Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6111-6115, August 1989 Biochemistry Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication NAFEES AHMAD, RATAN K. MAITRA, AND SUNDARARAJAN VENKATESAN* Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Communicated by Bernard Moss, May 25, 1989 ABSTRACT The replication of human immunodeficiency of Nef expressed from a Rev- provirus that was nonrepli- virus type 1 (HIV-1) requires the concerted action of two cating and had repressed Tat function. Rev-induced loss of virus-encoded transactivator proteins, Tat and Rev, and is in Nef synthesis paralleled augmented expression of viral pro- turn moderated by the viral transcriptional repressor Nef. We teins, and the magnitude of the Rev effect could be reduced show here that the phenotype of a Rev- HIV-1 provirus was by addition of exogenous Nef. nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nefgenes (Rev-Nef ) was also nonreplicating but MATERIALS AND METHODS had normal Tat function. Trans-complementation of the Rev- Virus and Cell Cultures. CD4' (Leu-3+) A3.01 T lympho- mutant with Rev caused a decrease ofboth the steady-state level cytes (21) in RPMI 1640 medium with 10% fetal bovine serum and the rate of synthesis of Nef. This was accompanied by were used for infection. For most DNA transfections, SW480 enhanced synthesis of viral structural proteins. Rev induced (22) or HeLa S3 monolayers or A3.01 cells were used. A even greater levels of virus production from the Rev-Nefr recombinant HIV-1 strain, pNL432 (22), referred to as wild double mutant. In contrast, exogenous Rev did not augment type (wt), served as a standard. Particulate reverse transcrip- virus production from wild-type provirus. Virus production tase (RT) assays were as described (22) and the infectivity from Rev- and Rev-Nef mutants induced by Rev was re- titers were determined by end-point dilution. pressed by exogenous Nef. The repression induced by Nefcould cDNA Plasmids. Refer to Fig. 1B. We constructed an not be reversed by exogenous Rev. The ability of Rev to Okayama-Berg cDNA library of mRNAs from A3.01 lym- modulate Nef expression solely from the provirus, and thereby phocytes infected with wt virus. The library was screened to relieve the Nef-mediated inhibition of transcription from the obtain recombinants corresponding to the heterogeneously viral long terminal repeat, reveals a delicate balance of the spliced 1.8- to 2.0-kb mRNA and one of these, pV102 (12), functions of these two proteins that might underlie the switch had a 69-base-pair (bp) central exon fused to 1352 bp of between latency and reactivation. 3'-terminal exon with an intact nef. The HIV-1 insert of pV102 was recovered by partial cleavage at the flanking Intrinsic to the strategy of replication of human immunode- HindIII sites in the 5' and 3' LTRs and inserted between the ficiency virus type 1 (HIV-1) is the functional expression of corresponding HindIII sites ofthe wt provirus, yielding pHIV certain small viral regulatory proteins. Among these, Tat and Nef. An oligonucleotide with a methionine codon and an Nco Rev are essential for viral replication (1-9). Tat and Rev are I site was inserted in-frame (11 codons upstream of the rev encoded by two overlapping open reading frames (ORFs) ORF) at the lone BssHII site of pHIV Nef to yield pHIV within two exons of a heterogeneous class of multiply spliced RevNef. The nefin pHIV RevNef was annulled by filling in 2.0-kilobase (kb) transcripts (10) that also encodes another the lone Xho I site, generating pHIV Rev. The rev-containing protein, Nef, which is a negative regulator (11, 12). The Tat Nar I-Xho I fragment of pHIV Rev was blunt-end-ligated protein is absolutely required for viral replication and the between the Xba I and Hpa I sites of pCMV CAT in place of expression of genes linked to the HIV-1 long terminal repeat the chloramphenicol acetyltransferase (CAT) gene, down- (LTR). Rev-defective viruses are nonreplicating and fail to stream of the cytomegalovirus (CMV) immediate early pro- synthesize the HIV-1 gag and env gene products (5, 6, 13). moter (9), generating pCMV Rev. Alternatively, the HIV-1 Rev may enable viral replication by facilitating the nuclear cDNA sequence in pHIV Rev was recovered by cleaving transport and stabilization of large HIV-1 RNAs coding for with Bgl II in the 5' and 3' LTRs and cloned downstream of structural proteins (14-17). the Rous sarcoma virus (RSV) LTR in the vector pRSV.5 to It has been difficult to assign the functions of Rev in the yield pRSV