Department of Molecular Biology1 and Centre for Research in Virology, Department of Microbiology and Immunology2, The Gade Institute, University of Bergen, HIB, Post-box 7800, N-5020 Bergen, Norway
Author for correspondence: Anne Marie Szilvay.Fax +47 55 58 96 83. e-mail Anne.Szilvay{at}mbi.uib.no
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Site-directed mutagenesis of the 116 amino acid Rev protein has defined two functional domains in addition to shorter amino acid sequences and single amino acid residues which seem to be necessary for function. Mutations in a basic domain (amino acids 3550) compromise nuclear and nucleolar accumulation and specific binding of Rev to the RRE RNA in vitro (Malim et al., 1989a ; Cochrane et al., 1990
; Hope et al., 1990
; Olsen et al., 1990
; Kjems et al., 1991b
, 1992
; Böhnlein et al., 1991
; Malim & Cullen, 1991
; Zapp et al., 1991
). Oligomer formation of Rev molecules has been demonstrated both in vitro (Olsen et al., 1990
; Malim & Cullen, 1991
; Zapp et al., 1991
) and in vivo (Hope et al., 1992
; Bogerd & Greene, 1993
; Madore et al., 1994
; Szilvay et al., 1997
). The ability to form oligomers appears to be critical for Rev function and several amino acid residues in the N-terminal region of Rev have been demonstrated to be essential for oligomerization (Olsen et al., 1990
; Malim & Cullen, 1991
; Zapp et al., 1991
; Hope et al., 1992
; Szilvay et al., 1997
; Stauber et al., 1998
).
Rev was originally defined as a nuclear and nucleolar protein (Cullen et al., 1988 ). Subsequent studies revealed that the subcellular localization is more dynamic (Kalland et al., 1994b
) implying that Rev is a nucleocytoplasmic shuttle protein (Kalland et al., 1994a
; Meyer & Malim, 1994
; Richard et al., 1994
). The leucine-rich activation domain of HIV-1 Rev was the first discovered example of an amino acid motif that signals active nuclear export (Meyer & Malim, 1994
; Szilvay et al., 1995
; Wolff et al., 1995
). Later, it was shown that micro-injection of albumin conjugated to a synthetic peptide corresponding to this domain was exported from the nucleus of Xenopus laevis oocytes. The domain was consequently named NES (nuclear export signal) (Fischer et al., 1995
). Mutations within this domain of HIV-1 Rev generate trans-dominant negative mutants (Malim et al., 1989b
, 1991
; Mermer et al., 1990
; Venkatesh & Chinnadurai, 1990
; Hope et al., 1992
; Weichselbraun et al., 1992
) and the basis for the trans-dominant negative effect was found to be nuclear retention of the wild-type protein (Stauber et al., 1995
; Szilvay et al., 1995
).
In the present study the inhibitory effect of one such trans-dominant negative Rev mutant on the expression of structural viral proteins from plasmids encoding Rev and other HIV-1 proteins was investigated. In these experimental systems the same species of transcribed pre-mRNA is either spliced or remains unspliced. The relative cellular expression of Rev and the viral structural proteins (Env and Gag) encoded by the spliced and unspliced mRNAs, respectively, will then reflect the extent of splicing and nuclear export of the viral pre-mRNA. The results indicate that the trans-dominant negative mutant obstructs the Rev-dependent regulation of viral pre-mRNA splicing in addition to its reported inhibitory effect on nuclear export of Rev (Stauber et al., 1995 ; Szilvay et al., 1995
). This assumption was confirmed using in situ RNA hybridization and an RNase protection assay analysing the localization and relative amount of spliced versus unspliced viral mRNAs.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The reporter plasmid pDM128 provided by T. Hope contains the CAT gene flanked by HIV intron sequences and the 5' and 3' splice sites from the env gene. Most of the sequence for rev exon 2 is deleted (Hope et al., 1990 ). The plasmid used for generation of the probe protecting spliced and unspliced RNA from the reporter plasmid pDM128 was kindly provided by Y. Huang and G. C. Carmichael (Huang & Carmichael, 1997
).
Cell lines and transfection.
COS cells were seeded into 35 mm wells or 100 mm plates 1 day prior to transfection, grown to 6070% confluence and transfected by the lipofectamine procedure of Gibco BLR using 7 µl/50 µl lipofectamine per 35 mm well/100 mm plate. The (different) amount of plasmid DNA used in each experiment is indicated above each lane in the RNase protection and Western blot figures
Monoclonal antibodies.
