_Archives

Vfrology

Arch Virol (1991) 118:29-41

© Springer-Verlag 1991 Printed in Austria

Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies N. Kienzle l, M. Briiker 2, H.-P. Harthus 2, M. Enders 1, V. Erfle 3, M. Buck 1, and N. MiiHer-Lantzsch 1

1Abteilung Virologie, Institut ffir Medizinische Mikrobiologie und Hygiene, Homburg, 2Behringwerke AG, Marburg, and 3GSF-Abteilung f/Jr Molekulare Zellpathologie,Neuherberg, Federal Republic of Germany Accepted October 29, 1990

Summary. We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal mouse antibodies against these recombinant proteins. The rabbit antisera, one of the monoclonal antibodies as well as a serum from a HIV-1 infected patient recognized the nef protein with Mr 27 kDa in latently HIV-1 infected glioma cells in the immunoblot. In contrast, these antibodies could not detect nef in productively HIV-1 infected Molt-3 cells neither in immunoblot nor in indirect immunofluorescence assays. These results indicate the possible participation of nef in viral latency. The recombinant nef proteins were used as probes for anti-nef antibodies in human sera. We observed in 17 of 57 sera tested specific anti-nef antibodies. All of these anti-nef positive sera also contained antibodies directed against viral structural proteins. The NH2-terminal region of the recombinant nef was shown to be the major immunodominant antigenic site in the immunoblot assay. Introduction One of the at least nine genes of the human immunodeficiency virus type 1 (HIV-1) is the negative factor (nef) encoding gene which is absent from the normal retroviral genome [2, 12, 15]. Related genes are also found in HIV-2 and simian immunodeficiency virus [9, 11]. HIV mutants with deleted nef genes show an enhanced ratio of replication and altered cytopathic effects in vitro when compared to their standard counterparts [13, 25, 41]. The nef gene product acts as a transcriptional silencer or repressor and is involved in downregulation of virus replication. It may thus participate in the establishment and maintenance of latency of the HIV provirus in infected cells [1, 29], although recent reports called this hypothesis in question [18, 21]. The nef gene product is a myristylated protein of 27 kDa which is localized

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at the m e m b r a n e and in the cytoplasm [2, 14]. Purified nef protein expressed in bacteria shows GTP-binding, GTPase and a u t o p h o s p h o r y l a t i o n activities [17]. Sequence homologies of nef with oncogenes, including ras, pp60 sRc or erb-B, have also been described [37]. N e f can also serve as a target for cytotoxic effector cells [35]. The nef protein is able to elicit an i m m u n o r e s p o n s e in H I V infected individuals since antibodies against nef were detected in sera of H I V patients [5, 6]. N e f antibodies were f o u n d to be an i m p o r t a n t m a r k e r for the indication o f HIV-1 infection in individuals that otherwise lacked detectable levels of antibodies against virion structural proteins [3, 31]. We used r e c o m b i n a n t nef proteins as probes to detect antibodies directed against nef in sera of HIV-1 infected patients. F u r t h e r m o r e , we have derived polyclonal rabbit and m o n o c l o n a t mouse antibodies against recombinant nef and have used these antibodies for investigation of nef expression in cells which were productively or latently infected with HIV-1.

Materials and methods Preparation of the nef recombinant expression vectors The HIV-1 DNA clone pBH10-R3 [32] was cleaved with EcoRV and PstI. The EcoRVsite (bp 8775) is present downstream to the stop codon (bp 8744) within the nef gene. The PstI-site is derived from the polylinker region of the plasmid. The resulting 420bp EcoRV - PstI fragment, which included 216 bp of the C-terminal nef sequence, was isolated and ligated into the Sinai and PstI digested tryptophan regulated expression vector pATH 1t. After transformation of E.coli with the ligation products a clone containing the recombinant nef sequence in the correct orientation was selected and designated pAK-F 1. The inducible reading frame of pATH (T. J. K6rner, pers. comm.) is under control of the TrpE operon of E.coli and produces a 37 kDa part of the anthranilate synthetase (TrpE) or a recombinant TrpE-fusion protein, respectively. Similar strategies were used to create a 690 bp X h o I - SalI fragment of plasmid pBTI containing the HIV-I(LAV) DNA [42] in the SalI cleaved expression vector pATH1. XhoI cut at position 8454 and the SalI-site was present in the polylinker of the plasmid. The recombinant vector obtained was called pAK-F2 harboring the 512 C-terminal bps of nef. To obtain the entire nef gene, plasmid pBT1 including the HIV-I(LAV) DNA [42] was digested with BamHI which cut at position 8032 and downstream from a Sinai site within the polylinker region of the plasmid. The 1120bp fragment received was ligated into the BamHI cleaved ml3mpl8 and pUC19 vectors (BRL Gibco) to obtain the plasmids mBB and pUB, respectively. A new Sinai site was created at position 8327 within the HIV-1 sequence of mBB by site directed mutagenesis (Amersham) using the oligonucleotide 3'GATCTTCTTATTCGGGCCCGAACCTTTCCT-5' as a primer. The 810bp SmaI fragment of such an altered clone was ligated into ml3mpl9 and sequenced by the dideoxymethod [38] with a'7 Sequencing Kit (Pharmacia). The 145bp PstI/XhoI fragment from this plasmid was used to replace the 422 bp PstI/XhoI fragment of plasmid pUB. By this procedure, we obtained plasmid pUF, which included the complete nef gene flanked by SmaI and BamHI sites. To construct pAK-F3, the 810 bp BamHI fragment of pUF was ligated to pATH10 digested with BamHI. To construct pMB302, the 543 bp HaeIII DNA fragment from the cloned HIV-1 isolate BH10 (bp 8413-8956) [32] coding for a nef gene with an internal stop codon was ligated

