_Archives
Vi rology
Arch Virol (1992) 126:293-301
© Springer-Verlag 1992 Printed in Austria
Expression of the H I V - 1 N e f protein in the baculovirns system: investigation of a n t i - N e f antibodies response in human sera and subcellular localization of N e f Brief R e p o r t
N. Kienzle1, M. Enders1, M. Buck 1, H. Siakkou2, S. Jahn3, G. Petzold4, K. E. Schneweis5, M. Bachmann6, W. E. G. Miiller6 and N. Mfiiler-Lantzsch~ ~Abtcilung Virologic, Institut fiir Medizinische Mikrobiologie und Hygiene, Homburg Institute ffir 2Virologic und fiir 3Immunologie der Charit6, Berlin 4Zentralinstitut fiir Hygiene, Mikrobiologie und Epidemiologie, Berlin 5Institut fiir Medizinische Mikrobiologie und Immunologie, Bonn 6Institut ffir Physiologische Chemie, Mainz, Federal Republic of Germany Accepted February 25, 1992
Summary. The nef gene of HIV-1 was expressed in insect cells using the eucaryotic baculovirus system. The recombinant Nef protein frequently reacted with seropositive sera ofHIV- 1 and HIV-2 infected patients. Anti-Nef antibodies in HIV-1 seronegative high risk groups individuals were only occasionally seen. Confocal laser scanning microscopy demonstrated that Nef is present both in the cytoplasm and in the nucleus, indicating that Nef might directly function on gene expression.
The nef gene is a unique feature of the primate lentiviruses simian and human immunodeficiency virus (SIV and HIV). The HIV-1 nefgene encodes a protein of 206 amino acids (aa) with an apparent molecular mass of 27 kDa. Nef is myristylated and phosphorylated [11] and mainly localized in the cytoplasm [6]. Although Nef was not essential for HIV replication [29], the function of Nef has as yet not been determined. Depending on its genetic background, Nef increased, decreased or could not alter, respectively, the amount and the kinetics of virus replication [1, 5, 12, 15, 28]. The Nef protein elicits humoral immune responses in HIV infected individuals. Using procaryotically expressed Nef proteins or synthetic oligopeptides, Nef specific antibodies were found to be an important marker for the identification of HIV- 1 infection since the immune
294
N. Kienzle et al.
response to Nef was induced before seroconversion against the structural virus proteins E2, 8, 22, 23]. In contrast to these results, other authors reported that the appearance of Nef antibodies before seroconversion is a rare event [-4, 24]. In order to perform further investigations on the presence of Nef specific antibodies in human sera and on the intracellular localization of Nef, we expressed Nef in the eucaryotic Autographa californica nuclear polyhedrosis virus (AcNPV) system. For construction of recombinant baculo-transfervectors, the BamHI fragment of plasmid pUF [14] containing the complete nef gene of HIV1LAV-BRU [30] was cloned into a pAcYM1 non-fusion vector [19] as well as into a pAc360 fusion vector [17]. The two neftransfer vectors encoded different Nef proteins: (0 a non-fused, 206 aa long Nef (AYF3) and (ii) a N-terminal fused Nef protein (D3F3), which additionally carried 16 aa from the linker- and the N-terminal polyhedrin-sequence and 9 aa of the 5' upstream vicinity of the nefsequence. The insect line SF158 was cotransfected with AcNPV DNA and a neftransfer vector as outlined [27]. Recombination between AcNPV sequences present in the transfer vector and viral DNA resulted in nef gene-positive but polyhedrin-negative virions which were identified as described recently [-7, 21]. In indirect immunofluorescence assays performed as described in [7], insect cells producing Nef could brilliantly be stained with anti-Nef monoclonal antibody (mab) (Fig. 1 A). We have developed a series of murine anti-Nef mabs as well as polyclonal rabbit anti-Nef sera E14; Siakkou etal., submitted]. In immunoblot assay done as outlined recently [14], the 32 kDa D3F3 Nef fusion protein and the 26 kDa non-fused AYF3 Nef protein were observed (Fig. 1 B, lanes 2 and 3). The AYF3 protein showed a slightly higher mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis when compared to the 27 kDa Nefmade in HIV-1 infected glioma cells [31] (Fig. 1 B, lane 4) or to the 27 kDa Nef produced in stably transfected B-cells E13] (data not shown). Although other authors described a 27kDa Nef produced by recombinant baculovirus [-18], this difference might be due to altered posttranslational modifications or different nef genes as mentioned recently [20]. Expression of AYF3 yielded an additional Nef protein of 31 kDa (Fig. 1B, lane 2). Since different non-fusion nef baculovirus clones produced the 31 kDa protein, this finding is not due to a single clone event. It might be a result of posttranslational modifications or of a further recombination process during generation of the nef-baculovirus. The cell extracts of AYF3 as well as of D3F3 revealed a lower molecular mass Nef protein (Fig. 1 B). This could be a degradation product, since frequent freezing and thawing of the cell extracts increased this band. The recombinant Nef proteins were produced in large quantities, which in the case of D3F3 even allowed detection by Coomassie blue staining (data not shown). Human sera were studied with respect to antibody responses to Nef derived from baculoviruses. The data are summarized in Table 1. Either the whole protein extract of nef-baculovirus infected cells or the
Nef expressed in insect cells
