A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (11). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.The genetic information of human immunodeficiency virus (HIV), which causes AIDS, is contained in a 9-kb genome. Elaborate differential splicing of the full-length initial transcript places the various reading frames into position for subsequent translation, resulting in three size classes of HIV mRNAs (about 9, 4, and 2 kb) (20,23,41). The two larger-size classes of HIV mRNA, which are incompletely spliced, cannot be expressed as Gag/Pol and Env proteins, respectively, unless an HIV regulatory protein, Rev, is present (16,35,44,47,50). Rev itself and Tat (11), another essential HIV protein, are made from the doubly spliced mRNA species which are expressed independently of Rev. When HIV transcription is activated in the infected cell, the full-length mRNA is spliced two times by default and translated into Tat and Rev proteins. Tat boosts transcription from the HIV promoter by positive feedback so that larger amounts of Tat and Rev are made. When Rev accumulates above a threshold level in the cell, the unspliced mRNAs (gagipol) and the singly spliced mRNAs (env) become preferentially expressed (38). The HIV life cycle thus consists of an early phase characterized by increasing Tat and Rev production from doubly spliced mRNAs and a late phase with favored expression of viral structural proteins from unspliced and singly spliced HIV mRNAs (28). The hallmark of the Rev-dependent HIV mRNAs is that they contain two types of cis-acting sequences, the Rev response element (RRE) and several cis-acting repressive sequences (42). The RRE is a sequence of 234 ribonucleotides present in the env region of the HIV mRNA between the two exons of Tat and Rev (13,36). The RRE folds into a complex stem-and-loop structure in vitro and binds avidly and specifically to the basic domain of the Rev protein (reviewed in references 11 and 23). The cis-acting repressive sequences are less well characterized but are present both in the gag/pol region and in the env region (10,32,42,46 (15,43). Apparently discrepant results have identified the host cell RNA splicing machinery, the mRNA transport system, or cytoplasmic events of mRNA expression as important targets fo...

Oligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a new in vivo assay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev.

A floatation centrifugation procedure is described by which antibodies and non-specific rubella virus haemagglutination inhibitors can be completely separated. Using this method it was shown that false positive reactions occur with the conventional pretreatments of sera for removing inhibitors, especially with the heparin-McCl2 procedure. Other methods for removal of inhibitors were examined in view of recent knowledge about the chemical nature of the inhibitors. Treatment with phospholipase C proved useful for disclosure of false positive reactions, while floatation centrifugation was the most sensitive method for demonstration of low antibody concentrations.

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...

The human immunodeficiency virus type 1 (HIV-1) Rev (regulator of virion protein expression) protein exemplifies a new type of posttranscriptional regulation. One main function of Rev is to increase the cytoplasmic expression of unspliced and incompletely spliced retroviral mRNAs from which viral structural proteins are made. In that way, Rev is essential in order to complete the retroviral life cycle. The biology of Rev in the host cell has remained elusive. In this study, a complex distribution of Rev in single cells was found. Rev was found in the cytoplasm, in a perinuclear zone, in the nucleoplasm, and in the nucleoli. In the nucleoplasm, Rev colocalized in a speckled pattern with host cell factors known to assemble on nascent transcripts. Those factors are involved in the processing of heterogeneous RNA to spliced mRNA in the nucleoplasm of all cells. The distribution of Rev was dependent only on Rev and host cell interactions, since neither the Rev target RNA nor other HIV proteins were expressed in the cells. Rev was found in the same subcellular compartments of cells treated for extended periods with cycloheximide, an inhibitor of protein synthesis. This finding implies that Rev shuttles continuously between cytoplasmic and nucleoplasmic compartments. The results suggest a potential role for Rev both in the RNA-splicing process and in the nucleocytoplasmic transport of Rev-dependent HIV mRNA.