Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1

Molecular and Cellular Probes (1990) 4, 6 3-72

Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1 Reinhard Voll,* Christian Aepinus,*$ Friedel Krapf,t Martin Herrmannt, Joachim R. Kaldent and Bernhard Fleckenstein* *Institut für Klinische und Molekulare Virologie and tinstitut für Klinische Immunologie und Rheumàtologie, Universität Erlangen-Nürnberg, Loschgestraße 7, D-8520 Erlangen, Federal Republic of Germany

(Received 20 September 1989, Accepted 22 September 1989) The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm . The rev protein was expressed in Escherichia coli as a (3galactosidase fusion protein with a cleavage site for proteinase factor X .. The rev-specific fragment was isolated to immunize mice . Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA . Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid .

KEYWORDS : monoclonal antibodies, human immunodeficiency virus, rev-protein, (3-galactosidase fusion protein, proteinase factor X . .

INTRODUCTION HIV-1 gene expression is regulated by viral and cellular factors . 1 ' z The rev (art/trs) protein is one of the important viral regulatory proteins . This polypeptide, encoded by a double spliced message,', ' is a phosphoprotein of approximately 20 kDa 5 that has been shown to be essential for viral structural protein expression .' There is now evidence that rev functions by supporting transport to cytoplasm of single-spliced and unspliced (i .e . genomic) HIV-1 transcripts . 4-' ° Furthermore, a role in mRNA stabilization and translation of these messages has been demonstrated ."" A cis-acting element that is essential and sufficient to mediate the rev effect has been mapped first in the 3'-half of the env mRNA . 11,12 More precisely, it corresponds to a Styl-Sau3A fragment of 210 nt coding for the N-terminus of the transmem$ Author to whom correspondence should be sent .

0890-8508/90/010063 + 10 $03 .00/0

63

© 1990 Academic Press Limited



R. Voll et al .

64

brane glycoprotein gp 41 10 ; the target has been termed cis-acting art/rev responsive sequence (CAR) 72 or rev responsive element (RRE) . 11 The rev effects are independent of splice donor/acceptor sites' and intron or exon localization of the CAR/RRE .10 To be active, the cis-acting element needs to be in sense orientation of the message . 10 The rev protein accumulates in the nucleus, particularly in nucleoli .9,13 Perkins et 14 al . recently characterized the nuclear targeting sequence of rev . However, a certain limitation of these experiments lies in the fact that the localization of rev was only achieved in cells transfected by eukaryotic expression cassettes, but not in HIV-1 infected T-cell lines . For a more detailed study of the intranuclear localization and possible interaction with RNA, we intended to use monoclonal antibodies . This paper describes prokaryotic expression of the rev polypeptide, raising of monoclonal antibodies, and their application in localization of rev .

MATERIALS AND METHODS Cloning procedures and purification of rev peptide A Hinfl-fragment was isolated from cDNA clone pCV-1 3 that was kindly provided by Flossie Wong-Staal . The recessed ends were filled in with Klenow fragment and cloned in the inducible prokaryotic expression vector pROS 15 (Fig . 1) . The resulting 0

200

600

400

800

1000

1200 1400

1600 1800

bp

I I 1 I I I I I I

nef

tat rev 287

5358

5625 7956

\ \~ Sac I Sol I

FXa-Cleavage Region

Hint

Bo

mH I

Hinf I

Xho I

pCV-1

Kpn I

Sac I

Multiple Cloning Site

pROS

3842 by

Fig. 1 . Construction of prokaryotic rev expression vector pRVA. A Hinfl fragment of 266 by coding for the amino acids 18 to 106 of the rev protein was isolated from cDNA clone pCV-1 3 The fragment was ligated in frame in a single Stul site of expression vector pROS tS



