Comparative Analysis of UL16 Mutants Derived from Multiple Strains of Herpes Simplex Virus 2 (HSV-2) and HSV-1 Reveals Species-Specific Requirements for the UL16 Protein.

Orthologs of the herpes simplex virus (HSV) UL16 gene are conserved throughout the Herpesviridae Because of this conservation, one might expect that the proteins perform similar functions for all herpesviruses. Previous studies on a UL16-null mutant derived from HSV-2 strain 186 revealed a roughly 100-fold replication defect and a critical role for UL16 in the nuclear egress of capsids. These findings were in stark contrast to what has been observed with UL16 mutants of HSV-1 and pseudorabies virus, where roughly 10-fold replication deficiencies that were accompanied by defects in the secondary envelopment of cytoplasmic capsids were reported. One possible explanation for this discrepancy is that HSV-2 strain 186 is not representative of the HSV-2 species. To address this possibility, multiple UL16-null mutants were constructed in multiple HSV-2 and HSV-1 strains by CRISPR/Cas9 mutagenesis, and their phenotypes were characterized side by side. This analysis showed that all the HSV-2 UL16 mutants had 50- to 100-fold replication deficiencies that were accompanied by defects in the nuclear egress of capsids, as well as defects in the secondary envelopment of cytoplasmic capsids. By contrast, most HSV-1 UL16 mutants had 10-fold replication deficiencies that were accompanied by defects in secondary envelopment of cytoplasmic capsids. These findings indicated that UL16 has HSV species-specific functions. Interestingly, HSV-1 UL16 could promote the nuclear egress of HSV-2 UL16-null strains, suggesting that, unlike HSV-1, HSV-2 lacks an activity that can promote nuclear egress in the absence of UL16.IMPORTANCE HSV-2 and HSV-1 are important human pathogens that cause distinct diseases in their hosts. A complete understanding of the morphogenesis of these viruses is expected to reveal vulnerabilities that can be exploited in the treatment of HSV disease. UL16 is a virion structural component that is conserved throughout the Herpesviridae and functions in virus morphogenesis; however, previous studies have suggested different roles for UL16 in the morphogenesis of HSV-2 and HSV-1. This study sought to resolve this apparent discrepancy by analyzing multiple UL16 mutant viruses derived from multiple strains of HSV-2 and HSV-1. The data indicate that UL16 has HSV species-specific functions, as HSV-2 has a requirement for UL16 in the escape of capsids from the nucleus whereas both HSV-2 and HSV-1 require UL16 for final envelopment of capsids at cytoplasmic membranes.