295
Fig. 1. A Indirect immunofluorescence. Cells producing either the D3F3 Nef protein (1) or wild-type AcNPV (2) were mixed with uninfected cells at a ratio of 1 : 50. The fixed cells were stained with anti-Nef mab# 158. B Immunoanalysis of Nef. 1-4 Total protein extracts from cells, which were infected with wild-type AcNPV (1) or with baculoviruses expressing the non-fusion Nef protein AYF3 (2) or the fusion Nef protein D3F3 (3), were separated on 12.5% SDS-PAGE and immunoblotted using anti-Nef mab # 158. 4 A total protein extract from glioma cells infected with HIV-1 was immunoblotted. 5-10 Cellular extracts from cells producing either the Nef proteins AYF3 (5, 8) and D3F3 (6, 9) or wild-type AcNPV (7, 10) were immunoprecipitated with an anti-Nef positive human serum (5-7) or a HIV-1 negative human serum (8-10). The precipitated samples were analyzed by 12.5% SDS-PAGE and following immunoblot assay with anti-Nef mab # 158. The apparent molecular masses of the marker proteins are indicated in kDa
partially purificated A Y F 3 and D3F3 proteins, p e r f o r m e d as described in [25], were used in i m m u n o b l o t assay to detect anti-Nef antibodies in h u m a n sera. F u r t h e r m o r e , we p e r f o r m e d non-radiactive i m m u n o p r e c i p i t a t i o n assays. The cell lysis using a NP40-buffer and the i m m u n o p r e c i p i t a t i o n with h u m a n sera were p e r f o r m e d as described recently [9]. The precipitated samples were analyzed by i m m u n o b l o t assay. As shown in Fig. 1 B, lanes 5-10, h u m a n sera specifically reacted with the r e c o m b i n a n t N e f proteins. Despite the low degree of a m i n o acid h o m o l o g y between HIV-1 N e f and its c o u n t e r p a r t HIV-2 N e f [10], we could d e m o n s t r a t e that HIV-2 positive h u m a n sera reacted with the HIV-1 N e f p r o t e i n s m a d e in the baculovirus system. The sera reacted with D3F3 and A Y F 3 but also revealed cross-immunoreactions with proteins f r o m the r e c o m b i n a n t as well as wild-type baculovirus infected cells (Fig. 2 A). This is possibly due to the partially purification of the N e f
296
N, Kienzle et al. Table 1. Antibody response to Nef protein of HIV-1 a
Serum sourceb
No. of sera tested
No. of Nef positive sera
% Nef positive sera
HIV-1 + Stage°
68 II: 31; III: 7 IV: 15; U: 15 50 20 11
34 II: 20; III: 4 IV: 4; U: 6 2 0 9
50
HIV-1 ?a HIV-1 HIV-2 +
4 0 82
a The test systems were immunofluorescence, immunoblot or immunoprecipitation of SF158 cells infected with recombinant Nef baculovirus bAll sera were derived from different individuals. The evaluation of infection and seroconversion against HIV proteins was performed by commercial immunoblots or antibody detection kits ~Numbers of the CDC classified clinical stages of disease of the HIV-1 + serum donors; U unknown dThe sera were either seronegative or exhibited an uncertain seroconversion status. All serum donors belonged to high risk groups including hemophiliacs, homosexuals, partners of HIV-1 infected patients and i.v. drug abusers
proteins. However, immunoprecipitations confirmed the presence o f anti-Nef antibodies (Fig. 2 B), The specific HIV-2 reactivity of the sera was analyzed and confirmed in commercial HIV-1 and HIV-2 protein i m m u n o b l o t assays (Pasteur) (data n o t shown). We could exclude the possibility of superinfection with HIV-1, since these sera exhibited only a weak cross-reactivity with HIV-1 proteins in i m m u n o b l o t tests. We could demonstrate that 50% of a total of 68 h u m a n sera, which had developed full seroconversion to HIV-1 proteins contained antibodies to N e f (Table 1). This is in agreement with recent reports [2, 22]. We did n o t find any correlation between the appearance of N e f antibodies a n d the clinical stages o f the serum donors. In c o m p a r i s o n with bacterially expressed N e f fusion proteins [14], the baculo N e f proteins were specifically recognized by a higher n u m b e r of h u m a n sera. Screening of HIV-1 seronegative high risk group samples observed N e f specific antibodies in only 2 o u t of 50 HIV-1 negative cases (Table 1). Both serum donors exhibited seroconversion to HIV-1 in later serum samples. These results were s u p p o r t e d by recent reports [4, 24] indicating that the detection of N e f antibodies usually coincides with the detection of antibodies directed against structural viral proteins and that the early appearance of N e f antibodies after HIV-1 infection is a rare event. We investigated, whether HIV-1 strains differing in their pathogenicity have any influence on the appearance o f N e f antibodies in h u m a n sera. We analyzed a total of 14 and 15 sera, respectively, which were derived from two collectives
Nef expressed in insect cells
297
Fig. 2. A Immunoblot. Either a mixture of the partially purified recombinant Nef proteins D3F3 and AYF3 (a) or the whole cell extract of wild-type AcNPV infected cells (b) were separated on 12.5% SDS-PAGE and immunoblottedwith HIV-2 positive human sera (111) or an anti-Nef mab (K+). B Immunoprecipitation. The whole extracts from cells producing wild-type AcNPV (a) or the Nef proteins AYF3 (b) or D3F3 (c) were immunoprecipitated with either an anti-Nef rabbit serum (K+) or human sera as in A (2, 8, 9) and subsequently immunoblotted as outlined in Fig. 1 B of HIV-1 seropositive individuals representing either low or high cytopathic HIV-1 strains. The cytopathic effects of the viruses isolated from the patients were examined in vitro [26]. We could not find any linkage between the detection of N e f antibodies and the in vitro virus pathogenicity (data not shown). Since the Nef expressing cells revealed brilliant immunofluorescence (Fig. 1 A), we studied the subcellular presence of Nef using the reliable and sensitive technique of confocal laser scanning microscopy as outlined recently
