and transdominant (TD) Rev, which results in the inhibition of the nucleocytoplasmic transport of Rev (Hope et al, 1992;Stauber et al, 1995;Szilvay et al, 1995). Kubota et al (1992) proposed that a Rev mutant lacking the N-terminal Arg-rich domain was transdominant by retaining WT Rev in the cytoplasm, thereby suggesting a new mechanism of transdominance.…”

In Vitro Interaction between Human Immunodeficiency Virus Type 1 Rev Protein and Splicing Factor ASF/SF2-associated Protein, p32

“…The Rev protein is quite small (116 amino acids), and two main functional domains have been defined by mutational analysis. A cluster of leucine residues around positions 78 -83 constitutes the nuclear export signal (7)(8)(9)(10), which interacts directly with nuclear proteins implicated in RNA export (11)(12)(13). A highly basic region at positions 34 -50 is responsible for specific RNA binding (14 -18), nuclear localization, and contributes to the oligomerization process (18 -20).…”