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Coexpression of biologically active simian immunodeficiency virus (SIV) rev and env in an SV40 system: The SIV rev gene regulates Env expression
VIROLOGY
177,816-819
(1990)
Coexpression
of Biologically
Active
Simian lmmunodeficiency
in an SV40 System: The SIV rev Gene Regulates
Virus (SW) Rev and Env Env Expression
SHEAU-MEI CHENG,*’ MARLENE BLUME,* SHAW-GUANG LEE,* PAUL P. HUNG,* VANESSA M. HIRscH,t AND PHILIP R. JOHNSON+ *Biotechnology
and Microbiology Division, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087; and tRetrovira/ Pathogenesis Division of Molecular Virology and Immunology, Georgetown University, Rockville, Maryland 20852 Received February
Section,
1, 1990; accepted April 13, 1990
The coexpression of biologically active simian immunodeficiencyvirus (SIV) Rev and Env gene products was obtained in COS-I cells from a single SIV subgenomic segment (which contains both exons of rev and the entire env gene) cloned into a SV40-directed vector. The SlVsm Rev trans-activated the expression of the full-length env mRNA and was required for the production of envelope glycoproteins. Furthermore, the alignment of the structural conservation of the Rev functional domains among all HIV and SIV was analyzed. 0 1990 Academic Press, Inc.
COS-1 cells were transfected with pBC17, pBD21, or pMLSV5 (control) and analyzed for the expression of SIVsm-specific glycoproteins (Fig. 2). Only cells transfected with pBC17 (rev+env+) contained SIVsm-specific glycoproteins (Fig. 2, lane 3). No SlVsm glycoproteins were detected in cells transfected with pMLSV5 or pBD21 (rev-env+) (Fig. 2, lanes 1 and 2). The precursor envelope glycoprotein (gpl60) expressed in COS1 cells transfected with pBC17 was properly processed into gpl20 and gp40 (Figs. 2, lane 3).
Simian immunodeficiency virus (SW) infection of macaques is a relevant and valuable animal model for AIDS vaccine studies (7, 2), because the infected macaques develop a human AIDS-like symptom (3). One approach to vaccine development involves the expression of the Env glycoprotein (gpl60) of SIV in live viral vectors such as vaccinia virus (4-6) or adenovirus (7). An important step in the development of such expression systems is an understanding of the regulation of SW envelope gene expression in cultured cells. The regulation of HIV-1 gene expression is complex and not fully understood (reviewed in Ref. (8)). HIV-1 has at least six novel genes (vif, vpr, tat, rev, vpu, and nef) when compared to prototypic retroviruses. The rev protein appears to post-transcriptionally enhance the production of the viral structural proteins Gag and Env (9-I I). SIV and HIV-1 share a similar, but not identical, genome organization (12- 16). SIV does possess the rev gene, but its protein shares only 30% amino acid identity with the HIV-1 Rev (16). Relatively little work has been reported on the regulation of SIV gene expression. To assess the role of rev in the expression of the SlVsm env gene, we generated two transient expression vectors (Fig. 1): (i) pBC17 contained a single subgenomic segment of SIV (smH-3, a molecular clone of SIV from sooty magabeys (75, 76)) that included both exons of rev and the entire env gene; (ii) pBD21 was derived from pBC17 by deleting most of the first rev exon. Thus, pBC17 (rev+env+) could potentially encode Rev and Env proteins, while pBD21 (rev-env+) could only encode the envelope glycoprotein.
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To assess the levels and forms of RNA transcribed from pBC17 and pBD21, three identical RNA blots (described below) were hybridized with one of the following probes (Figs. 3A, 3B. and 3C): probe A was specific to the first exon of rev (rev-only probe); probe B was specific for envelope transcripts (env-only probe); probe C was derived from the second exon of rev and therefore would recognize rev and env sequences (rev/ env probe). In cells transfected with pBC17 (rev+env+), three RNA species were detected (Figs. 3A, 3B, and 3C, lane 1): a 4-kb transcript (detected with all three probes); a 1.3-kb transcript (probes A and C); and a 0.9kb species (probe A only). The 4-kb RNA species represented the predicted unspliced mRNA initiating from the SV40 late promoter and terminating in the SV40 early polyadenylation sequence. Since this transcript hybridized with all three probes, it contained the first rev exon and the env gene, and was the only RNA species capable of encoding this envelope protein. Interestingly, the envelope protein would initiate from the second ATG of this mRNA since the first ATG belongs to the first exon of rev (15). The 1.3-kb transcript was the spliced RNA resulting from the predicted splice sites in rev and terminating in the SV40 early polyadenylation sequence (Fig. 1). This species represented
Copyright 0 1990 Academic Press, Inc. All rights of reproduction in any form reserved.
