Functional dissection of an alternatively spliced herpesvirus gene by splice site mutagenesis

Herpesviruses have large and complex DNA genomes. The largest among the herpesviruses, the cytomegaloviruses, encode over 170 genes. Although most herpesvirus gene products are expressed from unspliced transcripts, a substantial number of viral transcripts are spliced. Some viral transcripts are subject to alternative splicing, which leads to the expression of several proteins from a single gene. Functional analysis of individual proteins derived from an alternatively spliced gene is difficult as deletion and nonsense mutagenesis, both common methods used in the generation of viral gene knockout mutants, affect several or all gene products at the same time. Here we show that individual gene products of an alternatively spliced herpesvirus gene can be inactivated selectively by mutagenesis of the splice donor or acceptor site and by intron deletion or substitution mutagenesis. We used this strategy to dissect the essential M112/113 gene of murine cytomegalovirus (MCMV), which encodes the MCMV Early 1 (E1) proteins. The expression of each of the four E1 protein isoforms was inactivated individually, and the requirement of each isoform for MCMV replication was analyzed in fibroblasts, endothelial cells, and macrophages. We show that the E1 p87, but not the p33, p36, and p38 isoforms, are essential for viral replication in cell culture. Moreover, the presence of one of the two medium-sized isoforms (p36 or p38) and the presence of intron 1, but not its specific sequence, are required for viral replication. This study demonstrates the usefulness of splice site mutagenesis for the functional analysis of alternatively spliced herpesvirus genes.

IMPORTANCE: Herpesviruses encode up to 170 genes on their DNA genomes. The functions of most viral gene products remain poorly defined. The construction of viral gene knockout mutants has thus been an important tool for functional analysis of viral proteins. However, this strategy is of limited use when viral gene transcripts are alternatively spliced leading to the expression of several proteins from a single gene. In this study we showed, as a proof of principle, that each protein product of an alternatively spliced gene can be eliminated individually by splice site mutagenesis. Mutant viruses lacking individual protein products displayed different phenotypes, demonstrating that the products of alternatively spliced genes have non-redundant functions.