MHV-68 Open Reading Frame 20 is a nonessential gene delaying lung viral clearance

Recently, it has been demonstrated that the MHV-68 ORF20-encoded gene product induces cell-cycle arrest at the G2/M phase, followed by apoptosis. To study the role of this conserved gene in vivo, two independent ORF20-deficient MHV-68 viruses and their revertants were constructed. As the replication in vitro of both mutants followed similar kinetics to that of the wild-type and revertant viruses, ORF20 is therefore a nonessential virus gene. No cell cycle arrest could be observed upon infection of cells with wild type MHV-68 or mutant viruses. In addition, no major differences were detected between mock- and virus-infected cells when protein and inactivation levels of the mitotic promoter factor cdc2/cyclinB were analyzed. Following intranasal infection, the recovery of mutant, revertant and wild-type viruses in the lungs was similar. With the ORF20-deficient viruses, however, there was a significant delay of four days in clearance of virus from the lungs. Surprisingly, the magnitude and cell population distribution in the exudates of the lung was essentially similar to mice infected with wild-type, revertant or ORF20-deleted viruses. Subsequent establishment of latency was normal for both mutants, demonstrating that ORF20 does not play a critical role in establishment of a persistent infection. These results indicate that while expression of ORF20 may impact on the pathogenicity of the infection, the observed induction of G2/M arrest in ORF20-expressing cells may not be the primary function of ORF20 in the context of viral infection.