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pED21 SA
FIG. 1. Structure of the SlVsm env and rev gene expression vectors Subgenomic fragments isolated for psmH-3 were cloned into the unique Bglll site of the expression plasmid pMLSV5 (28). This vector contains the SV40 late promoter and polyadenylation region in the plasmid pML2. The polyadenylation region contains splice junctions derived from T, t, and early polydenylation sequences (29). Arrows represent transcriptional direction. The SV40 regulatory elements are represented by solid boxes and the sinusoidal lines are pML2 sequences (plasmid). pBC17 contains the SIV DNA insert starting from nucleotides 6402 to 9470 that includes the entire env coding sequence (open box) and both exons of the rev gene (shaded boxes). pBD21 is identical to pBCl7 except that 136 nt are deleted from the 5’ end (including the initiating ATG for rev) and 106 nt are deleted from the 3’ noncoding region of SIV DNA. Only 6 nt of the first rev exon remained (including the splice donor) in pBD21. The following are approximate lengths for elements of the vector: SV40 late promoter, 0.35 kb; env gene, 3.0 kb; spliced rev gene, 0.3 kb; SV40 polyadenylation region, 0.8 kb. SD, splice donor. SA, splice acceptor.
the rev transcript because it was observed only in cells transfected with pBC17 (rev+env+), and was hybridized with both rev probes but not with the env only probe. The small 0.9-kb transcript was detected only with probe A and probably represented an alternative splicing event between the splice donor site of the first exon of rev and the splice acceptor site in the SV40 polyadenylation region (29). In cells transfected with pBD21 (rev-env+), only one main RNA species was observed (Figs. 3A, 3B, and 3C, lane 2): a 1.1 -kb transcript detected only with probe C (rev/env probe). The 1 .I-kb transcript was consistent with a spliced RNA derived from the splice donor of the truncated first exon of rev in pBD21 and the splice acceptor in the second exon of rev, and terminated in the SV40 early polyadenylation sequence (Fig. 1). No hybridizing RNA was detected in cells transfected with thevector pMLSV5 (Figs. 3A, 3B, and 3C, lane 3). The results of the RNA analyses can be summarized as follows: (i) a 4-kb unspliced RNA and a 1.3-kb spliced RNA were the envelope and rev transcripts, respectively; (ii) the coexpression of these two RNA species was observed only in cells transfected with pBCl7 (rev+env+). (iii) Cells transfected with pBD21 (rev-env+) contained only a spliced RNA containing the second exon of rev and the SV40 polyadenylation region. These data, considered with the protein analyses described above, suggest that Rev facilitates the accumulation of unspliced envelope RNA transcripts that are translated into the envelope glycoprotein.
817
One biologic activity of the SIV envelope glycoprotein is the induction of syncytia in CD4+ lymphocytes susceptible to exogeneous infection by SIV (17). Significant syncytium formation was only observed when COS-1 cells transfected with pBC17 (rev+env+) were cocultured with MT-2 cells (a human CD4+ T-lymphocyte line transformed by HTLV-1) (Fig. 4). To examine the specificity of syncytium induction, blocking experiments were performed. Syncytium formation was completely inhibited by the mouse monoclonal antibody OKT4A; but not by an irrelevant monoclonal antibody OKT3 (data not shown). This indicated a specific interaction between the biologically active SlVsm envelope glycoprotein expressed in COS-1 cells and the CD4 molecule on MT-2 cells. Our data is the first to show that the SIV rev gene is functionally similar to the HIV-1 rev gene (18-21). The SlVsm rev gene acted to increase the level of unspliced mRNA capable of encoding the SlVsm envelope glycoprotein. This effect was rev-specific because the SV40 constructs used contained only the rev and env genes, and expression was driven by a heterologous SV40 late promoter rather than the viral LTR. Thus, only cells transfected with the rev and env genes expressed sig-
-P66 r,“g -6P40 -p32
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analysis of SIVsm-specific glycoproteins from FIG. 2. lmmunoblot transfected COS-1 cells. COS-1 cells were transfected with pMLSV5 (lane l), pBD21 (lane 2) or pBC17 (lane 3). harvested at 60 hr posttransfection, and solubilized. Lysates were adsorbed to agarose beads conjugated to lentil-lectin (Sigma, St. Louis, MO), washed, and eluted in cY-methylmannoside, as previously described (15). The eluted glycoproteins were separated by electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. Reaction with SlVsm immune serum and detection of bound antibodies were performed as previously described (75). Lanes 4 and 5 were included as controls Lane 4 represents glycoproteins extracted from CEM cells persistently infected with SIVsm. Lane 5 is ceil-free (partially purified) SIVsm; note the absence of gpl60 from purified virions. Positions of molecular weight markers are shown on the left, and molecular weights of SlVsm polypeptides are shown on the right.
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FIG. 3. Northern blot analyses of SlVsm RNA species isolated from transfected COS-1 cells. Total RNA was collected at 60 hr posttransfection, and 10.rg RNA aliquots were loaded on each lane. Lane 1 represents RNA isolated from cells transfected with pBC17, Lane 2 is pBD21, and Lane 3 is pMLSV5 (control). The sizes for the rRNA (28 S and 18 S) are indicated by arrows. After electrophoresis through a 1.3% formaldehyde-agarose gel, RNAs were transferred to nitrocellulose and hybridized with the following probes. (A) A 59. mer. derived from the N-terminal of the first rev exon (nt 6481-6539 (75)); (B) The EcoRI-Ncol DNA fragment (nt 7746-8433 (15)); (C) a 76.mer. derived from the C-terminal of the second rev exon (nt 89249009 (75)). Probes A and C were chemically synthesized and 5’endlabeled. Probe B was nick-translated. For loading controls, equal intensities of rRNA bands were observed after the gel was stained with acridine orange (data not shown). The transfection efficiency was assayed by cotransfection of cells with pXGH5, a plasmid that contains a reporter gene (human growth hormone) (30).
