ANALYSIS OF ADENOASSOCIATED VIRUS (AAV) WILD-TYPE AND MUTANT REP PROTEINS FOR THEIR ABILITIES TO NEGATIVELY REGULATE AAV P(5) AND P(19) MESSENGER-RNA LEVELS

The rep gene of adeno-associated virus type 2 (AAV) encodes four overl apping Rep proteins that are involved in gene regulation and replicati on of the virus. We studied here the regulation of mRNA transcribed fr om the AAV p(5) and p(19) promoters, using transient expression in hum an 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p(5) transcript encodes the larger Rep proteins, Rep78 and Rep68, whi le the p(19) transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber muta tion in the rep gene accumulated higher levels of p(5) and p(19) mRNA than a plasmid containing the wild-type AAV genome. Addition of increa sing amounts of the wild-type rep gene in trans from a heterologous pr omoter inhibited p(5)- and p(19) mRNA accumulation from pNTC3, indicat ing that the levels of both transcripts were decreased by the Rep prot eins. Cotransfections with plasmids producing individual wild-type Rep proteins in trans showed that p(5) and p(19) mRNA accumulation was in hibited 5- to 10-fold by Rep78 and Rep68 and 2- to 3-fold by Rep52 and Rep40. Analysis of carboxyl-terminal truncation mutants of Rep78 show ed that the ability of Rep78 to decrease p(5) and p(19) mRNA levels wa s lost when 159 or more amino acids were deleted. Rep78 and Rep68 muta nts deleted for the methionine at residue 225 showed decreased abiliti es to down-regulate both p(5) and p(19) transcript levels, while mutan ts containing a substitution of glycine for the methionine resembled t he wild-type Rep78. A Rep78 protein,vith a mutation in the putative nu cleoside triphosphate binding site inhibited expression from p(5) but not from p(19), suggesting that the regulation of p(5) transcript leve ls by Rep78 and Rep68 differs from that of p(19). A deletion analysis of AAV cis sequences revealed that an intact terminal repeat was not r equired for negative regulation of p(5) and p(19) transcript levels an d that the regulation of p(19) mRNA levels by Rep78 did not require th e presence of the p(5) promoter.