Transposon-based insertional mutagenesis screens have assessed how disruption of numerous human cytomegalovirus (HCMV) open reading frames (ORFs) impacts viral replication. Insertional mutagenesis of the HCMV U30 gene was previously found to substantially inhibit production of viral progeny. However, there are a number of putative U30-associated ORFs, and it is unclear how they impact viral replication. Here, we report on the contributions of the eight U30-associated ORFs to infection. We find that deletion of the canonically annotated U30 ORF substantially reduces production of infectious virus at both high and low multiplicities of infection (MOI). This deletion likely has complex effects on viral replicatio... More
Transposon-based insertional mutagenesis screens have assessed how disruption of numerous human cytomegalovirus (HCMV) open reading frames (ORFs) impacts viral replication. Insertional mutagenesis of the HCMV U30 gene was previously found to substantially inhibit production of viral progeny. However, there are a number of putative U30-associated ORFs, and it is unclear how they impact viral replication. Here, we report on the contributions of the eight U30-associated ORFs to infection. We find that deletion of the canonically annotated U30 ORF substantially reduces production of infectious virus at both high and low multiplicities of infection (MOI). This deletion likely has complex effects on viral replication, as we find that it reduces the expression of neighboring non-U30-associated ORFs. Mutation of the initiating methionine of the canonical U30 ORF indicated that it is dispensable for high- and low-MOI infection in the highly passaged AD169 strain, although it is important for low-MOI infection in the less-passaged TB40/E strain. Comutation of eight methionines in the U30 region results in a low-MOI viral replication defect, as does mutation of the TATA box responsible for the most abundant U30 transcript, which is found to be necessary for the accumulation of multiple U30-associated protein isoforms during infection. In total, our data indicate the importance of the U30-associated ORFs during low-MOI HCMV infection and further highlight the difficulty associated with the functional interrogation of broadly disruptive mutations: e.g., large deletions or transposon insertions. Viral genes and their products are the critical determinants of viral infection. Human cytomegalovirus (HCMV) encodes many gene products whose roles during viral infection have not been assessed. Elucidation of the contributions that various HCMV gene products make to infection provides insight into the infectious program, which could potentially be used to limit HCMV-associated morbidity, a major issue during congenital infection and in immunosuppressed populations. Here, we explored the role of HCMV's U30-associated gene products and found that they are important for HCMV replication. Future work elucidating the mechanisms through which they contribute to viral infection could highlight novel avenues for therapeutic intervention.