Monoclonal antibodies against HIV-1-rev

65

expression plasmid was sequenced to ensure correct ligation . The (3-galactosidase fusion protein is composed of the 375 N-terminal amino acids of Escherichia coli (3galactosidase, a factor X a (FX a ) cleavage site, followed by a rev-specific sequence of 89 amino acids . The cleavage product has 17 unrelated amino acids attached to the N-terminus of rev polypeptide . Cloning procedures followed Maniatis et al ., 16 sequencing was performed by dideoxy chain termination 17 using Sequenase' kit (United States Biochemicals, Cleveland) . Fusion protein was purified from transformed E. coli BMH 71-18 18 as published by Nagai et al." In order to remove remaining bacterial proteins, the fusion protein was isolated from a preparative SDS-polyacrylamide gel by electro-elution ." Cleavage with proteinase FX a, a gift from Sylvia Ellinger, was performed as described by Ellinger et al." The pure rev peptide was prepared from stained SIDS-polyacrylamide gel bands as reported by 21 Weintraub & Raymond For eukaryotic expression of rev polypeptide, the human cytomegalovirus enhancer/promoter22 was placed in front of the rev reading frame that was taken from the plasmid pH3-art, 12 kindly provided by William Haseltine. The resulting plasmid was termed pCMV-REV .

Immunization procedure BALB/c mice pretreated with pristane were immunized intraperitoneally at weekly intervals with 20 lag rev peptide using 200 lag poly(A) •p oly(U) as an adjuvant. 23 After seven inoculations, a last boost was given intravenously with about 50 gg antigen 3 days prior to removal of the spleens .

Procedures for production of monoclonal antibodies Splenocytes were prepared and fused with SP2 /O, derived from X63-Ag8 X BALB/c, 24 using polyethylene glycol 1500 (Boehringer, Mannheim) as fusogenic agent . 25 Hybridomas were selected in DMEM supplemented with HAT (100 µnn hypoxanthin, 0. 4 gm aminopterin and 16 gm thymidine) . Culture wells- showing cell growth were tested for rev-specific antibody production by ELISA . Hybridomas producing reactive antibodies were repeatedly recloned by limiting dilution . Immunoglobulin isotypes were determined by direct immunofluorescence using an antiserum kit (Southern Biotechnology Associates) .

ELISA Enriched fusion protein was dissolved in phosphate buffered saline (PBS) at a concentration of 40 gg ml - ' . Microtitre plates were coated with 50 gl well - ' and subsequently blocked with 1 % bovine serum albumine in PBS . Protein obtained from bacteria transformed with the expression vector pROS served as controls . Supernatants from hybridomas were incubated for 3 hours at room temperature . A goat anti-mouse serum conjugated with alkaline phosphatase (Medac, Hamburg) was used as second antibody ."



66

R. Voll et al.

Western blot assay To confirm rev-specifity, Western blots were performed with FXa cleaved protein and with uncleaved fusion protein. The proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred from polyacrylamide gels onto nitrocellulose (NC) sheets as reported by Towbin et al ." The NC-stripes were blocked in PBS containing 1 % bovine serum albumin and incubated with hybridoma supernatants (diluted 1 :20 in blocking buffer) . Blots were stained with rabbit anti-mouse immunoglobulin that was conjugated with horseradish peroxidase . Diaminobenzidine was used as the substrate .

Immunofluorescence Hela cells were transfected with the eukaryotic rev-expression plasmid pCMV-REV and a control plasmid by the calcium phosphate procedure . 28 Cells were fixed 48 hours post-transfection and were incubated with antibodies as published by Ruben et al . 29 Fluorescein isothiocyanate-conjugated rabbit anti-mouse F(ab) 2-fragment (Dakopatts, Denmark) was used for staining .

RESULTS Prokaryotic expression of rev protein Large amounts of the expected rev/ß-galactosidase fusion protein of about 63 kDa were obtained without appreciable degradation . The fusion protein was enriched to about 80 percent of total protein . The cleavage of the fusion protein by FX a provided sufficient amounts of intact rev peptide (Fig . 2). The resulting protein should consist of 106 amino acids including a rev-specific sequence of 89 residues . The N-terminus has 17 amino acids attached that are coded by the vector pROS between FX a cleavage region and the Stul site (NH 2 -Lys-Gly-Gly-Gly-Thr-Ser-Asp-Pro-Ser-Thr-ArgAsp-Ile-Gly-ALa-Ser-Gly) . This rev peptide- migrated in SDS-PAGE like a peptide of about 16 kDa, 4 kDa higher than the calculated molecular mass . This is probably due to unusual amino acids composition . 30 Mice were immunized with rev peptide of more than 95% purity .

