Rev-induced modulation of Nef protein underlies temporal regulation of human immunodeficiency virus replication.

Abstract

The replication of human immunodeficiency virus type 1 (HIV-1) requires the concerted action of two virus-encoded transactivator proteins, Tat and Rev, and is in turn moderated by the viral transcriptional repressor Nef. We show here that the phenotype of a Rev-HIV-1 provirus was nonreplicating and was distinguished by accumulation of Nef protein and reduced Tat function. Provirus defective in both the rev and nef genes (Rev-Nef ) was also nonreplicating but had normal Tat function. [14][15][16][17]. It has been difficult to assign the functions of Rev in the HIV-1 life cycle. For instance, the Rev molecular studies used chimeric reporter plasmids containing the putative target HIV-1 sequences for Rev fused to indicator genes (13) or subgenomic HIV-1 plasmids linked to HIV-1 LTR or heterologous promoters (16-18). On the other hand, studies of the role of Rev in viral replication used a standard provirus that had a premature termination codon in the nefORF (5,19,20). We recently demonstrated that Nef is a trans-acting transcriptional inhibitor of the HIV-1 LTR (12). Studies of Rev function would therefore be incomplete without addressing the nature of the functional interactions between Rev and Nef. In this report, we show that Rev induced a marked decrease in both the steady-state levels and rates of synthesis of Nef expressed from a Rev-provirus that was nonreplicating and had repressed Tat function. Rev-induced loss of Nef synthesis paralleled augmented expression of viral proteins, and the magnitude of the Rev effect could be reduced by addition of exogenous Nef. MATERIALS AND METHODSVirus and Cell Cultures. CD4' (Leu-3+) A3.01 T lymphocytes (21) in RPMI 1640 medium with 10% fetal bovine serum were used for infection. For most DNA transfections, SW480 (22) or HeLa S3 monolayers or A3.01 cells were used. A recombinant HIV-1 strain, pNL432 (22), referred to as wild type (wt), served as a standard. Particulate reverse transcriptase (RT) assays were as described (22) and the infectivity titers were determined by end-point dilution.cDNA Plasmids. Refer to Fig. 1B. We constructed an Okayama-Berg cDNA library of mRNAs from A3.01 lymphocytes infected with wt virus. The library was screened to obtain recombinants corresponding to the heterogeneously spliced 1.8-to 2.0-kb mRNA and one of these, pV102 (12), had a 69-base-pair (bp) central exon fused to 1352 bp of 3'-terminal exon with an intact nef. The HIV-1 insert of pV102 was recovered by partial cleavage at the flanking HindIII sites in the 5' and 3' LTRs and inserted between the corresponding HindIII sites ofthe wt provirus, yielding pHIV Nef. An oligonucleotide with a methionine codon and an Nco I site was inserted in-frame (11 codons upstream of the rev ORF) at the lone BssHII site of pHIV Nef to yield pHIV RevNef. The nef in pHIV RevNef was annulled by filling in the lone Xho I site, generating pHIV Rev. The rev-containing Nar I-Xho I fragment of pHIV Rev was blunt-end-ligated between the Xba I and Hpa I sites of pCMV CAT in place of the chloramphenicol...

Pathogenesis of Human Immunodeficiency Virus Infection

“…Moreover, with interviral recombinants resistant or sensitive to Nef, resistance was mapped to a region encompassing the second exon of Rev (159). Observations on the effect of Rev on Nef expression also support these conclusions (9). Most probably, Nef functions through an interaction with cellular proteins, probably via phosphorylation events (414,415,647,945,1294) that have yet to be defined.…”

Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells

“…Because of its capacity to down-regulate viral expression, the nef protein has been classified among the regulatory proteins that control HIV expression, possibly acting like a signal transducing protein (Guy et al, 1987;Gallo et al, 1988). In accord with this, recent results from different laboratories suggest that the nefprotein acts as a specific repressor of HIV transcription and might be critical for maintenance of HIV-I latency (Luciw et al, 1987;Ahmad & Venkatesan, 1988;Ahmad et al, 1989;Niederman et al, 1989). In general however, a functional nefgene does not abolish virus expression completely but seems to retard the rate of viral replication (Terwilliger et al, 1986).…”

Mechanism of translation of monocistronic and multicistronic human immunodeficiency virus type 1 mRNAs.

“…This may reflect a requirement for higher production of Tat than of Rev protein or, alternatively, a requirement for expression of Tat in the absence of Rev. Since the Rev protein has a negative effect on the expression of Tat, Rev, and Nef through a feedback mechanism (2,16,60), it may be beneficial for the virus to inhibit the expression of Rev at an early stage of the viral life cycle.…”