nificant amounts of the SlVsm envelope glycoprotein. These data are similar to a recent report describing the effects of rev on expression of HIV-1 envelope glycoprotein ( 19, 2 1). Because of the functional similarities between the SlVsm and HIV-l rev genes, we compared the rev amino acid sequence of SIVsm, HIV-l, and four other primate lentiviruses in search of structural similarities suggestive of conserved functional domains. As seen
in Fig. 5, the amino-terminal half of rev is highly conserved (50% of the residues present in at least five of the six sequences) among all the primate lentiviruses. In contrast, the carboxy-terminal half is poorly conserved (less than 109’0 identity). This suggested that the amino-terminal half of the rev protein of primate lentiviruses might contain sequences important for rev function. Recently, Malim et al. (22) presented an elegant dissection of the functional domains of the HIV-1 rev protein. These workers identified two distinct functional regions in rev: an amino-terminal domain that serves as a nuclear localization signal and may participate in RNA binding; and a carboxy-terminal domain that may be similar to activation domains defined for other transcription factors. In the amino-terminal, the argininerich nuclear localization domain contains a four residue stretch (RRRW, residues 38-41 in the SlVsm sequence) that is conserved in all the primate and human sequences examined (Fig. 5). As might be expected for a highly conserved region, mutation of these 4 amino acids and surrounding residues ablates HIV-1 rev function and confers a recessive negative phenotype to the mutant protein (22). In the carboxy-terminal, the second functional domain defined by Malim et al, is not conserved. Deletion of this domain in HIV-l results in a mutant rev protein that acts as a trans-dominant inhibitor of rev function (22). The dichotomy of sequence conservation between the two functional domains of primate lentivirus rev proteins suggests that the poorly conserved carboxy-terminal domain may confer specificity to rev function. The rev protein has been proposed as a new class of sequence-specific RNA-binding protein, because of the presence of the highly conserved arginine-rich
FIG. 4. Syncytium formation in MT2 cells after cocultivation with transfected plasmids: (A) pBD21 (rev-en@) and (B) pBCl7 (rev*env+). Following transfection, were taken 48 hr after cocultivation.
COS-1 cells. COS-1 cells were cocultured
cells were transfected with the following with MT-2 cells, and photomicrographs
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FIG. 5. Alignment of the rev proteins of six primate lentiviruses. Multiple alignments were performed using the program Genalign (IntelliGenetics. Mountain View, CA). Dashed lines represent identity with the SlVsm sequence and periods represent gaps Introduced to optlmlze the alignment. All of the sequences (except SIVagm2) were obtained from the Los Alamos HIV Sequence Database. The sequences used were all derived from biologically active molecular clones: SIVsm, clone smH-4; SIVmac, clone MM1 42; HIV-2, clone NIH-Z; SIVagml, clone TYO-1; SIVagm2, clone 155-4 (P.R.J. and V.M.H., unpublished data); HIV-l, clone HXB2. Proposed exon junctlons are indicated above the sequences. Solid dots above residues indicate identity for at least five sequences. Under the HIV-1 sequence, notations regarding functional domains are indicated (22). Shaded bars represent clustered point mutations that ablate rev function. Deletions that also ablate rev function are spanned by lines interrupted by A.
RNA-binding motif (23-25). The RNA target sequence for Rev, named as a rev responsive element (RRE), has been mapped to a conserved region (about 230 bases long) of env that encodes the N-terminus of gp40 (1820, 26, 27). In SlVsm envelope sequences, the conserved RRE sequence was located between nt 8 154 to 8375 (numbered according to Ref. (16)). The SIV and HIV-2 RRE sequences shared about 90% identity with each other and about 69% with HIV-l (26). Recent reports demonstrated that the RNA sequences for all RRE examined can form a stable complex stem-loop structure (20,26,27). In HIV-1 RRE, a stem-loop II subdomain is sufficient to bind the trans-activator rev and is essential for its biologic activity (23). However, the cross-complementation between the rev protein of SIV (HIV-2) and the HIV-1 rev protein is nonreciprocal (26, 27). The HIV-1 rev works with the HIV-1 RRE as well as the SIV (HIV-2) RRE, but the SIV (HIV-2) rev does not work with the HIV-1 RRE. In addition, the HIV-2 rev protein does not work with a short homologous RRE (which shares 69% identity with HIV-l), but works with a larger HIV-2 fragment (which includes the short RRE sequences described above) (27). This suggests the rev trans-activator of SIV (HIV-2) is more discriminating than that of HIV-l. The detailed mechanisms for the interaction between SIV (HIV-2) rev and its homologous RRE are not clear at this point. REFERENCES 1. SHARP, P. M.. and LI, W.-H., Nature (London) 336, 315 (1988). 2. SMITH, T. F., SRINIVASAN, A., SCHOCHETMAN, G., MARCUS, M., and MYERS, G.. Nature (London) 333, 573-575 (1988).