The anti-Rev MAbs 8E7 and 1G7 have been described (Kalland et al., 1994b ). In short, MAb 8E7 (IgG2a) binds to the activation domain of Rev and consequently does not recognize TD Rev. MAb 1G7 (IgG2b) binds to an epitope in the C-terminal region of Rev that is deleted in the Rev mutant
12/14. The anti-Rev MAb 8E7 was also used to detect the Tev/Tnv protein. For detection of gp160/120 (Env) the anti-gp120 MAb ADP327 (IgG1) from C. Thiriart and C. Buck and supplied by H. C. Holmes, Medical Research Council, London, UK, was used (Thiriart et al., 1989
). For Western blot analysis the anti-gp120 MAb T7 kindly provided by L.
kerblom, was also used (
kerblom et al., 1990
).
Western blot analysis.
COS cells in 35 mm wells were collected in 150 µl of lysis buffer 48 h after transfection (Szilvay et al., 1995 ). The samples were separated by 7·5% or 15% SDSPAGE for detection of gp160/120 or Rev respectively. The separated proteins electrophoretically transferred to nitrocellulose (0·22 µm) were subjected to immunodetection by the appropriate antibodies as previously described using the ECL detection system (Amersham) (Szilvay et al., 1995
). Pre-stained molecular mass standards (Bio-Rad) and recombinant Rev protein (Intracel) were used as standards. The films were scanned using a Microtek Scanmaker IIX flatbed scanner and the figures were created using the program Adobe Photoshop version 3.0.
Immunofluorescence.
For the immunofluorescence assay cells were seeded onto 12 mm coverslips in 24-well plates 24 h after transfection. The next day the coverslips were washed with PBS and fixed in 4% formaldehyde followed by permeabilization in ice-cold methanol. The coverslips were stored in methanol at -20 °C. The assays were performed as previously described (Szilvay et al., 1995 ). For double labelling, isotype-specific secondary antibodies conjugated with FITC or Texas Red (Southern Biotechnology) were used. The samples were viewed at a magnification of 400x using a Nikon Microphot-SA with epifluorescence. Microscopic images were captured by a colour camera (Hamamatsu 3 chips cooled CCD camera with a Hamamatsu C5810 controller unit) and transferred by a frame grabber to a Macintosh Power PC where the images were stored digitally. The figures were created using Adobe Photoshop version 3.0.
Detection of p24 in the culture medium of COS cells transfected with pSVc21.
Cell culture medium from COS cells transfected with the proviral clone pSVc21 was collected 40 h after transfection. After addition of Triton X-100, the samples were stored at -20 °C. The p24 ELISA test was performed as previously described (Sundquist et al., 1989 ).
In situ hybridization.
Digoxigenin-labelled RNA probes were generated using an in vitro transcription kit (Boehringer Mannheim). The intron-probe was transcribed from a plasmid containing a 2006 bp fragment of the HIV-1 genome (nt 61278133 in GenBank locus HIVHXB2CG) using SP6 RNA polymerase. Cells were transfected and fixed as for the immunofluorescence assay. The hybridization protocol was performed as described (Bøe et al., 1998 ). After hybridization the RNA probe was labelled using a rhodamine-conjugated anti-digoxigenin Fab fragment. Simultaneous detection of Rev or gp160/120 was performed using the anti-Rev MAb 8E7 or the anti-gp120 MAb ADP327, respectively, as primary antibodies, biotin conjugated anti-mouse antibodies as secondary antibodies and then FITC-conjugated streptavidin (Pierce). The cells were examined as described using a confocal laser scanning microscope (Bøe et al., 1998
) Pseudo-colouring of images was performed using a Macintosh computer and Adobe Photoshop version 3.0.
RNase protection assay.
