A 40 kDa isoform of the type 5 adenovirus IVa2 protein is sufficient for virus viability

The multifunctional IVa2 protein is essential for adenovirus replication [J. Virol. 77 (2003) 3586], but the relative importance of the transcriptional and encapsidation functions is unknown. As part of a study of IVa2 function, we created a set of mutations in the IVa2 gene in the correct location in the viral genome. Unexpectedly, an opal stop codon at position 6 was recovered in virus twice. Isolate #2 showed defective viral replication, but produced late proteins at almost wild-type levels. Analysis of IVa2 mRNA showed an additional species, larger and more abundant than the equivalent wild-type species. It was a hybrid of the 5' UTR of L3 23 kDa attached to the IVa2 second exon, so that M75 is the 5' proximal methionine. This mRNA arises from a corresponding hybrid DNA, present in the virus stock. A protein of approximately 40 kDa, consistent with translation from the hybrid mRNA, was detected. It is able to bind to the packaging sequence and to the MLP downstream elements (DE1/2). Isolate #8 was more defective in replication than #2. No hybrid mRNA or DNA was detected, but it also produces a 40 kDa isoform, which is present in wild-type-infected cells. Mutational analysis of M75 and M101 revealed that the 40 kDa isoform is produced by initiation at Met75. This might be the origin of the previously unidentified 40 kDa factor present in the heterodimer DEF-A, which binds to DE1 and DE2a.