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3. LET~IN, N. L., DANIEL. M. D., SEHGAL, P. K., DESROSIERS, R. C., HUNG, R. D., WALDRON, L. M., MACKEY, J. J.. SCHMIDT, D. K., CHALIFOUX. L. V., and KING, N. W., Science 230,71-73 (1985). CHAKRABARTI, S.. ROBERT-GURAFF, M., WONG-STAAL, F., GALLO, R. C., and Moss, B.. Nature (London) 320,535-537 (1986). Hu, S.-L., KOSOWSKI, S. G., and DALRYMPLE. J. M., Nature (London) 320,537-540 (1987). KOENIG, S., HIRSCH, V. M., OLMSTED, R. A., POWELL, D., MAURY, W., RABSON, A., FAUCI, A. S., PURCELL, R. H., and JOHNSON, P. R., Proc. Nat/. Acad. Sci. USA 86, 2443-2447 (1989). 7. CHANDA, P., NATUK, R.. GREENBERG, L., MASON, B., BHAT, B., DHEER, S., MORIN, J., MOLNAR-KIMBER, K., MIZUTANI, S., LUBECK, M., DAVIS, A., and HUNG, P. P., In “Vaccines 89: Modern Approaches to New Vaccines Including Prevention of AIDS,” pp. 207-21 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989). 8. PETERLIN, B. M., and LUCIW, P. A., A/DS (Suppl. 1) 2, S29-S40 (1988). 9. FEINBERG, M. B., JARREITE, R. F., ALDOVINI, A., GALLO, R. C., and WONG-STAAL., F., Cell46, 807-817 (1986). 10. KNIGHT. D. M.. FLOMERFELT, F. A., and GHRAYEB. J., Science 236, 837-840 (1987). 11. SODROSKI, J., GOH, W. C., ROSEN, C., DAYTON, A.. TERWILLIGER, E., and HASELTINE, W. A., Nature (London) 321,412-417 (1986). 12. CHAKARABARTI, L., GUYADER, M., ALIZON. B., DANIEL, M. D., DESROSIER% R. C., TOI-LAIS, P., and SONIGO, P., Nature (London) 328,543-547 (1987). 13. FRANCHINI, G., GURGO. C., Guo, H-.G., GALLO, R. C., COLLATI, E., FARGNOLI, K. A., HALL, L. F., WONG-STAAL, F.. and REITZ, M. S., JR., Nature (London) 328, 539-543 (1987). 14. HIRSCH, V. M., REIDEL, N., and MULLINS. 1. I., Cell 49, 307-319 (1987). 15. HIRSCH, V. M.. DAPOLITO, G., MCGANN. C., OLMSTED, R. A., PURCELL, R. H.. and JOHNSON, P. R., /. Med. Primarol. 18,279-288 (1989). 16. HIRSCH, V. M.. OLMSTED, R. A., MURPHEY-CROB, M., PURCELL, R. H., and JOHNSON, P. R., Nature (London) 339, 389-392 (1989). 17. SATTENTAU, Q. J., and WEISS, R. A., Cell52, 631-633 (1988). 18. HADZOPOULOU-CLADARAS, M., FELEIER, B. K., CLADARAS, C., ATHANASSOPOULOS, A., TSE, A., and PAVLAKIS. G. N., /. Virol. 63, 1265-1274(1989). 19. HAMMARSKJOLD, M.-L.. HEIMER. J., HAMMARSKJOLD, B., SANGWAN, I., ALBERT, L., and REKOSH, D., 1. Hro/. 63, 1959-l 966 (1989). 20. MALIM, M. H., HAUBER, J., LE, S.-Y., MAIZEL, J. V., and CULLEN, B. R., Nature (London) 338, 254-257 (1989). 21. REKOSH. D., NYGREN, A., FLOOSY, P., HAMMARSKIOLD, M.-L., and WIGZELL, H., Proc. Nat/. Acad. Sci. USA 85, 334-338 (1988). 22. MALIM, M. H., BOHLEIN, S., HAUBER, J., and CULLEN, B. R., Cell58, 205-214(1989). 23. MALIM, M. H., TILEY, L. S., MCCARN, D., RUSCHE. J., HAUBER, J., and CULLEN, B. R., Cell60, 675-683 (1990). 24. ZAPP, M., L., and GREEN, M. Nature (London) 342, 714-716 (1989). 25. DALY, T. J., COOK, K. S., GRAY, G., MAIONE, T. E., and RUSCHE, J. R., Nature (London) 342, 816-819 (1989). 26. MALIM, M. H., BOHNLEIN, S., FENRICK, R., LE. S.-Y., MAIZEL, J. V., and CULLEN, B. R.. Proc. Nat/. Acad. SC;. USA 86, 8222-8226 (1989). 27. LEWIS, N., WILLIAMS, J.. REKOSH, D., and HAMMARSKJOLD, M.-L., J. t’irol. 64, 1690-1697(1990). 28. CHENG, S. M., LEE, S. G., KALYAN, N. K., AND MCCLOUD, S., LEVNER, M., and HUNG, P. P., Gene 58, 229-303 (1987). 29. REDDY, V. B., GHOSH, P. K., LEBOWITZ. P., PIATAK, M., and WEISSMAN, S. M., J. Viral. 30, 279-296 (1979). 30. SELDEN, R. F., HOWIE, K. B., ROWE, M. E. GOODMAN, H. M., and MOORE, D., Mol. Cell. Biol. 6, 3173%3179(1986).