COS cells were seeded in 10 cm tissue culture plates 1 day prior to transfection. Transfections were then carried out using 50 µl lipofectamine with 2·5 µg pDM128, 5 µg pcrev and 10 µg TD rev. Total cellular RNA was isolated as described by Chomczynski & Sacchi (1987) 48 h after transfection. RNase protection was performed using the RPA III kit from Ambion. Probes were generated by in vitro transcription using T3 RNA polymerase in the presence of [
-32P]UTP. As template for the in vitro transcription a plasmid provided by Y. Huang and C. C. Carmichael was used (Huang & Carmichael, 1997
). Labelled probes were hybridized to target RNA in the hybridization buffer supplied in the kit at 43 °C for 4 h. After digestion with RNase A and T1 the protected fragments were resolved on a 5% acrylamide8 M urea gel and visualized by autoradiography. The 415 nt probe protected 185 nt of spliced mRNA and 397 nt of unspliced mRNA (Huang & Carmichael, 1997
) The films were scanned using an Agfa Snap Scan 600 flatbed scanner using the program Color-It version 3.0 for Macintosh Power PC. Adobe Photoshop version 3.0 was used when designing the figure. Protected bands were quantified using a Fuji FLA-2000 PhosphoImager. The background was subtracted using regions of identical size located immediately below or above the bands to be quantified. 32P-labelled restriction fragments (
X174/HinfI) was used as a molecular size marker (Promega).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hence, 1·0 µg of the rev plasmids pcrev or TD rev was cotransfected with decreasing concentrations of pcDNA1E7. To the samples which received no rev plasmid, 1 µg of the control plasmid pGL2 was added.
Addition of 1·0 µg pcrev did not significantly change the levels of gp160/120 compared to those expressed from pcDNA1E7 with pGL2 (Fig. 1 A; compare lanes 13 with lanes 46).
When TD rev was added, a marked decrease of gp160/120 production was observed (Fig. 1 A
; compare lanes 13 with lanes 79).
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When the proviral construct pSVc21 was used in cotransfection experiments with TDrev, not only the amount of Rev protein increased. Higher levels of the Tev/Tnv protein translated from an alternatively spliced HIV mRNA was observed also (D'Agostino et al., 1992 ; Salfeld et al., 1990
). An interpretation of these observations may be that both the host cell splicing machinery and the nuclear export of intron-containing HIV mRNAs are targets for normal Rev activity. This assumption was confirmed using an RNase protection assay. It was shown that the relative amount of spliced versus unspliced viral mRNA was shifted towards spliced mRNA when Rev was inhibited by co-expressed TD Rev. Analysis of single cells cotransfected with pcDNA1E7 and TD rev using in situ hybridization demonstrated both nuclear confinement and a decrease of unspliced mRNA (Figs 4
and 5
). Neither HIV RNA nor Rev accumulated at any peripheral site of the nucleus. Instead, intron-containing mRNA and Rev were distributed throughout the nucleoplasm in cells containing detectable amounts of unspliced mRNA. These findings suggest that the trans-dominant negative effect is executed in the nucleoplasm and not at the nuclear membrane. There are several feasible means by which Rev may increase the amount of unspliced viral mRNAs. The effect can be achieved indirectly by removing the viral pre-mRNA at the sites of transcription before splicing occurs. However, since not only unspliced HIV pre-mRNA, but also singly spliced HIV mRNAs, are subject to Rev regulation, it is obvious that Rev can act on an mRNA that has already been committed to a splicing pathway. This indicates an association of Rev with the splicing machinery. Furthermore, double labelling utilizing in situ hybridization and indirect immunofluorescence has shown that the HIV pre-mRNA and Rev preferentially localize to the periphery of nuclear speckles in regions of the weak SC-35 staining (Bøe et al., 1998
). These areas correspond to sites of active transcription and splicing (Wansink et al., 1993
; Misteli et al., 1997
). Oligomerization is essential for Rev function and complexes consisting of wild-type Rev and TD Rev have been demonstrated in the cytoplasm, during nuclear import and in the nucleus (Hope et al., 1992
