Truncation of the human adenovirus type 5 L4 33-kDa protein: Evidence for an essential role of the carboxy-terminus in the viral infectious cycle

The subgroup C human adenovirus L4 33-kDa protein is a nuclear phosphoprote in that plays a direct, but dispensable, role in virion assembly. The r-str and open reading frame (ORF) for this protein lies opposite to the 5' end o f the I-strand E2 early (E2E) transcription units. To facilitate studies of regulation of E2E transcription, we wished to construct a mutant virus in which the 33-kDa ORF was truncated to serve as a background into which spec ific E2E mutations could be introduced without also altering the 33-kDa pro tein. We constructed viral DNA (vDNA) containing within the 33-kDa ORF two tandem, premature stop codons that should prevent translation of the C-term inal 47 amino acids of the protein (Delta 47). We report here the unanticip ated lethality of such truncation of the L4 33-kDa protein. Viral DNA harbo ring the Delta 47 mutations did not produce infectious virus when transfect ed into cultured cells. In contrast, infectious virus was recovered upon tr ansfection of revertant vDNA, indicating that the Delta 47 mutations were r esponsible for the observed phenotype. The Delta 47 mutations did not affec t E2E transcription or production of the E2 DNA-binding protein. Transfecte d Delta 47 vDNA was replicated and directed the production of early and lat e viral proteins, including hexon protein in the trimer conformation. Howev er, no virus particles of any kind were produced. We propose that truncatio n of the adenovirus 33-kDa protein results in a lethal, late block in the i nfectious cycle during the assembly of progeny virions and discuss the impl ications of this phenotype for the mechanism of virion assembly. (C) 2001 A cademic Press.