Characteristics of the monoclonal antibodies against the rev polypeptide Three hybridoma cell lines (rev111, rev348, rev529) were established producing revspecific monoclonal antibodies of IgG-1 subclass . Two additional stable hybridomas (rev159, rev398) secreted antibodies of IgM class . All monoclonal antibodies recognize the rev peptide from prokaryotic expression in ELISA (data not shown) and Western blot analysis (Fig . 3).



Monoclonal antibodies against HIV-1-rev

67

123M

f p

a•-

ß-gal

-

66 K

- 45 K - 36 K

- 29 K - 24 K

- 20 K

rev -- 14 K Fig. 2. Prokaryotic expression, purification, and cleavage of fusion protein with a factor X a recognition sequence between viral (rev) and prokaryotic (0-gal) part. Proteins were separated by SDS-PAGE (12 .5% acrylamide gel) and stained with Coomassie Brilliant Blue . Lane 1, fusion protein (fp) enriched of bacterial lysat according to the protocol of Nagai et ai .; 19 lane 2, fusion protein electro-eluted from a band of preparative polyacrylamide gel ; lane 3, same protein as in lane 2, cleaved with factor X,.

Subcellular localization of rev by indirect immunofluorescence The plasmid pCMV-REV was transfected into Hela cells for transient expression . The rev polypeptides were visualized by indirect immunofluorescence with the three IgG-1 monoclonal antibodies (Fig . 4) . There was a brilliant accentuated nucleolar staining . The specifity of nucleolar localization was confirmed by phase contrast microscopy . In addition, a specific relatively weak hazy fluorescence through the nuclei became apparent . Some cells showed a densely speckled granular staining



68

R. Voll et ai .

M

2

3

4

5

kd 66 45 36 29 24 -

20 -

14 -

Fig. 3. Western blot analysis of monoclonal antibodies against rev peptide . Cleaved fusion protein was separated by SDS-PAGE and electrophoretically transferred onto nitrocellulose (NC) filters . Lane M, amido black stained sheet showing molecular size standards and, on the right side, 13-galactosidase (about 45 kDa) (44 ) and rev-peptide (about 16 kDa) (4-) . Western blot stripes were incubated with supernatants of hybridoma clones . Lanes : 1, revlll (IgG-1); 2, rev348 (IgG-1); 3, rev529 (IgG-1); 4, rev159 (IgM); 5, rev398- (LgM).

pattern over the entire nucleus . Only some of the fluorescence could be related to the nucleoli in phase contrast mode .

DISCUSSION This study demonstrates that a peptide expressed in E. coli as a cleavable fusion protein and purified by polyacrylamide gel electrophoresis is a useful immunogen for the production of monoclonal antibodies . It recognizes the eukaryotic protein by indirect immunofluorescence in the nuclei of cells that were transiently expressing rev. It has been reported previously that the rev protein is predominantly localized in the nucleus . 13 Recently, more specific experiments using monospecific anti-peptide sera suggested particular nucleolar localization ."' Our fluorescence data with monoclonal antibodies are consistent with these results . However, it appears that the speckled immunofluorescence is not restricted to the nucleoli . All these experiments have been performed using transfected cultures . Human T-cell leukemia virus type I (HTLV-I), a pathogenic human retrovirus,

Monoclonal antibodies against HIV-1-rev

Fig . 4 . Indirect immunofluorescence of Hela cells expressing rev protein . Hela cells were transfected with plasmid pCMV-REV and fixed 48 hours post transfection . Staining was performed after incubation with monoclonal rev348 using FITC-conjugated rabbit anti-mouse F(ab) Z fragment. Matched fluorescent and phase contrast photomicrographs are shown . Arrows point to the cells that show rev-specific nuclear staining by immunofluorescence .

69



70

R. Voll et al .

synthesizes a 27 kDa phosphoprotein (pp27reX) that is required for the formation of single-spliced and unspliced mRNA . 31,32 Thus, the HTLV-1 rex protein appears to be the functional equivalent of rev in HIV-1 . Moreover, pp27rex can functionally replace the HIV-1 rev gene product . 33 However, the RNA region essential for the pp27feX effects seems to be different from the CAR sequence (W . A . Haseltine, personal communication) . The functional substitution between HIV-1 rev and pp27 1e" is remarkable, since appreciable homologies in primary sequences are lacking . Both types of regulator proteins, HIV-1 rev and pp27feX of HTLV-1, show a preferential localization in the nucleoli . This supports the notion of equivalent functions in virus replication . 34 The pronounced nucleolar staining of rev protein after transient expression does not exclude important functions of rev in the nuclear matrix or in the cytoplasm, if it is present there in lower amounts . The predominant localization of a protein does not need to coincide with the most important point of action . There are some indications of rev acting in mRNA stabilization and increasing translation efficiency of HIV-1 genomic RNA and env-transcripts 9'" Until now, it has been unclear whether the effects of rev on mRNA transport are fully explained by its nucleolar localization . The rev specific monoclonal antibodies described here should help to analyse in more detail the mechanisms of rev effects .

