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

作者：KYOSTIO SRM^*; OWENS RA; WEITZMAN MD; ANTONI BA; CHEJANOVSKY N; CARTER BJ

来源：Journal of Virology, 1994, 68(5): 2947-2957.

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(5) and p(19) promoters, using transient expression in human 293 cells followed by Northern (RNA) blot analysis of the mRNA. The p(5) transcript encodes the larger Rep proteins, Rep78 and Rep68, while the p(19) transcript encodes the smaller proteins, Rep52 and Rep40. A plasmid (pNTC3) containing the entire AAV genome with an amber mutation 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 increasing amounts of the wild-type rep gene in trans from a heterologous promoter inhibited p(5)- and p(19) 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 p(5) and p(19) mRNA accumulation was inhibited 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 showed that the ability of Rep78 to decrease p(5) and p(19) mRNA levels was lost when 159 or more amino acids were deleted. Rep78 and Rep68 mutants deleted for the methionine at residue 225 showed decreased abilities to down-regulate both p(5) and p(19) transcript levels, while mutants containing a substitution of glycine for the methionine resembled the wild-type Rep78. A Rep78 protein,vith a mutation in the putative nucleoside triphosphate binding site inhibited expression from p(5) but not from p(19), suggesting that the regulation of p(5) transcript levels 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 required for negative regulation of p(5) and p(19) transcript levels and that the regulation of p(19) mRNA levels by Rep78 did not require the presence of the p(5) promoter.

• 出版日期1994-5