Rev. HIV-1 life cycle. For instance, the Rev molecular studies Proviral Plasmids. The infectious provirus pNL432 (wt) has used chimeric reporter plasmids containing the putative been described (19). The EcoRI-BamHI fragment of wt was target HIV-1 sequences for Rev fused to indicator genes (13) mutagenized (23) to introduce a single termination codon or subgenomic HIV-1 plasmids linked to HIV-1 LTR or after the first two residues in the rev ORF. This fragment was heterologous promoters (16-18). On the other hand, studies then transferred to wt to generate the Rev- mutant provirus. ofthe role ofRev in viral replication used a standard provirus The wt provirus was also cleaved at the lone Xho I site in the that had a premature termination codon in the nefORF (5, 19, nef ORF, blunt-ended with T4 DNA polymerase, and self- 20). We recently demonstrated that Nef is a trans-acting ligated to yield the Nef- mutant (12). A Rev-Nef double transcriptional inhibitor of the HIV-1 LTR (12). Studies of mutant was constructed from the Nef- provirus by replacing Rev function would therefore be incomplete without address- its own wt rev ORF with the mutagenized version. ing the nature of the functional interactions between Rev and DNA Transfections. Subconfluent monolayers in T25 flasks Nef. In this report, we show that Rev induced a marked were transfected with 10 ,ug of total DNA (adjusted with decrease in both the steady-state levels and rates of synthesis Abbreviations: CAT, chloramphenicol acetyltransferase; HIV-1, The publication costs of this article were defrayed in part by page charge human immunodeficiency virus type 1; LTR, long terminal repeat; payment. This article must therefore be hereby marked "advertisement" ORF, open reading frame; RT, reverse transcriptase; wt, wild type. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 6111 Downloaded by guest on September 29, 2021 6112 Biochemistry: Ahmad et al. Proc. Natl. Acad. Sci. USA 86 (1989) A Immunological Procedures. Viral proteins in the tranfec- 1 2 3 4 5 6 7 8 9 kb tants were detected by immunoblotting (25) using pooled HBsPH Bc K K EHK HBaXK H AIDS patient sera, rabbit antibodies against E. coli fusion C Bg B BiH S B Be Bg Reyel 0 L nS± protein corresponding to the p24 gag protein, and antisera JR eITs l te.2N fI raised against synthetic peptides corresponding to residues }Tate tx ORF. Rabbit anitserum against t ~~~~~~~rev nef wt 67-77 or 196-206 of the nef WTGGCAGGAAGAAGC . .GCATCTCGAGAC. AAACAT. AkCAAGT.. ORF was supplied by Bryan Cullen H6 Is M AG R S . A S RD . K I . residues 27-51 of the rev I rev RevT (Duke University). Radiolabeled cell extracts were pro- MHBs TGGCATGAAGAAGC BaPvK H cessed for immunoprecipitation (26) and the immunoreactive M A * IInefIc Nef- proteins were resolved by SDS/PAGE and visualized by WTGGCAGGAAGAAGC -*GCATCTCGATCG . AAAACA.. AAGTAG.. IM A G R S A S R S - K T K fluorography. IHBI s 33 35 36 40 4 CAT Assay. Cell lysates of CAT gene transfectants were rev nef Il Rev Nef- AAAACA... AAGTAG.. assayed as described (27). Typically, assays were run for 20 TGGCATGAAGAAGC * GCATCTCGATCG K M A * A S R S - K T min in duplicate with three different volumes of each lysate, which were adjusted to constant levels of p-galactosidase B HBs H BaXK H expression. For each set of plasmids, the values were cal- pV102 culated from six independent transfections. CAT activity, H. 1 Ss H Ba XK H [::w'------- ------------------------------ iev-I ----- -Lill pHIVNef expressed as percent acetylated chloramphenicol, was de- |GCGCGCAC ACTG GGAAGAAGC.. S A H T G R S. termined by scintillation counting. HNc H Bn XK H pHIVRevNef 13-Galactosidase Assay. Two different volumes of cell ex- TGGGCGCGCAC ACT.. GAAGC.. tracts were assayed in duplicate in 96-well microtiter plates G A H T G S M R BaPv H as Reac- pHIVRev with o-nitrophenyl f3-D-galactoside substrate (28). -li----------------------------------r-re n e tinns were monitored hv Asnc with an automated ELISA ¶ATGGCGCGCAC ACT AGAAG1AC.. GCATCTCGATCG .. AAACA.. AAGTAG.. M G A H T IG R S ..A S R S - K T K reader. (Na)Nc Ba( X)Ba revi~h w pCMV Rev C (Xb) AGAAGC.. (Hp) ACT. G R S RESULTS ATGGG-CG4CGCAC V0plAst M G A H. .T. SV40 polyA site Rev Induces Enhanced Virus Production from Rev- and Xb(Bg) H Nc Ba Pv (g) Rev-Nef Mutants. The functional role(s) of Rev protein ---------------------- -------------rev pRSV Rev rGGAAGAAGC (Ba)S. during infection was evaluated in a transient virus-production RSV (a) G R S ATGGGCGCGCAC .... ACM.. SV40 poly A site assay using wt, Rev-, and Rev-Nef proviruses (Fig.