[3]. Nef is mainly located in the cytoplasm and at the cell membranes (Fig. 3 A, panels 1-3), which has already been shown [6]. The membrane association of baculo Nef indicates a possible myristylation as reported recently [18]. Moreover, Nef protein could also unequivocally be visualized in the nucleus. Since the immunoreacting material present in the nucleus is predominantly visualized in the middle sections of the cells, possible superpositions with cytoplasmic material could be excluded. Figure 3 B, panels 1 and 2, presents a further example for the different localization of N e f within the cells. Besides a partially clustered occurrence in the cytoplasm, Nef revealed a dispersal appearance in the nucleus. As already shown in Fig. 1 A, wild-type baculovirus infected cells could not be stained with the anti N e f mab.
298
N. Kienzle etal.
Fig. 3. Intracellular localization analysis of Nef applying confocal laser scanning microscopy. Fixed cells producing the AYF3 Nef protein were stained with anti-Nef mab # 158 and analyzed with a LSM10 microscope. The distance between the optical sections (A 1-3; B 1 and 2) performed at the z-distance through the entire cell is 1.5 gm. The corresponding phase-contrast images (DIC procedure) are given in panels 4 and 3 of A and B, respectively. c Cytoplasm; n nucleus A l t h o u g h our cell system is n o t the natural environment of Nef, our data are s u p p o r t e d by recent studies demonstrating that N e f is also present in the nucleus of HIV-1 infected cells [-16, 20]. Moreover, we obtained a similar localization pattern of N e f in B-cells stably transfected with a N e f expression plasmid [13]. Therefore, a direct function of N e f on gene expression should be considered.
Nef expressed in insect cells
299
Acknowledgements We are grateful to G. Enders (Stuttgart, F.R.G.), L. Giirtler (Mfinchen, F.R.G.), M. Forsgren (Stockholm, Sweden) and D. Vejlsgaard (Copenhagen, Denmark) for providing sera. We thank V. Erfle (Miinchen, F.R.G.) for the gift of HIV-1 infected glioma cell extract. We are indebted to B. Frech and F. Gr/isser for critically reading the manuscript as well as to E. Vollmer for experimental assistance. These studies were supported by the BMFT grant II-074-88.
References 1. Ahmad N, Venkatesan S (1988) NEF protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:1481-1485 2. Ameisen J-C, Guy B, Chamaret S, Loche M, Mouton Y, Neyrinck J-L, Khalife J, Leprevost C, Beaucaire G, Boutillon C, Gras-Masse H, Maniez M, Kieny M-P, Laustriat D, Berthier A, Mach B, Montagnier L, Lecocq J-P, Capron A (1989) Antibodies to the nef protein and to nef peptides in HIV-1-infected seronegative individuals. AIDS Res Human Retroviruses 5:279--291 3. Bachmann M, Chang SE, Slor H, Kukulies J, Miiller WEG (1990) Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes. Exp Cell Res 191:171-180 4. Cheingsong-Popov R, Panagiotidi C, Munaf A, Bowcock S, Watkins P, Aronstam A, Wassef M, Weber J (1990) Antibodies to HIV-1 nef (p27): prevalence, significance and relationship to seroconversion. AIDS Res Human Retroviruses 6:1099-1105 5. Cheng-Mayer C, Iannello P, Shaw K, Luciw PA, Levy JA (1989) Differential effects of nef on HIV replication: implications for viral pathogenesis in the host. Science 246: 1629-1632 6. Franchini G, Robert-Guroff M, Ghrayeb J, Chang NT, Wong-Staal F (1986) Cytoplasmic localization of the HTLV-III 3' off protein in cultured T cells. Virology 155: 593-599 7. Frech B, Zimber-Strobl U, Suentzenich K-O, Pavlish O, Lenoir GM, Bornkamm GW, Miiller-Lantzsch N (1990) Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera. J Virol 64:2759-2767 8. Gombert FO, Blecha W, T/ithinen M, Ranki A, Pfeifer S, Tr6ger W, Braun R, MfillerLantzsch N, Jung G, Rfibsamen-Waigmann H, Krohn K (1990) Antigenic epitopes of NEF proteins from different HIV-1 strains as recognized by sera from patients with manifest and latent HIV infection. Virology 176:458-466 9. Gr/isser FA, Haiss P, GSttel S, Mfiller-Lantzsch N (1991) Biochemical characterization of Epstein-Barr virus nuclear antigen 2A. J Virol 65:377%3788 10. Guyader M, Emerman M, Sonigo P, Clavel F, Montagnier L, Alizon M (1987) Genome organization and transactivation of the human immunodeficiencyvirus type 2. Nature 326:662-669 11. Guy B, Kieny MP, Riviere Y, Le Peuch C, Dott K, Girad M, Montagnier L, Lecocq JP (t987) HIV F/3' off encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330:266-269 12. Kestler III HW, Ringler D J, Mori K, Panicali DL, Seghal PK, Daniel MD, Desrosiers RC (1991) Importance of the NEF gene for maintenance of high virus loads and for development of AIDS. Cell 65:651-662 13. Kienzle N, Bachmann M, Miiller WEG, Mfilter-Lantzsch N (1992) Expression and cellular localization of the Nef protein from human immunodeficiencyvirus- 1 in stably transfected B-cells. Arch Virol 124:123-t32
