The rep gene of adeno-associated virus type 2 (AAV) encodes four overlapping Rep proteins that are involved in gene regulation and replication of the virus. We studied here the regulation of mRNA transcribed from the AAV p. and pig promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p5 transcript encodes the larger Rep proteins, Rep78 and Rep68, while the plg transcript encodes the smaller proteins, Rep52 and Rep4O. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation in the rep gene accumulated higher levels of p5 and p19 mRNA than a plasmid containing the wild-type AAV genome. Addition of increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p5 and plg mRNA accumulation from pNTC3, indicating that the levels of both transcripts were decreased by the Rep proteins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that P5 and P19 mRNA accumulation was inhibited 5to 10-fold by Rep78 and Rep68 and 2-to 3-fold by Rep52 and Rep4O. Analysis of carboxyl-terminal truncation mutants of

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep4O) required for AAV DNA replication and AAV gene regulation. In addition, the Rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular Rep78 may mediate a negative regulatory effect. We analyzed the effects of the AAV rep gene on human immunodeficiency virus type 1 (HIV-1) gene expression. The rep gene proteins of AAV type 2 (AAV2) inhibited the trans-activating ability of HIV-1. Constructs containing the AAV2 rep gene (pHIVrep) or a CAT gene (pBennCAT) expressed from the 5' HIV-1 long terminal repeat were inducible for Rep78 and Rep68 or CAT expression, respectively, when cotransfected with a plasmid containing the HIV-1 tat gene (pARtat). When equivalent amounts of pHIVrep and pBennCAT were cotransfected with increasing amounts of pARtat, expression of CAT activity was decreased. The pHIVrep construct was more inhibitory than plasmids expressing rep from the wild-type AAV2 p5 transcription promoter. rep expression from pHIVrep almost completely inhibited the replication of an HIV-1 proviral clone as measured by reverse transcriptase activity and p24 protein levels. Inhibition of HIV-1 production by Rep protein was also seen at the transcriptional level in that all HIV-1 transcripts were decreased when pHIVrep was present. The inhibitory effects of pHIVrep appear to be mediated primarily by Rep78 and perhaps Rep68. These results suggest that a transacting protein from a heterologous virus might be used to inhibit HIV-1 growth.

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