177,816-819
(1990)
Coexpression
of Biologically
Active
Simian lmmunodeficiency
in an SV40 System: The SIV rev Gene Regulates
Virus (SW) Rev and Env Env Expression
SHEAU-MEI CHENG,*’ MARLENE BLUME,* SHAW-GUANG LEE,* PAUL P. HUNG,* VANESSA M. HIRscH,t AND PHILIP R. JOHNSON+ *Biotechnology
and Microbiology Division, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087; and tRetrovira/ Pathogenesis Division of Molecular Virology and Immunology, Georgetown University, Rockville, Maryland 20852 Received February
Section,
1, 1990; accepted April 13, 1990
The coexpression of biologically active simian immunodeficiencyvirus (SIV) Rev and Env gene products was obtained in COS-I cells from a single SIV subgenomic segment (which contains both exons of rev and the entire env gene) cloned into a SV40-directed vector. The SlVsm Rev trans-activated the expression of the full-length env mRNA and was required for the production of envelope glycoproteins. Furthermore, the alignment of the structural conservation of the Rev functional domains among all HIV and SIV was analyzed. 0 1990 Academic Press, Inc.
COS-1 cells were transfected with pBC17, pBD21, or pMLSV5 (control) and analyzed for the expression of SIVsm-specific glycoproteins (Fig. 2). Only cells transfected with pBC17 (rev+env+) contained SIVsm-specific glycoproteins (Fig. 2, lane 3). No SlVsm glycoproteins were detected in cells transfected with pMLSV5 or pBD21 (rev-env+) (Fig. 2, lanes 1 and 2). The precursor envelope glycoprotein (gpl60) expressed in COS1 cells transfected with pBC17 was properly processed into gpl20 and gp40 (Figs. 2, lane 3).
Simian immunodeficiency virus (SW) infection of macaques is a relevant and valuable animal model for AIDS vaccine studies (7, 2), because the infected macaques develop a human AIDS-like symptom (3). One approach to vaccine development involves the expression of the Env glycoprotein (gpl60) of SIV in live viral vectors such as vaccinia virus (4-6) or adenovirus (7). An important step in the development of such expression systems is an understanding of the regulation of SW envelope gene expression in cultured cells. The regulation of HIV-1 gene expression is complex and not fully understood (reviewed in Ref. (8)). HIV-1 has at least six novel genes (vif, vpr, tat, rev, vpu, and nef) when compared to prototypic retroviruses. The rev protein appears to post-transcriptionally enhance the production of the viral structural proteins Gag and Env (9-I I). SIV and HIV-1 share a similar, but not identical, genome organization (12- 16). SIV does possess the rev gene, but its protein shares only 30% amino acid identity with the HIV-1 Rev (16). Relatively little work has been reported on the regulation of SIV gene expression. To assess the role of rev in the expression of the SlVsm env gene, we generated two transient expression vectors (Fig. 1): (i) pBC17 contained a single subgenomic segment of SIV (smH-3, a molecular clone of SIV from sooty magabeys (75, 76)) that included both exons of rev and the entire env gene; (ii) pBD21 was derived from pBC17 by deleting most of the first rev exon. Thus, pBC17 (rev+env+) could potentially encode Rev and Env proteins, while pBD21 (rev-env+) could only encode the envelope glycoprotein.
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To assess the levels and forms of RNA transcribed from pBC17 and pBD21, three identical RNA blots (described below) were hybridized with one of the following probes (Figs. 3A, 3B. and 3C): probe A was specific to the first exon of rev (rev-only probe); probe B was specific for envelope transcripts (env-only probe); probe C was derived from the second exon of rev and therefore would recognize rev and env sequences (rev/ env probe). In cells transfected with pBC17 (rev+env+), three RNA species were detected (Figs. 3A, 3B, and 3C, lane 1): a 4-kb transcript (detected with all three probes); a 1.3-kb transcript (probes A and C); and a 0.9kb species (probe A only). The 4-kb RNA species represented the predicted unspliced mRNA initiating from the SV40 late promoter and terminating in the SV40 early polyadenylation sequence. Since this transcript hybridized with all three probes, it contained the first rev exon and the env gene, and was the only RNA species capable of encoding this envelope protein. Interestingly, the envelope protein would initiate from the second ATG of this mRNA since the first ATG belongs to the first exon of rev (15). The 1.3-kb transcript was the spliced RNA resulting from the predicted splice sites in rev and terminating in the SV40 early polyadenylation sequence (Fig. 1). This species represented
Copyright 0 1990 Academic Press, Inc. All rights of reproduction in any form reserved.