; Szilvay et al., 1997
; Stauber et al., 1998
). The nuclear retention of wild-type Rev by TD Rev was suggested to be caused by generation of export-deficient mixed multimers (Stauber et al., 1995
; Szilvay et al., 1995
; Wolff et al., 1995
). Support for this assumption is that the nuclear export of the human T-lymphotropic virus type I protein Rex, which does not interact with Rev (Bogerd & Greene, 1993
), is not affected by a trans-dominant negative Rev mutant (Stauber et al., 1995
). However, inhibition of Rex function by a trans-dominant negative Rev mutant in an HIV-based system has been demonstrated (Solomin et al., 1990
). These apparently opposing results concerning the Rex protein indicate that at least two different inhibitory mechanisms are mediated by TD Rev. One of these could be caused by competition by TD Rev for binding to the target RNA leading to nuclear confinement of the RRE RNA. Direct competition for the cellular cofactors CRM1 (exportin 1), eIF5a or hRip (Rab) seems unlikely since they have been shown to interact with the activation domain or NES, which is deleted in TD Rev (Ruhl et al., 1993
; Bogerd et al., 1995
; Ossareth-Nazari et al., 1997
). However, the proposed formation of mixed multimers may cause imperfect binding to any of these factors or with other unknown cellular cofactors. Such defective interactions may take place at any of the steps along the shuttling pathway of Rev. One consequence might therefore be that Rev simply does not reach the nuclear targets for Rev regulation. Whatever the underlying mechanism of inhibition, the effect of trans-dominant inhibition is an increase of viral pre-mRNA splicing leading to higher levels of protein products from spliced and alternatively spliced mRNAs. The results therefore provide new evidence to support the assumption that Rev regulates the splicing of viral pre-mRNA besides facilitating the export of singly spliced and unspliced viral mRNAs. The recent publication by Huang et al. (1999)
confirms this. It was shown that RNA transport elements from intronless cellular and viral mRNAs act as polyadenylation enhancers and inhibitors of splicing. These elements were further demonstrated to functionally replace Rev and RRE in a Rev dependent expression system (Huang et al., 1999
). These findings support the suggestion that Rev regulates more than the nuclear export step of viral mRNAs.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arrigo, S. J. & Chen, I. S. Y. (1991). Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr and env/vpu 2 RNAs. Genes & Development 5, 808819.[Abstract]
Benko, D. M., Schwartz, S., Pavlakis, G. N. & Felber, B. K. (1990). A novel human immunodeficiency virus type 1 protein, Tev, shares sequences with Tat, Env, and Rev proteins. Journal of Virology 64, 2505-2518.[Medline]
Bøe, S. O., Bjørndal, B., Røsok, B., Szilvay, A. M. & Kalland, K.-H. (1998). Sub-cellular localization of human immunodeficiency virus type 1 RNAs, Rev and the splicing factor SC-35. Virology 244, 473-482.[Medline]
Bogerd, H. & Greene, W. C. (1993). Dominant negative mutants of human T-cell leukemia virus type 1 Rex and human immunodeficiency virus type 1 Rev fail to multimerize in vivo. Journal of Virology 67, 2496-2502.[Abstract]
Bogerd, H. P., Fridell, R. A., Madore, S. & Cullen, B. R. (1995). Identification of a novel cellular cofactor for the Rev/Rex class of retroviral regulatory proteins. Cell 82, 485-494.[Medline]
Böhnlein, E., Berger, J. & Hauber, J. (1991). Functional mapping of the human immunodeficiency virus type 1 Rev RNA binding domain: new insights into the domain structure of Rev and Rex. Journal of Virology 65, 7051-7055.[Medline]
Chang, D. D. & Sharp, P. A. (1989). Regulation by HIV Rev depends upon recognition of splice sites. Cell 59, 789-795.[Medline]
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanatephenolchloroform extraction. Analytical Biochemistry 162, 156-159.[Medline]
Cochrane, A. W., Perkins, A. & Rosen, C. A. (1990). Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function. Journal of Virology 64, 881-885.[Medline]
Cochrane, A. W., Jones, K. S., Beidas, S., Dillon, P. J., Skalka, A. M. & Rosen, C. A. (1991). Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. Journal of Virology 62, 5305-5313.