300
N. Kienzle et al.
14. Kienzle N, Br6ker M, Harthus H-P, Enders M, Erfle V, Buck M, Mfiller-Lantzsch N (1991) Immunological study of the Nef protein from HIV-1 by polyclonal and monoclonal antibodies. Arch Virol 118:29-41 15. Kim S, Ikeuchi K, Byrn R, Groopman J, Baltimore D (1989) Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 86:9544-9548 16. Krohn K, Ovod V, Lagerstedt A (1991) Expression kinetics and cellular localization of HIV Tat and Nef proteins in relation to the expression of viral structural mRNA and viral particles. In: Chanock RM, Ginsberg HS, Brown F, Lerner RA (eds) Vaccines. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 97-101 17. Luckow VA, Summers MD (1988) Trends in the development of baculovirus expression vectors. Bio/Technology 6:47-55 18. Matsuura Y, Maekawa M, Hattori S, Ikegami N, Hayashi A, Yamazaki S, Morita C, Takebe Y (1991) Purification and characterization of human immunodeficiency virus tpye t nefgene product expressed by a recombinant baculovirus. Virology 184: 580586 19. Matsuura Y, Possee RD, Overton HA, Bishop DHL (1987) Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol 68:1233-1250 20. Ovod V, Lagerstedt A, Ranki A, Gombert FO, Spohn R, T/ihtinen M, Jung G, Krohn KJE (1992) Immunological variation and immunohistochemical localization of HIV1 Nef demonstrated with monoclonal antibodies. AIDS 6:25-34 21. Pen J, Welling GW, Welting-Wester S (1989) An efficient procedure for the isolation of recombinant baculovirus. Nucleic Acid Res 17:451 22. Ranki A, J/irvinen K, Valle S-L, Nurmilaakso P, Krohn K (1990) Antibodies to recombinant HIV-1 Nef detected in HIV-1 infection as well in nonrisk individuals. J AIDS 3:348-355 23. Ranki A, Krohn M, Allain JP, Franchini G, Valle SL, Antonen J, Leuther M, Krohn K (1987) Long latency precedes overt seroconversion in sexually transmitted humanimmunodeficiency-virus infection. Lancet ii: 58%593 24. Reiss P, deRonde A, Lange JMA, deWolf F, Dekker J, Debouck C, Goudsmit J (1989) Antibody response to the viral negative factor (net) in HIV-1 infection: a correlate of levels of HIV-1 expression. AIDS 3:227-233 25. Sauter M, Miiller-Lantzsch N (1987) Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera. Virus Res 8:141-152 26. Schneweis KE, Kleim JP, Bailly E, Niese D, Wagner N, Brackmann HH (1990) Graded cytopathogenicity of the human immunodeficiencyvirus (HIV) in the course of HIV infection. Med Microbiol Immunol 179:193-2133 27. Summers MD, Smith GE (1987) A manual of methods for baculovirus vectors and insect culture procedures. Tex Agric Exp Stat Bull no 1555 28. Terwilliger EF, Langhoff E, Gabuzda D, Zazopoulis E, Haseltine WA (1991) Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 88:10971-10975 29. Terwilliger E, Sodroski J, Rosen CA, Haseltine WA (1986) Effects of mutations within the 3' off open reading frame region of human T-cell lymphotropic virus type III (HTLVIII/LAV) on replication and cytopathogenicity. J Virol 60:754-760 30. Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M (1985) Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17 31. Werner T, Ferroni S, Saermark T, Brack-Werner R, Banati RB, Mager R, Steinaa L, Kreutzberg GW, Erfle V (1991) HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K + channels. AIDS 5:1301-1308
Nef expressed in insect cells
301
Authors' address: N. Mtiller-Lantzsch, Abteilung fiir Virologie, Institut ftir Medizinische Mikrobiologie und Hygiene, Haus 47, D-W-6650 Homburg, Federal Republic of Germany. Received December 9, 1991
Vi rology
Arch Virol (1992) 126:293-301
© Springer-Verlag 1992 Printed in Austria
Expression of the H I V - 1 N e f protein in the baculovirns system: investigation of a n t i - N e f antibodies response in human sera and subcellular localization of N e f Brief R e p o r t
N. Kienzle1, M. Enders1, M. Buck 1, H. Siakkou2, S. Jahn3, G. Petzold4, K. E. Schneweis5, M. Bachmann6, W. E. G. Miiller6 and N. Mfiiler-Lantzsch~ ~Abtcilung Virologic, Institut fiir Medizinische Mikrobiologie und Hygiene, Homburg Institute ffir 2Virologic und fiir 3Immunologie der Charit6, Berlin 4Zentralinstitut fiir Hygiene, Mikrobiologie und Epidemiologie, Berlin 5Institut fiir Medizinische Mikrobiologie und Immunologie, Bonn 6Institut ffir Physiologische Chemie, Mainz, Federal Republic of Germany Accepted February 25, 1992
Summary. The nef gene of HIV-1 was expressed in insect cells using the eucaryotic baculovirus system. The recombinant Nef protein frequently reacted with seropositive sera ofHIV- 1 and HIV-2 infected patients. Anti-Nef antibodies in HIV-1 seronegative high risk groups individuals were only occasionally seen. Confocal laser scanning microscopy demonstrated that Nef is present both in the cytoplasm and in the nucleus, indicating that Nef might directly function on gene expression.