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FIG. 1. Structure of the SlVsm env and rev gene expression vectors Subgenomic fragments isolated for psmH-3 were cloned into the unique Bglll site of the expression plasmid pMLSV5 (28). This vector contains the SV40 late promoter and polyadenylation region in the plasmid pML2. The polyadenylation region contains splice junctions derived from T, t, and early polydenylation sequences (29). Arrows represent transcriptional direction. The SV40 regulatory elements are represented by solid boxes and the sinusoidal lines are pML2 sequences (plasmid). pBC17 contains the SIV DNA insert starting from nucleotides 6402 to 9470 that includes the entire env coding sequence (open box) and both exons of the rev gene (shaded boxes). pBD21 is identical to pBCl7 except that 136 nt are deleted from the 5’ end (including the initiating ATG for rev) and 106 nt are deleted from the 3’ noncoding region of SIV DNA. Only 6 nt of the first rev exon remained (including the splice donor) in pBD21. The following are approximate lengths for elements of the vector: SV40 late promoter, 0.35 kb; env gene, 3.0 kb; spliced rev gene, 0.3 kb; SV40 polyadenylation region, 0.8 kb. SD, splice donor. SA, splice acceptor.
the rev transcript because it was observed only in cells transfected with pBC17 (rev+env+), and was hybridized with both rev probes but not with the env only probe. The small 0.9-kb transcript was detected only with probe A and probably represented an alternative splicing event between the splice donor site of the first exon of rev and the splice acceptor site in the SV40 polyadenylation region (29). In cells transfected with pBD21 (rev-env+), only one main RNA species was observed (Figs. 3A, 3B, and 3C, lane 2): a 1.1 -kb transcript detected only with probe C (rev/env probe). The 1 .I-kb transcript was consistent with a spliced RNA derived from the splice donor of the truncated first exon of rev in pBD21 and the splice acceptor in the second exon of rev, and terminated in the SV40 early polyadenylation sequence (Fig. 1). No hybridizing RNA was detected in cells transfected with thevector pMLSV5 (Figs. 3A, 3B, and 3C, lane 3). The results of the RNA analyses can be summarized as follows: (i) a 4-kb unspliced RNA and a 1.3-kb spliced RNA were the envelope and rev transcripts, respectively; (ii) the coexpression of these two RNA species was observed only in cells transfected with pBCl7 (rev+env+). (iii) Cells transfected with pBD21 (rev-env+) contained only a spliced RNA containing the second exon of rev and the SV40 polyadenylation region. These data, considered with the protein analyses described above, suggest that Rev facilitates the accumulation of unspliced envelope RNA transcripts that are translated into the envelope glycoprotein.
817
One biologic activity of the SIV envelope glycoprotein is the induction of syncytia in CD4+ lymphocytes susceptible to exogeneous infection by SIV (17). Significant syncytium formation was only observed when COS-1 cells transfected with pBC17 (rev+env+) were cocultured with MT-2 cells (a human CD4+ T-lymphocyte line transformed by HTLV-1) (Fig. 4). To examine the specificity of syncytium induction, blocking experiments were performed. Syncytium formation was completely inhibited by the mouse monoclonal antibody OKT4A; but not by an irrelevant monoclonal antibody OKT3 (data not shown). This indicated a specific interaction between the biologically active SlVsm envelope glycoprotein expressed in COS-1 cells and the CD4 molecule on MT-2 cells. Our data is the first to show that the SIV rev gene is functionally similar to the HIV-1 rev gene (18-21). The SlVsm rev gene acted to increase the level of unspliced mRNA capable of encoding the SlVsm envelope glycoprotein. This effect was rev-specific because the SV40 constructs used contained only the rev and env genes, and expression was driven by a heterologous SV40 late promoter rather than the viral LTR. Thus, only cells transfected with the rev and env genes expressed sig-
-P66 r,“g -6P40 -p32
-p26
analysis of SIVsm-specific glycoproteins from FIG. 2. lmmunoblot transfected COS-1 cells. COS-1 cells were transfected with pMLSV5 (lane l), pBD21 (lane 2) or pBC17 (lane 3). harvested at 60 hr posttransfection, and solubilized. Lysates were adsorbed to agarose beads conjugated to lentil-lectin (Sigma, St. Louis, MO), washed, and eluted in cY-methylmannoside, as previously described (15). The eluted glycoproteins were separated by electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. Reaction with SlVsm immune serum and detection of bound antibodies were performed as previously described (75). Lanes 4 and 5 were included as controls Lane 4 represents glycoproteins extracted from CEM cells persistently infected with SIVsm. Lane 5 is ceil-free (partially purified) SIVsm; note the absence of gpl60 from purified virions. Positions of molecular weight markers are shown on the left, and molecular weights of SlVsm polypeptides are shown on the right.
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FIG. 3. Northern blot analyses of SlVsm RNA species isolated from transfected COS-1 cells. Total RNA was collected at 60 hr posttransfection, and 10.rg RNA aliquots were loaded on each lane. Lane 1 represents RNA isolated from cells transfected with pBC17, Lane 2 is pBD21, and Lane 3 is pMLSV5 (control). The sizes for the rRNA (28 S and 18 S) are indicated by arrows. After electrophoresis through a 1.3% formaldehyde-agarose gel, RNAs were transferred to nitrocellulose and hybridized with the following probes. (A) A 59. mer. derived from the N-terminal of the first rev exon (nt 6481-6539 (75)); (B) The EcoRI-Ncol DNA fragment (nt 7746-8433 (15)); (C) a 76.mer. derived from the C-terminal of the second rev exon (nt 89249009 (75)). Probes A and C were chemically synthesized and 5’endlabeled. Probe B was nick-translated. For loading controls, equal intensities of rRNA bands were observed after the gel was stained with acridine orange (data not shown). The transfection efficiency was assayed by cotransfection of cells with pXGH5, a plasmid that contains a reporter gene (human growth hormone) (30).
