Comparative Analysis of UL16 Mutants Derived from Multiple Strains of HSV-2 and HSV-1 Reveals Species-Specific Requirements for the UL16 Protein

Orthologs of the herpes simplex virus (HSV) gene are conserved throughout the . Because of this conservation, one might expect that these proteins perform similar functions for all herpesviruses. Previous studies on a 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 mutants of HSV-1 and pseudorabies virus where roughly 10-fold replication deficiencies were reported that were accompanied by defects in the secondary envelopment of cytoplasmic capsids. One possible explanation for this discrepancy is that the HSV-2 186 strain is not representative of the HSV-2 species. To address this possibility, multiple null mutants were constructed in multiple HSV-2 and HSV-1 strains by CRISPR/Cas9 mutagenesis and their phenotypes characterized side-by-side. This analysis showed that all the HSV-2 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 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 null strains, suggesting that, unlike HSV-1, HSV-2 lacks an activity that can compensate for nuclear egress in the absence of UL16.