The nef gene is a unique feature of the primate lentiviruses simian and human immunodeficiency virus (SIV and HIV). The HIV-1 nefgene encodes a protein of 206 amino acids (aa) with an apparent molecular mass of 27 kDa. Nef is myristylated and phosphorylated [11] and mainly localized in the cytoplasm [6]. Although Nef was not essential for HIV replication [29], the function of Nef has as yet not been determined. Depending on its genetic background, Nef increased, decreased or could not alter, respectively, the amount and the kinetics of virus replication [1, 5, 12, 15, 28]. The Nef protein elicits humoral immune responses in HIV infected individuals. Using procaryotically expressed Nef proteins or synthetic oligopeptides, Nef specific antibodies were found to be an important marker for the identification of HIV- 1 infection since the immune
294
N. Kienzle et al.
response to Nef was induced before seroconversion against the structural virus proteins E2, 8, 22, 23]. In contrast to these results, other authors reported that the appearance of Nef antibodies before seroconversion is a rare event [-4, 24]. In order to perform further investigations on the presence of Nef specific antibodies in human sera and on the intracellular localization of Nef, we expressed Nef in the eucaryotic Autographa californica nuclear polyhedrosis virus (AcNPV) system. For construction of recombinant baculo-transfervectors, the BamHI fragment of plasmid pUF [14] containing the complete nef gene of HIV1LAV-BRU [30] was cloned into a pAcYM1 non-fusion vector [19] as well as into a pAc360 fusion vector [17]. The two neftransfer vectors encoded different Nef proteins: (0 a non-fused, 206 aa long Nef (AYF3) and (ii) a N-terminal fused Nef protein (D3F3), which additionally carried 16 aa from the linker- and the N-terminal polyhedrin-sequence and 9 aa of the 5' upstream vicinity of the nefsequence. The insect line SF158 was cotransfected with AcNPV DNA and a neftransfer vector as outlined [27]. Recombination between AcNPV sequences present in the transfer vector and viral DNA resulted in nef gene-positive but polyhedrin-negative virions which were identified as described recently [-7, 21]. In indirect immunofluorescence assays performed as described in [7], insect cells producing Nef could brilliantly be stained with anti-Nef monoclonal antibody (mab) (Fig. 1 A). We have developed a series of murine anti-Nef mabs as well as polyclonal rabbit anti-Nef sera E14; Siakkou etal., submitted]. In immunoblot assay done as outlined recently [14], the 32 kDa D3F3 Nef fusion protein and the 26 kDa non-fused AYF3 Nef protein were observed (Fig. 1 B, lanes 2 and 3). The AYF3 protein showed a slightly higher mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis when compared to the 27 kDa Nefmade in HIV-1 infected glioma cells [31] (Fig. 1 B, lane 4) or to the 27 kDa Nef produced in stably transfected B-cells E13] (data not shown). Although other authors described a 27kDa Nef produced by recombinant baculovirus [-18], this difference might be due to altered posttranslational modifications or different nef genes as mentioned recently [20]. Expression of AYF3 yielded an additional Nef protein of 31 kDa (Fig. 1B, lane 2). Since different non-fusion nef baculovirus clones produced the 31 kDa protein, this finding is not due to a single clone event. It might be a result of posttranslational modifications or of a further recombination process during generation of the nef-baculovirus. The cell extracts of AYF3 as well as of D3F3 revealed a lower molecular mass Nef protein (Fig. 1 B). This could be a degradation product, since frequent freezing and thawing of the cell extracts increased this band. The recombinant Nef proteins were produced in large quantities, which in the case of D3F3 even allowed detection by Coomassie blue staining (data not shown). Human sera were studied with respect to antibody responses to Nef derived from baculoviruses. The data are summarized in Table 1. Either the whole protein extract of nef-baculovirus infected cells or the
Nef expressed in insect cells
295
Fig. 1. A Indirect immunofluorescence. Cells producing either the D3F3 Nef protein (1) or wild-type AcNPV (2) were mixed with uninfected cells at a ratio of 1 : 50. The fixed cells were stained with anti-Nef mab# 158. B Immunoanalysis of Nef. 1-4 Total protein extracts from cells, which were infected with wild-type AcNPV (1) or with baculoviruses expressing the non-fusion Nef protein AYF3 (2) or the fusion Nef protein D3F3 (3), were separated on 12.5% SDS-PAGE and immunoblotted using anti-Nef mab # 158. 4 A total protein extract from glioma cells infected with HIV-1 was immunoblotted. 5-10 Cellular extracts from cells producing either the Nef proteins AYF3 (5, 8) and D3F3 (6, 9) or wild-type AcNPV (7, 10) were immunoprecipitated with an anti-Nef positive human serum (5-7) or a HIV-1 negative human serum (8-10). The precipitated samples were analyzed by 12.5% SDS-PAGE and following immunoblot assay with anti-Nef mab # 158. The apparent molecular masses of the marker proteins are indicated in kDa