nificant amounts of the SlVsm envelope glycoprotein. These data are similar to a recent report describing the effects of rev on expression of HIV-1 envelope glycoprotein ( 19, 2 1). Because of the functional similarities between the SlVsm and HIV-l rev genes, we compared the rev amino acid sequence of SIVsm, HIV-l, and four other primate lentiviruses in search of structural similarities suggestive of conserved functional domains. As seen
in Fig. 5, the amino-terminal half of rev is highly conserved (50% of the residues present in at least five of the six sequences) among all the primate lentiviruses. In contrast, the carboxy-terminal half is poorly conserved (less than 109’0 identity). This suggested that the amino-terminal half of the rev protein of primate lentiviruses might contain sequences important for rev function. Recently, Malim et al. (22) presented an elegant dissection of the functional domains of the HIV-1 rev protein. These workers identified two distinct functional regions in rev: an amino-terminal domain that serves as a nuclear localization signal and may participate in RNA binding; and a carboxy-terminal domain that may be similar to activation domains defined for other transcription factors. In the amino-terminal, the argininerich nuclear localization domain contains a four residue stretch (RRRW, residues 38-41 in the SlVsm sequence) that is conserved in all the primate and human sequences examined (Fig. 5). As might be expected for a highly conserved region, mutation of these 4 amino acids and surrounding residues ablates HIV-1 rev function and confers a recessive negative phenotype to the mutant protein (22). In the carboxy-terminal, the second functional domain defined by Malim et al, is not conserved. Deletion of this domain in HIV-l results in a mutant rev protein that acts as a trans-dominant inhibitor of rev function (22). The dichotomy of sequence conservation between the two functional domains of primate lentivirus rev proteins suggests that the poorly conserved carboxy-terminal domain may confer specificity to rev function. The rev protein has been proposed as a new class of sequence-specific RNA-binding protein, because of the presence of the highly conserved arginine-rich
FIG. 4. Syncytium formation in MT2 cells after cocultivation with transfected plasmids: (A) pBD21 (rev-en@) and (B) pBCl7 (rev*env+). Following transfection, were taken 48 hr after cocultivation.
COS-1 cells. COS-1 cells were cocultured
cells were transfected with the following with MT-2 cells, and photomicrographs
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FIG. 5. Alignment of the rev proteins of six primate lentiviruses. Multiple alignments were performed using the program Genalign (IntelliGenetics. Mountain View, CA). Dashed lines represent identity with the SlVsm sequence and periods represent gaps Introduced to optlmlze the alignment. All of the sequences (except SIVagm2) were obtained from the Los Alamos HIV Sequence Database. The sequences used were all derived from biologically active molecular clones: SIVsm, clone smH-4; SIVmac, clone MM1 42; HIV-2, clone NIH-Z; SIVagml, clone TYO-1; SIVagm2, clone 155-4 (P.R.J. and V.M.H., unpublished data); HIV-l, clone HXB2. Proposed exon junctlons are indicated above the sequences. Solid dots above residues indicate identity for at least five sequences. Under the HIV-1 sequence, notations regarding functional domains are indicated (22). Shaded bars represent clustered point mutations that ablate rev function. Deletions that also ablate rev function are spanned by lines interrupted by A.
RNA-binding motif (23-25). The RNA target sequence for Rev, named as a rev responsive element (RRE), has been mapped to a conserved region (about 230 bases long) of env that encodes the N-terminus of gp40 (1820, 26, 27). In SlVsm envelope sequences, the conserved RRE sequence was located between nt 8 154 to 8375 (numbered according to Ref. (16)). The SIV and HIV-2 RRE sequences shared about 90% identity with each other and about 69% with HIV-l (26). Recent reports demonstrated that the RNA sequences for all RRE examined can form a stable complex stem-loop structure (20,26,27). In HIV-1 RRE, a stem-loop II subdomain is sufficient to bind the trans-activator rev and is essential for its biologic activity (23). However, the cross-complementation between the rev protein of SIV (HIV-2) and the HIV-1 rev protein is nonreciprocal (26, 27). The HIV-1 rev works with the HIV-1 RRE as well as the SIV (HIV-2) RRE, but the SIV (HIV-2) rev does not work with the HIV-1 RRE. In addition, the HIV-2 rev protein does not work with a short homologous RRE (which shares 69% identity with HIV-l), but works with a larger HIV-2 fragment (which includes the short RRE sequences described above) (27). This suggests the rev trans-activator of SIV (HIV-2) is more discriminating than that of HIV-l. The detailed mechanisms for the interaction between SIV (HIV-2) rev and its homologous RRE are not clear at this point. REFERENCES 1. SHARP, P. M.. and LI, W.-H., Nature (London) 336, 315 (1988). 2. SMITH, T. F., SRINIVASAN, A., SCHOCHETMAN, G., MARCUS, M., and MYERS, G.. Nature (London) 333, 573-575 (1988).
