MUTATIONS IN THE ADENOVIRUS-ENCODED SINGLE-STRANDED-DNA BINDING-PROTEIN THAT RESULT IN ALTERED ACCUMULATION OF EARLY AND LATE VIRAL RNAS

Adenovirus encodes a 72-kDa single-stranded DNA binding protein (DBP) that is necessary for Viral DNA replication and is involved in control ling viral RNA metabolism. Studies of temperature-sensitive (fs) and s ite-directed mutants of the DBP have identified at least four regions of the protein involved in binding to single-stranded DNA and hence in DNA replication. Two of the ts mutants, Ad2ts111A and Ad2ND1(+)ts23, are deficient in DNA binding and in supporting in vitro DNA replicatio n. Their effects on viral RNA metabolism are presented here. At early and late times of infection, accumulation of RNAs from the viral E3 re gion is increased up to sevenfold in Ad2ts111A- and Ad2ND1(+)ts23-infe cted cells relative to wild-type virus. This effect is temperature-ind ependent and seems to involve nuclear RNA stability. Steady-state leve ls of the viral E1B and E4 RNAs increase following the onset of viral DNA replication in cells infected by the wild-type virus, but not in c ells infected by ts111A or ts23. The increase in E1B and E4 RNAs al la te limes of infection is due to a stabilization of the mRNA. The stead y-state levels of L3 and L5 RNAs are two- to fourfold higher in cells infected with ts23 and ts111A than with wildtype virus. None of these differences were observed following infection of cells with a temperat ure-independent revertant of ts111, indicating that the mutations in t he DBP were responsible for the phenotypes. However, for the E3 effect , the change brought about by the mutations in the DBP does not seem t o be the result of a change in the normal function of the protein, as an essentially DBP-negative virus (Ad5d/802) shows no differences in E 3 RNA metabolism compared to wildtype virus at early times of infectio n. These results demonstrate that mutation of amino acids 280 and 282 of the DBP significantly perturbs the normal regulation of viral RNA m etabolism. These effects clearly differ from the phenotypes observed w ith adenoviruses containing mutations in other early genes and from th ose ascribed to the ts125 mutation by others. These results are discus sed in terms of the mechanisms by which early and late viral RNA metab olism are controlled and the possible effects of the DBP mutations on them. (C) 1995 Academic Press, Inc.