partially purificated A Y F 3 and D3F3 proteins, p e r f o r m e d as described in [25], were used in i m m u n o b l o t assay to detect anti-Nef antibodies in h u m a n sera. F u r t h e r m o r e , we p e r f o r m e d non-radiactive i m m u n o p r e c i p i t a t i o n assays. The cell lysis using a NP40-buffer and the i m m u n o p r e c i p i t a t i o n with h u m a n sera were p e r f o r m e d as described recently [9]. The precipitated samples were analyzed by i m m u n o b l o t assay. As shown in Fig. 1 B, lanes 5-10, h u m a n sera specifically reacted with the r e c o m b i n a n t N e f proteins. Despite the low degree of a m i n o acid h o m o l o g y between HIV-1 N e f and its c o u n t e r p a r t HIV-2 N e f [10], we could d e m o n s t r a t e that HIV-2 positive h u m a n sera reacted with the HIV-1 N e f p r o t e i n s m a d e in the baculovirus system. The sera reacted with D3F3 and A Y F 3 but also revealed cross-immunoreactions with proteins f r o m the r e c o m b i n a n t as well as wild-type baculovirus infected cells (Fig. 2 A). This is possibly due to the partially purification of the N e f
296
N, Kienzle et al. Table 1. Antibody response to Nef protein of HIV-1 a
Serum sourceb
No. of sera tested
No. of Nef positive sera
% Nef positive sera
HIV-1 + Stage°
68 II: 31; III: 7 IV: 15; U: 15 50 20 11
34 II: 20; III: 4 IV: 4; U: 6 2 0 9
50
HIV-1 ?a HIV-1 HIV-2 +
4 0 82
a The test systems were immunofluorescence, immunoblot or immunoprecipitation of SF158 cells infected with recombinant Nef baculovirus bAll sera were derived from different individuals. The evaluation of infection and seroconversion against HIV proteins was performed by commercial immunoblots or antibody detection kits ~Numbers of the CDC classified clinical stages of disease of the HIV-1 + serum donors; U unknown dThe sera were either seronegative or exhibited an uncertain seroconversion status. All serum donors belonged to high risk groups including hemophiliacs, homosexuals, partners of HIV-1 infected patients and i.v. drug abusers
proteins. However, immunoprecipitations confirmed the presence o f anti-Nef antibodies (Fig. 2 B), The specific HIV-2 reactivity of the sera was analyzed and confirmed in commercial HIV-1 and HIV-2 protein i m m u n o b l o t assays (Pasteur) (data n o t shown). We could exclude the possibility of superinfection with HIV-1, since these sera exhibited only a weak cross-reactivity with HIV-1 proteins in i m m u n o b l o t tests. We could demonstrate that 50% of a total of 68 h u m a n sera, which had developed full seroconversion to HIV-1 proteins contained antibodies to N e f (Table 1). This is in agreement with recent reports [2, 22]. We did n o t find any correlation between the appearance of N e f antibodies a n d the clinical stages o f the serum donors. In c o m p a r i s o n with bacterially expressed N e f fusion proteins [14], the baculo N e f proteins were specifically recognized by a higher n u m b e r of h u m a n sera. Screening of HIV-1 seronegative high risk group samples observed N e f specific antibodies in only 2 o u t of 50 HIV-1 negative cases (Table 1). Both serum donors exhibited seroconversion to HIV-1 in later serum samples. These results were s u p p o r t e d by recent reports [4, 24] indicating that the detection of N e f antibodies usually coincides with the detection of antibodies directed against structural viral proteins and that the early appearance of N e f antibodies after HIV-1 infection is a rare event. We investigated, whether HIV-1 strains differing in their pathogenicity have any influence on the appearance o f N e f antibodies in h u m a n sera. We analyzed a total of 14 and 15 sera, respectively, which were derived from two collectives
Nef expressed in insect cells
297
Fig. 2. A Immunoblot. Either a mixture of the partially purified recombinant Nef proteins D3F3 and AYF3 (a) or the whole cell extract of wild-type AcNPV infected cells (b) were separated on 12.5% SDS-PAGE and immunoblottedwith HIV-2 positive human sera (111) or an anti-Nef mab (K+). B Immunoprecipitation. The whole extracts from cells producing wild-type AcNPV (a) or the Nef proteins AYF3 (b) or D3F3 (c) were immunoprecipitated with either an anti-Nef rabbit serum (K+) or human sera as in A (2, 8, 9) and subsequently immunoblotted as outlined in Fig. 1 B of HIV-1 seropositive individuals representing either low or high cytopathic HIV-1 strains. The cytopathic effects of the viruses isolated from the patients were examined in vitro [26]. We could not find any linkage between the detection of N e f antibodies and the in vitro virus pathogenicity (data not shown). Since the Nef expressing cells revealed brilliant immunofluorescence (Fig. 1 A), we studied the subcellular presence of Nef using the reliable and sensitive technique of confocal laser scanning microscopy as outlined recently
[3]. Nef is mainly located in the cytoplasm and at the cell membranes (Fig. 3 A, panels 1-3), which has already been shown [6]. The membrane association of baculo Nef indicates a possible myristylation as reported recently [18]. Moreover, Nef protein could also unequivocally be visualized in the nucleus. Since the immunoreacting material present in the nucleus is predominantly visualized in the middle sections of the cells, possible superpositions with cytoplasmic material could be excluded. Figure 3 B, panels 1 and 2, presents a further example for the different localization of N e f within the cells. Besides a partially clustered occurrence in the cytoplasm, Nef revealed a dispersal appearance in the nucleus. As already shown in Fig. 1 A, wild-type baculovirus infected cells could not be stained with the anti N e f mab.