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3. LET~IN, N. L., DANIEL. M. D., SEHGAL, P. K., DESROSIERS, R. C., HUNG, R. D., WALDRON, L. M., MACKEY, J. J.. SCHMIDT, D. K., CHALIFOUX. L. V., and KING, N. W., Science 230,71-73 (1985). CHAKRABARTI, S.. ROBERT-GURAFF, M., WONG-STAAL, F., GALLO, R. C., and Moss, B.. Nature (London) 320,535-537 (1986). Hu, S.-L., KOSOWSKI, S. G., and DALRYMPLE. J. M., Nature (London) 320,537-540 (1987). KOENIG, S., HIRSCH, V. M., OLMSTED, R. A., POWELL, D., MAURY, W., RABSON, A., FAUCI, A. S., PURCELL, R. H., and JOHNSON, P. R., Proc. Nat/. Acad. Sci. USA 86, 2443-2447 (1989). 7. CHANDA, P., NATUK, R.. GREENBERG, L., MASON, B., BHAT, B., DHEER, S., MORIN, J., MOLNAR-KIMBER, K., MIZUTANI, S., LUBECK, M., DAVIS, A., and HUNG, P. P., In “Vaccines 89: Modern Approaches to New Vaccines Including Prevention of AIDS,” pp. 207-21 1. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989). 8. PETERLIN, B. M., and LUCIW, P. A., A/DS (Suppl. 1) 2, S29-S40 (1988). 9. FEINBERG, M. B., JARREITE, R. F., ALDOVINI, A., GALLO, R. C., and WONG-STAAL., F., Cell46, 807-817 (1986). 10. KNIGHT. D. M.. FLOMERFELT, F. A., and GHRAYEB. J., Science 236, 837-840 (1987). 11. SODROSKI, J., GOH, W. C., ROSEN, C., DAYTON, A.. TERWILLIGER, E., and HASELTINE, W. A., Nature (London) 321,412-417 (1986). 12. CHAKARABARTI, L., GUYADER, M., ALIZON. B., DANIEL, M. D., DESROSIER% R. C., TOI-LAIS, P., and SONIGO, P., Nature (London) 328,543-547 (1987). 13. FRANCHINI, G., GURGO. C., Guo, H-.G., GALLO, R. C., COLLATI, E., FARGNOLI, K. A., HALL, L. F., WONG-STAAL, F.. and REITZ, M. S., JR., Nature (London) 328, 539-543 (1987). 14. HIRSCH, V. M., REIDEL, N., and MULLINS. 1. I., Cell 49, 307-319 (1987). 15. HIRSCH, V. M.. DAPOLITO, G., MCGANN. C., OLMSTED, R. A., PURCELL, R. H.. and JOHNSON, P. R., /. Med. Primarol. 18,279-288 (1989). 16. HIRSCH, V. M.. OLMSTED, R. A., MURPHEY-CROB, M., PURCELL, R. H., and JOHNSON, P. R., Nature (London) 339, 389-392 (1989). 17. SATTENTAU, Q. J., and WEISS, R. A., Cell52, 631-633 (1988). 18. HADZOPOULOU-CLADARAS, M., FELEIER, B. K., CLADARAS, C., ATHANASSOPOULOS, A., TSE, A., and PAVLAKIS. G. N., /. Virol. 63, 1265-1274(1989). 19. HAMMARSKJOLD, M.-L.. HEIMER. J., HAMMARSKJOLD, B., SANGWAN, I., ALBERT, L., and REKOSH, D., 1. Hro/. 63, 1959-l 966 (1989). 20. MALIM, M. H., HAUBER, J., LE, S.-Y., MAIZEL, J. V., and CULLEN, B. R., Nature (London) 338, 254-257 (1989). 21. REKOSH. D., NYGREN, A., FLOOSY, P., HAMMARSKIOLD, M.-L., and WIGZELL, H., Proc. Nat/. Acad. Sci. USA 85, 334-338 (1988). 22. MALIM, M. H., BOHLEIN, S., HAUBER, J., and CULLEN, B. R., Cell58, 205-214(1989). 23. MALIM, M. H., TILEY, L. S., MCCARN, D., RUSCHE. J., HAUBER, J., and CULLEN, B. R., Cell60, 675-683 (1990). 24. ZAPP, M., L., and GREEN, M. Nature (London) 342, 714-716 (1989). 25. DALY, T. J., COOK, K. S., GRAY, G., MAIONE, T. E., and RUSCHE, J. R., Nature (London) 342, 816-819 (1989). 26. MALIM, M. H., BOHNLEIN, S., FENRICK, R., LE. S.-Y., MAIZEL, J. V., and CULLEN, B. R.. Proc. Nat/. Acad. SC;. USA 86, 8222-8226 (1989). 27. LEWIS, N., WILLIAMS, J.. REKOSH, D., and HAMMARSKJOLD, M.-L., J. t’irol. 64, 1690-1697(1990). 28. CHENG, S. M., LEE, S. G., KALYAN, N. K., AND MCCLOUD, S., LEVNER, M., and HUNG, P. P., Gene 58, 229-303 (1987). 29. REDDY, V. B., GHOSH, P. K., LEBOWITZ. P., PIATAK, M., and WEISSMAN, S. M., J. Viral. 30, 279-296 (1979). 30. SELDEN, R. F., HOWIE, K. B., ROWE, M. E. GOODMAN, H. M., and MOORE, D., Mol. Cell. Biol. 6, 3173%3179(1986).