Antibodies against nef of HIV- 1

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into the expression vector pBD2-UK, linearized with StuI. This plasmid is a derivative of pBD2 [7], which carries a truncated lacZ gene coding for the aminoterminal 375 amino acids of 13gal.

Expression and partial purification of the proteins E.coli C600 [4] containing the vector pATH1 or the recombinant trpE-nef vectors were cultured and protein expression was induced as described [40]. The TrpE protein and the different fusion proteins PAK-F1, PAK-F2, and PAK-F3 (Fig. 1) were recovered from SDS-polyacrylamide gels as described recently [39]. The expression of [~galactosidase-nef ([3gal::nef; Fig. 1) fusion protein from plasmid pMB302 was induced in E.coli BMH71-18 [22] with t m M IPTG. After sonication of the harvested bacteria the debris containing the insoluble fusion protein was treated with buffer containing increasing amounts of urea. Finally, the recombinant nef was solubilized in 8 M urea and dialyzed against 10raM Tris HC1 pH 7.5 and 0.1% SDS.

Generation of specific polyclonal rabbit antisera In order to generate polyclonat rabbit sera the lyophilized fusion proteins PAK-F1, PAKF2, and PAK-F3 were dissolved in PBS and used for immunization (300 ~tg protein per rabbit) by intradermal injection [43]. In the case of 13gal::nef, the rabbits were injected three times sub-cutaneously with 100 gg of the fusion protein in incomplete Freund's adjuvant in 14 days intervalls.

Preparation and purification of monoclonal antibodies Mouse monoclonal antibodies (mabs) directed against the pMB302 encoded fusion protein were generated according to standard procedures [19] in Balb/c mice using SP2/0 cells for the cell fusion. Screening for nef specific mabs was performed by ELISA technique using microtiter plates either coated with the recombinant [3gal::nef or with [3gal. Antibodies bound to the solid phase were detected by a second rabbit anti-mouse antibody labelled with horseradish peroxidase. Cell lines producing anti-nef antibodies were propagated as ascites fluids using IFAprimed Balb/c mice. Immunoglobulins were affinity purified using Protein A-Sepharose (Pharmacia/LKB) according to the procedure recommended by the manufactures. Isotypes were determined by double diffusion techniques (Miles).

Polyacrylamide gel electrophoresis and immunoblotting Electrophoresis in 8-12% SDS-polyacrylamide gels (SDS-PAGE) was performed as described [23, 27] using low molecular weight protein markers (Pharmacia) to determine the apparent molecular masses. Protein samples were prepared by resuspending cells or bacterial proteins in SDS-PAGE sample buffer and boiling for 10 rain. About 10 gg of isolated proteins or 60 gg of complete cellular protein extracts as judged by Lowry protein determination [24] or optical density(zs0nm) measurement were applied on each track of the gel. The separated proteins were transferred electrophoretically to nitrocellulose sheets or Immobilon membranes (Millipore) as described [8] and blotted with the following modifications [28]: For indirect peroxidase staining peroxidase-labeled goat anti-rabbit-, goat anti-mouse-, or goat anti-human-IgG sera was employed. Diamino benzidine (0.5 mg/ml in PBS containing 0.02% H202) served as substrate for the enzyme reaction. In additon, alkaline phosphatase coupled to antimouse or anti-rabbit antibodies was used as conjugate with Fast Blue B salt (Serva) and Naphtol AS-MX Phosphate (Sigma) as substrates. For immunostaining the anti-nef sera were diluted 100 fold.

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Results

Construction of recombinant nef fusion proteins The complete reading frame, two C-terminal regions and an internal sequence of the nef gene from HIV-1 were expressed in procaryotic fusion vectors as described in Materials and methods. Induction of plasmids pAK-F 1, pAK-F2, pAK-F3 and the vector pATH1 yielded the three fusion proteins PAK-F1, PAK-F2, and PAK-F3 with molecular apparent sizes of 43 kDa, 54 kDa, and 60 kDa, respectively, and the 37 kDa TrpE protein. All fusion proteins consisted of a 37 kDa TrpE part at their NH2-terminus. Plasmid pMB302 was able to produce the 62kDa fusion protein 13gal::nef, which carried a 40 kDa part of 13gal at its NHz-terminus. Figure 1 gives an illustration of the constructed recombinant nef proteins. The fusion proteins PAK-F1, PAK-F2, PAK-F3, [3gal::nef as well as the TrpE protein were enriched as described in Materials and methods.

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Immunological study of the nef protein from HIV-1 by polyclonal and monoclonal antibodies.

We constructed and expressed different overlapping fusion proteins with the nef gene of HIV-1 and generated specific polyclonal rabbit and monoclonal ...
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