298
N. Kienzle etal.
Fig. 3. Intracellular localization analysis of Nef applying confocal laser scanning microscopy. Fixed cells producing the AYF3 Nef protein were stained with anti-Nef mab # 158 and analyzed with a LSM10 microscope. The distance between the optical sections (A 1-3; B 1 and 2) performed at the z-distance through the entire cell is 1.5 gm. The corresponding phase-contrast images (DIC procedure) are given in panels 4 and 3 of A and B, respectively. c Cytoplasm; n nucleus A l t h o u g h our cell system is n o t the natural environment of Nef, our data are s u p p o r t e d by recent studies demonstrating that N e f is also present in the nucleus of HIV-1 infected cells [-16, 20]. Moreover, we obtained a similar localization pattern of N e f in B-cells stably transfected with a N e f expression plasmid [13]. Therefore, a direct function of N e f on gene expression should be considered.
Nef expressed in insect cells
299
Acknowledgements We are grateful to G. Enders (Stuttgart, F.R.G.), L. Giirtler (Mfinchen, F.R.G.), M. Forsgren (Stockholm, Sweden) and D. Vejlsgaard (Copenhagen, Denmark) for providing sera. We thank V. Erfle (Miinchen, F.R.G.) for the gift of HIV-1 infected glioma cell extract. We are indebted to B. Frech and F. Gr/isser for critically reading the manuscript as well as to E. Vollmer for experimental assistance. These studies were supported by the BMFT grant II-074-88.
References 1. Ahmad N, Venkatesan S (1988) NEF protein of HIV-1 is a transcriptional repressor of HIV-1 LTR. Science 241:1481-1485 2. Ameisen J-C, Guy B, Chamaret S, Loche M, Mouton Y, Neyrinck J-L, Khalife J, Leprevost C, Beaucaire G, Boutillon C, Gras-Masse H, Maniez M, Kieny M-P, Laustriat D, Berthier A, Mach B, Montagnier L, Lecocq J-P, Capron A (1989) Antibodies to the nef protein and to nef peptides in HIV-1-infected seronegative individuals. AIDS Res Human Retroviruses 5:279--291 3. Bachmann M, Chang SE, Slor H, Kukulies J, Miiller WEG (1990) Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes. Exp Cell Res 191:171-180 4. Cheingsong-Popov R, Panagiotidi C, Munaf A, Bowcock S, Watkins P, Aronstam A, Wassef M, Weber J (1990) Antibodies to HIV-1 nef (p27): prevalence, significance and relationship to seroconversion. AIDS Res Human Retroviruses 6:1099-1105 5. Cheng-Mayer C, Iannello P, Shaw K, Luciw PA, Levy JA (1989) Differential effects of nef on HIV replication: implications for viral pathogenesis in the host. Science 246: 1629-1632 6. Franchini G, Robert-Guroff M, Ghrayeb J, Chang NT, Wong-Staal F (1986) Cytoplasmic localization of the HTLV-III 3' off protein in cultured T cells. Virology 155: 593-599 7. Frech B, Zimber-Strobl U, Suentzenich K-O, Pavlish O, Lenoir GM, Bornkamm GW, Miiller-Lantzsch N (1990) Identification of Epstein-Barr virus terminal protein 1 (TP1) in extracts of four lymphoid cell lines, expression in insect cells, and detection of antibodies in human sera. J Virol 64:2759-2767 8. Gombert FO, Blecha W, T/ithinen M, Ranki A, Pfeifer S, Tr6ger W, Braun R, MfillerLantzsch N, Jung G, Rfibsamen-Waigmann H, Krohn K (1990) Antigenic epitopes of NEF proteins from different HIV-1 strains as recognized by sera from patients with manifest and latent HIV infection. Virology 176:458-466 9. Gr/isser FA, Haiss P, GSttel S, Mfiller-Lantzsch N (1991) Biochemical characterization of Epstein-Barr virus nuclear antigen 2A. J Virol 65:377%3788 10. Guyader M, Emerman M, Sonigo P, Clavel F, Montagnier L, Alizon M (1987) Genome organization and transactivation of the human immunodeficiencyvirus type 2. Nature 326:662-669 11. Guy B, Kieny MP, Riviere Y, Le Peuch C, Dott K, Girad M, Montagnier L, Lecocq JP (t987) HIV F/3' off encodes a phosphorylated GTP-binding protein resembling an oncogene product. Nature 330:266-269 12. Kestler III HW, Ringler D J, Mori K, Panicali DL, Seghal PK, Daniel MD, Desrosiers RC (1991) Importance of the NEF gene for maintenance of high virus loads and for development of AIDS. Cell 65:651-662 13. Kienzle N, Bachmann M, Miiller WEG, Mfilter-Lantzsch N (1992) Expression and cellular localization of the Nef protein from human immunodeficiencyvirus- 1 in stably transfected B-cells. Arch Virol 124:123-t32