ACKNOWLEDGEMENTS We thank Burkhard Trusen and Martina Geithner for assistance with fusion procedure. This work was supported by Bundesministerium für Forschung und Technologie, Projektträger PBE/12 /DV 0318836 A and AIDS II-100-89 .

REFERENCES 1 . Haseltine, W . A . (1988) . Replication and pathogenesis of the AIDS virus . Journal of Aquired Immune Deficiency Syndromes 1, 217-40 . 2 . Kawakami, K ., Scheidereit, C . & Roeder, R . G. (1988) . Identification and purification of a human immuno-globulin-enhancer-binding protein (NF-kB) that activates transcription from a human immunodeficiency virus type 1 promotor in vitro . Proceedings of the National Academy of Sciences, USA 85, 4700-04 . 3 . Arya, S . K., Guo, C., Josephs, S . F. & Wong-Staal, F . (1985) . Trans-activator gene of human Tlymphotropic virus type III (HTLV-111). Science 229, 69-73 . 4 . Sodroski, J ., Goh, W . C., Rosen, C., Dayton, A ., Terwilliger, E ., & Haseltine, W. A . (1986) . A second post-transcriptional trans-activator gene required for HTLV-III replication . Nature 321, 412-7 . 5 . Hauber, J ., Bouvier, M., Malim, M . H . & Cullen, B . R . (1988). Phosphorylation of the rev gene product of human immunodeficiency virus type 1 . journal of Virology 62, 4801-4. 6 . Feinberg, M. B., Jarrett, R. F ., Aldovini, A ., Gallo, R. C . & Wong-Stäal, F . (1986). HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA . Cell 46, 807-17 . 7 . Knight, D . M., Flomerfelt, F . A . & Ghrayeb, J . (1987) . Expression of the art/trs protein of HIV and study of its role in viral envelope synthesis . Science 236, 837-40. 8 . Hammarskjöld, M.-L ., Heimer, J ., Hammarskjöld, B ., Sangwan, I ., Albert, L . & Rekosh, D . (1989) . Regulation of human immunodeficiency virus env expression by the rev gene product . Journal of Virology 63, 1959-66 . 9 . Felber, B . K ., Hadzopoulou-Cladaras, M., Cladaras, C., Copeland, T . & Pavlakis, G . N. (1989) . rev



