A murine cytomegalovirus cell cycle regulator (m54.5p) evolved within the conserved viral DNA polymerase gene

Ribosome profiling (Ribo-seq) and transcription start site profiling recently unveiled hundreds of novel viral gene products in lytic murine cytomegalovirus (MCMV) infection. One of these is the highly expressed m54.5 open reading frame (ORF) located within the highly conserved viral DNA polymerase locus (M54). Here, we show that m54.5 encodes a nuclear protein (m54.5p) that contributes to cell cycle regulation during MCMV infection. m54.5p interacts with the anaphase-promoting complex/cyclosome (APC/C) and protein phosphatase-6 (PP6). Ectopic m54.5p expression resulted in nuclear accumulation of PP6C and multiple APC/C subunits, accompanied by increased nuclear levels of the APC/C substrates. Accordingly, ectopic m54.5p expression resulted in G1 cell cycle arrest in nocodazole mitotic arrest and serum starvation assays. While an m54.5p-null mutant virus replicated with wild-type kinetics in vitro, it was mildly attenuated in mouse lungs by 14 days post-infection. Our findings highlight the surprising plasticity of herpesvirus genomes, facilitating the evolution of a > 200 aa viral open reading frame within the highly conserved viral DNA polymerase gene locus.