Cullen, B. R. (1992). Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiological Reviews 56, 375-394.[Abstract]
Cullen, B. R., Hauber, J., Campbell, K., Sodroski, J. G., Haseltine, W. A. & Rosen, C. A. (1988). Subcellular localization of the human immunodeficiency virus trans-acting art gene product. Journal of Virology 62, 2498-2501.[Medline]
D'Agostino, D. M., Felber, B. K., Harrison, J. E. & Pavlakis, G. N. (1992). The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. Molecular and Cellular Biology 12, 1375-1386.[Abstract]
Dayton, A. I., Terwilliger, E. F., Potz, J., Kowalski, M., Sodroski, J. G. & Haseltine, W. A. (1988). Cis-acting sequences responsive to the Rev gene product of the human immunodeficiency virus. Journal of AIDS 1, 441-452.[Medline]
Dayton, E. T., Douglas, M., Powell, D. & Dayton, A. (1989). Functional analysis of CAR, the target sequence for the Rev protein of HIV-1. Science 246, 1625-1629.[Medline]
Emerman, M., Vazeux, R. & Peden, K. (1989). The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell 57, 1155-1165.[Medline]
Favaro, J. P. & Arrigo, S. J. (1997). Characterization of Rev function using subgenomic and genomic constructs in T and COS cells. Virology 228, 29-38.[Medline]
Felber, B. K., Hadzopoulou-Cladaras, M., Cladaras, C., Copeland, T. & Pavlakis, G. N. (1989). Rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proceedings of the National Academy of Sciences, USA 86, 1495-1499.[Abstract]
Fischer, U., Meyer, S., Teufel, M., Heckel, C., Lührmann, R. & Rautmann, G. (1994). Evidence that HIV-1 Rev directly promotes the nuclear export of unspliced RNA. EMBO Journal 13, 4105-4112.[Abstract]
Fischer, U., Huber, J., Bolens, W. C., Mattaj, I. W. & Luhrmann, R. (1995). The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475-483.[Medline]
Green, M. R. (1993). Molecular mechanisms of Tat and Rev. AIDS Research Reviews 3, 41-55.
Hammarskjöld, M. L., Li, H., Rekosh, D. & Prasad, S. (1994). Human immunodeficiency virus env expression becomes Rev-independent if the env region is not defined as an intron. Journal of Virology 68, 951-958.[Abstract]
Haseltine, W. A. (1991). Regulation of HIV-1 replication. In Genetic Structure and Regulation of HIV-1, pp. 1-42. Edited by W. A. Haseltine & F. Wong-Staal. New York: Raven Press.
Hope, T. J., Huang, X., McDonald, D. & Parslow, T. G. (1990). Steroid-receptor fusion of the human immunodeficiency virus type 1 Rev transactivator: mapping cryptic functions of the arginine-rich motif. Proceedings of the National Academy of Sciences, USA 87, 7787-7791.[Abstract]
Hope, T. J., Klein, N. P., Elder, M. E. & Parslow, T. G. (1992). Trans-dominant inhibition of human immunodeficiency virus type 1 Rev occurs through formation of inactive protein complexes. Journal of Virology 66, 1849-1855.[Abstract]
Huang, Y. & Carmichael, G. C. (1997). The mouse histone H2a gene contains a small element that facilitates cytoplasmic accumulation of intronless gene transcripts and of unspliced HIV-1 related mRNAs. Proceedings of the National Academy of Sciences, USA 94, 10104-10109.
Huang, Y., Wimler, K. M. & Carmichael, G. C. (1999). Intronless mRNA transport elements may affect multiple steps of pre-mRNA processing. EMBO Journal 18, 1642-1652.
Kalland, K. H., Langhoff, E., Bos, H. J., Göttlinger, H. & Haseltine, W. A. (1991). Rex-dependent nucleolar accumulation of HTLV-I mRNA. New Biologist 3, 389-397.[Medline]
Kalland, K. H., Szilvay, A. M., Brokstad, K. A., Sætrevik, W. & Haukenes, G. (1994a). The human immunodeficiency virus type 1 (HIV-1) Rev protein shuttles between the cytoplasm and nuclear compartments. Molecular and Cellular Biology 14, 7436-7444.[Abstract]
Kalland, K. H., Szilvay, A. M., Langhoff, E. & Haukenes, G. (1994b). Subcellular distribution of human immunodeficiency virus type 1 Rev and colocalization of Rev with RNA splicing factors in a speckled pattern in the nucleoplasm. Journal of Virology 68, 1475-1485.[Abstract]