Monoclonal antibodies against HIV-1-rev

71

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, 1495-9 . 10 . Malim, M . H ., Hauber, J ., Le, S .-Y ., Maizel, J . V . & Cullen, B . R . (1989) . The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA . Nature 338, 254-7. 11 . Hadzopoulou-Cladaras, M ., Felber, B . K ., Cladaras, C ., Athanassopoulos, A ., Tse, A . & Pavlakis, G . N . (1989) . The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region . Journal of Virology 63, 1265-74 . 12 . Rosen, C. A ., Terwilliger, E., Dayton, A ., Sodroski, J . G. & Haseltine, W . A . (1988). Intragenic cisacting art gene-responsive sequences of the human immunodefiiciency virus . Proceedings of the National Academy of Sciences, USA 85, 2071-5 . 13 . 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, 2498-501 . 14. Perkins, A ., Cochrane, A . W., Ruben, S . M . & Rosen, C . A . (1989). Structural and functional characterization of the human immunodeficiency virus rev protein . Journal of Acquired Immune Deficiency Syndromes 2, 256-63 . 15 . Ellinger, S ., Glockshuber, R., Jahn, G . & Plückthun, A . (1989) . Cleavage of prokaryotically expressed human immunodeficiency virus fusion proteins with factor X a and application in Western blot (immunoblot) assays . Journal of Clinical Microbiology 27, 971-6. 16 . Maniatis, T ., Fritsch, E . F ., & Sambrook J . (1982) . Molecular Cloning : A Laboratory Manual . 1st edn, Cold Spring Harbor: Cold Spring Harbor Laboratory . 17. Sanger, F ., Nicklen, S . & Coulson, A . R . (1977) . DNA sequencing with chain-terminating inhibitors . Proceedings of the National Academy of Sciences, USA 74, 5463-7 . 18. Koenen, M ., Rüther, U . & Müller-Hill, B . (1982) . Immunoenzymatic detection of expressed gene fragments cloned in lac Z gene of E. coli . EMBO Journal 1, 509-12 . 19. Nagai, K. & Thogersen, H . C . (1987). Synthesis and sequence specific proteolysis of hybrid proteins produced in E. coli. Methods in Enzymology 153, 461-81 . 20. Spiker, S . & Isenberg, I . (1983). Preparative polyacrylamide gel electrophoresis . Methods in Enzymology 91, 214-26 . 21 . Weintraub, M . & Raymond, S . (1963) . Antiserums prepared with acrylamide gel used as adjuvant . Science 142, 1677-8 . 22 . Boshart, M ., Weber, F ., Jahn, G ., Dorsch-Häsler, K ., Fleckenstein, B . & Schaffner, W. (1985) . A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus . Cell 41, 521-30 . 23 . Hovanessian, A . G ., Galabru, J ., Riviere, Y . & Montagnier, L . (1988) . Efficiency of poly(A) •poly(U) as an adjuvant . Immunology Today 9, 161-2 . 24. Shulman, M., Wilde, C . D . & Köhler, G . (1978). A better cell line for making hybridomas secreting specific antibodies . Nature 276, 269-70 . 25 . Fazekas de St . Groth, S . & Scheidegger, D . (1980) . Production of monoclonal antibodies : Strategy and tactics . Journal of Immunological Methods 35, 1-25. 26. Al Moudallal, Z., Altschuh, D ., Briand, J. P . & van Regenmortel, M . H . V . (1984). Comparative sensitivity of different ELISA procedures for detecting monoclonal antibodies . Journal of Immunological Methods 68, 35-43 . 27 . Towbin, H ., Staehelin, T . & Gordon, J . (1979) . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets : procedure and some applications . Proceedings of the National Academy of Sciences, USA 76, 4350-4 . 28 . Graham, F. L. & van der Eb, A . J . (1973) . A new technique for the assay of infectivity of human adenovirus 5 DNA . Virology 52, 456-67. 29 . Ruben, S ., Perkins, A ., Purcell, R ., Joung, K ., Sia, R., Burghoff, R ., Haseltine, W . A. & Rosen, C. A. (1989) . Structural and functional characterization of human immunodeficiency virus tat protein . Journal of Virology 63, 1-8 . 30. Goh, W . C ., Sodroski, J . G ., Rosen, C. A ., & Haseltine, W . A . (1987). Expression of the art gene protein of human T-lymphotropic virus type III (HTLV-III/LAV) in bacteria . Journal of Virology 61, 633-7 . 31 . Inoue, J .-I ., Yoshida, M., & Seiki, M . (1987) . Transcriptional (p40') and post-transcriptional (p27'- "')



72

R. Voll et al .

regulators are required for the expression and replication of human T-cell leukemia virus type I genes . Proceedings of the National Academy of Sciences, USA 84, 3653-7. 32 . Hidaka, M., Inoue, J ., Yoshida, M . & Seiki, M . (1988) . Post-transcriptional regulator (rex) of HTLV-1 initiates expression of viral structural proteins but suppresses expression of regulatory proteins . EMBO Journal 7, 519-23 . 33 . Rimsky, L ., Hauber, J ., Dukovich, M., Malim, M . H ., Langlois, A ., Cullen, B . R . & Greene, W . C . (1988) . Functional replacement of the HIV-1 rev protein by the HTLV-1 rex protein . Nature 335, 738-40 . 34. Siomi, H ., Shida, H ., Nam, S . H ., Nosaka, T., Maki, M . & Hatanaka, M. (1988) . Sequence requirements for nucleolar localization of human T cell leucemia virus type I pX protein, which regulates viral RNA processing . Cell 55, 197-220 .