300
N. Kienzle et al.
14. Kienzle N, Br6ker M, Harthus H-P, Enders M, Erfle V, Buck M, Mfiller-Lantzsch N (1991) Immunological study of the Nef protein from HIV-1 by polyclonal and monoclonal antibodies. Arch Virol 118:29-41 15. Kim S, Ikeuchi K, Byrn R, Groopman J, Baltimore D (1989) Lack of a negative influence on viral growth by the nef gene of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 86:9544-9548 16. Krohn K, Ovod V, Lagerstedt A (1991) Expression kinetics and cellular localization of HIV Tat and Nef proteins in relation to the expression of viral structural mRNA and viral particles. In: Chanock RM, Ginsberg HS, Brown F, Lerner RA (eds) Vaccines. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp 97-101 17. Luckow VA, Summers MD (1988) Trends in the development of baculovirus expression vectors. Bio/Technology 6:47-55 18. Matsuura Y, Maekawa M, Hattori S, Ikegami N, Hayashi A, Yamazaki S, Morita C, Takebe Y (1991) Purification and characterization of human immunodeficiency virus tpye t nefgene product expressed by a recombinant baculovirus. Virology 184: 580586 19. Matsuura Y, Possee RD, Overton HA, Bishop DHL (1987) Baculovirus expression vectors: the requirements for high level expression of proteins, including glycoproteins. J Gen Virol 68:1233-1250 20. Ovod V, Lagerstedt A, Ranki A, Gombert FO, Spohn R, T/ihtinen M, Jung G, Krohn KJE (1992) Immunological variation and immunohistochemical localization of HIV1 Nef demonstrated with monoclonal antibodies. AIDS 6:25-34 21. Pen J, Welling GW, Welting-Wester S (1989) An efficient procedure for the isolation of recombinant baculovirus. Nucleic Acid Res 17:451 22. Ranki A, J/irvinen K, Valle S-L, Nurmilaakso P, Krohn K (1990) Antibodies to recombinant HIV-1 Nef detected in HIV-1 infection as well in nonrisk individuals. J AIDS 3:348-355 23. Ranki A, Krohn M, Allain JP, Franchini G, Valle SL, Antonen J, Leuther M, Krohn K (1987) Long latency precedes overt seroconversion in sexually transmitted humanimmunodeficiency-virus infection. Lancet ii: 58%593 24. Reiss P, deRonde A, Lange JMA, deWolf F, Dekker J, Debouck C, Goudsmit J (1989) Antibody response to the viral negative factor (net) in HIV-1 infection: a correlate of levels of HIV-1 expression. AIDS 3:227-233 25. Sauter M, Miiller-Lantzsch N (1987) Characterization of an Epstein-Barr virus nuclear antigen 2 variant (EBNA 2B) by specific sera. Virus Res 8:141-152 26. Schneweis KE, Kleim JP, Bailly E, Niese D, Wagner N, Brackmann HH (1990) Graded cytopathogenicity of the human immunodeficiencyvirus (HIV) in the course of HIV infection. Med Microbiol Immunol 179:193-2133 27. Summers MD, Smith GE (1987) A manual of methods for baculovirus vectors and insect culture procedures. Tex Agric Exp Stat Bull no 1555 28. Terwilliger EF, Langhoff E, Gabuzda D, Zazopoulis E, Haseltine WA (1991) Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1. Proc Natl Acad Sci USA 88:10971-10975 29. Terwilliger E, Sodroski J, Rosen CA, Haseltine WA (1986) Effects of mutations within the 3' off open reading frame region of human T-cell lymphotropic virus type III (HTLVIII/LAV) on replication and cytopathogenicity. J Virol 60:754-760 30. Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M (1985) Nucleotide sequence of the AIDS virus, LAV. Cell 40:9-17 31. Werner T, Ferroni S, Saermark T, Brack-Werner R, Banati RB, Mager R, Steinaa L, Kreutzberg GW, Erfle V (1991) HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K + channels. AIDS 5:1301-1308
Nef expressed in insect cells
301
Authors' address: N. Mtiller-Lantzsch, Abteilung fiir Virologie, Institut ftir Medizinische Mikrobiologie und Hygiene, Haus 47, D-W-6650 Homburg, Federal Republic of Germany. Received December 9, 1991