Kjems, J. & Sharp, P. A. (1993). The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. Journal of Virology 67, 4769-4776.[Abstract]
Kjems, J., Frankel, A. D. & Sharp, P. A. (1991a). Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell 67, 169-178.[Medline]
Kjems, J., Brown, M., Chang, D. D. & Sharp, P. A. (1991b). Structural analysis of the interaction between the human immunodeficiency virus Rev protein and the Rev responsive element. Proceedings of the National Academy of Sciences, USA 88, 683-687.[Abstract]
Kjems, J., Calnan, B. J., Frankel, A. D. & Sharp, P. A. (1992). Specific binding of a basic peptide from HIV-1 Rev. EMBO Journal 11, 1119-1129.[Abstract]
Lu, X., Heimer, J., Rekosh, D. & Hammarskjöld, M. L. (1990). U1 small nuclear RNA plays a direct role in the formation of rev-regulated human immunodeficiency virus env mRNA that remains unspliced. Proceedings of the National Academy of Sciences, USA 87, 7598-7602.[Abstract]
Madore, S. J., Tiley, L. S., Malim, M. M. & Cullen, B. R. (1994). Sequence requirements for Rev multimerization in vivo. Virology 202, 186-194.[Medline]
Maldarelli, F., Martin, M. A. & Strebel, K. (1991). Identification of posttranscriptionally active inhibitory sequences in human immunodeficiency virus type 1 RNA: novel level of gene regulation. Journal of Virology 65, 5732-5743.[Medline]
Malim, M. H. & Cullen, B. R. (1991). HIV-1 structural gene expression requires the binding of multiple Rev monomers to the viral RRE: implications for HIV-1 latency. Cell 65, 241-248.[Medline]
Malim, M. H. & Cullen, B. R. (1993). Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Molecular and Cellular Biology 13, 6180-6189.[Abstract]
Malim, M. H., Hauber, J., Le, S. Y., Maizel, J. V. & Cullen, B. R. (1989a). The HIV-1 rev transactivator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature 338, 254-257.[Medline]
Malim, M. H., Böhnlein, S., Hauber, J. & Cullen, B. R. (1989b). Functional dissection of the HIV-1 Rev transactivator derivation of a trans-dominant repressor of Rev function. Cell 58, 205-214.[Medline]
Malim, M. H., Tiley, L. S., McCarn, D. F., Rusche, J. R., Hauber, J. & Cullen, B. R. (1990). HIV-1 structural gene expression requires binding of the rev trans-activator to its RNA target sequence. Cell 60, 675-683.[Medline]
Malim, M. H., McCarn, D. F., Tiley, L. S. & Cullen, B. R. (1991). Mutational definition of the human immunodeficiency virus type 1 rev activation domain. Journal of Virology 65, 4248-4254.[Medline]
Mermer, B., Felber, B. K., Campbell, M. & Pavlakis, G. N. (1990). Identification of trans-dominant HIV-1 Rev protein mutants by direct transfer of bacterially produced proteins into human cells. Nucleic Acids Research 18, 2037-2044.[Abstract]
Meyer, B. E. & Malim, M. H. (1994). The HIV-1 Rev trans-activator shuttles between the nucleus and the cytoplasm. Genes & Development 8, 1538-1547.[Abstract]
Misteli, T., Caceres, J. F. & Spector, D. L. (1997). The dynamics of a pre-mRNA splicing factor in living cells. Nature 387, 523-527.[Medline]
Nosaka, T., Takamatsu, T., Miyazaki, Y., Sano, K., Sato, A., Kubota, S., Sakurai, M., Ariumi, Y., Nakai, M., Fujita, S. & Hatanaka, M. (1993). Cytotoxic activity of Rev protein of human immunodeficiency virus type 1 by nucleolar dysfunction. Experimental Cell Research 209, 89-102.[Medline]
Olsen, H. S., Cochrane, A. W., Dillon, P. J., Nalin, C. M. & Rosen, C. A. (1990). Interaction of the human immunodeficiency virus type 1 Rev protein with a structured region in env mRNA is dependent on multimer formation mediated through a basic stretch of amino acids. Genes & Development 4, 1357-1364.[Abstract]
Ossareth-Nazari, B., Bachelerie, F. & Dargemont, C. (1997). Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science 278, 141-144.
Richard, N., Iacampo, S. & Cochrane, A. (1994). HIV-1 Rev is capable of shuttling between the nucleus and cytoplasm. Virology 204, 123-131.[Medline]
Rosen, C. A., Terwilliger, E., Dayton, A., Sodroski, J. & Haseltine, W. A. (1988). Intragenic cis-acting art-gene-responsive sequences of the human immunodeficiency virus. Proceedings of the National Academy of Sciences, USA 85, 2071-2075.[Abstract]
Ruhl, M., Himmelspach, M., Bahr, G. M., Hammerschmid, F., Jaksche, H., Wolff, B., Aschauer, H., Farrington, G. K., Probst, H., Bevec, D. & Hauber, J. (1993). Eukaryotic initiation factor 5A is a cellular target of the human immunodeficiency virus type 1 Rev activation domain mediating trans-activation. Journal of Cell Biology 123, 1309-1320.[Abstract]
Salfeld, J., Gottlinger, H. G., Sia, R., Park, R., Sodroski, J. G. & Haseltine, W. A. (1990). A tripartite HIV-1 TatEnvRev fusion protein. EMBO Journal 9, 965-970.[Abstract]
Schwartz, S., Felber, B. K. & Pavlakis, G. N. (1992). Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. Journal of Virology 66, 150-159.[Abstract]
Sodroski, J., Goh, W. C., Rosen, C. A., Dayton, A., Terwilliger, E. & Haseltine, W. A. (1986). A second post-transcriptional activator gene required for HTLV-III replication. Nature 321, 412-417.[Medline]
Solomin, L., Felber, B. K. & Pavlakis, G. N. (1990). Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1. Journal of Virology 64, 6010-6017.[Medline]
Stauber, R., Gaitanaris, G. A. & Pavlakis, G. N. (1995). Analysis of trafficking and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm. Virology 213, 439-449.[Medline]
Stauber, R., Alfonina, E., Gulnik, S., Erickson, J. & Pavlakis, G. N. (1998). Analysis of intracellular trafficking and interactions of cytoplasmic HIV-1 Rev mutants in living cells. Virology 251, 38-48.[Medline]
Stutz, F. & Rosbash, M. (1994). A functional interaction between Rev and yeast pre-mRNA is related to splicing complex formation. EMBO Journal 13, 4096-4104.[Abstract]
Sundquist, V. A., Albert, J., Ohlson, E., Hinkula, J., Fenyø, E. M. & Wahren, B. (1989). Human immunodeficiency virus type 1 p24 production and antigenic variation in tissue culture of isolates with various growth characteristics. Journal of Medical Virology 29, 170-175.[Medline]
Szilvay, A. M., Brokstad, K. A., Kopperud, R., Haukenes, G. & Kalland, K. H. (1995). Nuclear export of the nucleocytoplasmatic shuttle protein HIV-1 Rev is mediated by its activation domain and is blocked by transdominant negative mutants. Journal of Virology 69, 3315-3323.[Abstract]
Szilvay, A. M., Brokstad, K. A., Bøe, S. O., Haukenes, G. & Kalland, K. H. (1997). Oligomerization of HIV-1 Rev mutants in the cytoplasm and during nuclear import. Virology 235, 73-81.[Medline]
Thiriart, C., Francotte, M., Cohen, J., Collignon, C., Delers, A., Kummert, S., Molitor, C., Gilles, D., Roelants, P., van Wijnendaele, F. and others (1989). Several antigenic determinants exposed on the gp120 moiety of HIV-1 gp160 are hidden on the mature gp120. Journal of Immunology 143, 18321836.
Venkatesh, L. K. & Chinnadurai, G. (1990). Mutants in a conserved region near the carboxy-terminus of HIV-1 Rev identify functionally important residues and exhibit a dominant negative phenotype. Virology 178, 327-330.[Medline]
Wansink, D. G., Schul, W., van der Kraan, I., van Steensel, B., van Driel, R. & de Jong, L. (1993). Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. Journal of Cell Biology 122, 283-293.[Abstract]
Weichselbraun, I., Farrington, G. K., Rusche, J. R., Böhnlein, E. & Hauber, J. (1992). Definition of the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex protein activation domain by functional exchange. Journal of Virology 66, 2583-2587.[Abstract]
Wolff, B., Cohen, G., Hauber, J., Meshceryakova, D. & Rabeck, C. (1995). Nucelocytoplasmic transport of the Rev protein of human immunodeficiency virus type 1 is dependent on the activation domain of the protein. Experimental Cell Research 217, 31-41.[Medline]
Zapp, M. L., Hope, T. J., Parslow, T. G. & Green, M. R. (1991). Oligomerization and RNA binding domains of the type 1 human immunodeficiency virus Rev protein: a dual function for an arginine-rich binding motif. Proceedings of the National Academy of Sciences, USA 88, 7734-7738.[Abstract]
Received 25 February 1999;
accepted 16 April 1999.