The conserved carboxyl-terminal half of herpes simplex virus type 1 regulatory protein ICP27 is dispensable for viral growth in the presence of compensatory mutations

ICP27 is an essential herpes simplex virus type 1 (HSV-1) immediate-early p rotein that regulates viral gene expression by poorly characterized mechani sms. Previous data suggest that its carboxyl (C)-terminal portion is absolu tely required for productive viral infection. In this study, we isolated M1 6R, a second-site revertant of a viral ICP27 C-terminal mutant, M16R harbor s an intragenic re-version, as demonstrated by the fact that its cloned ICP 27 allele can complement the growth of an HSV-1 ICP27 deletion mutant. DNA sequencing demonstrated that the intragenic reversion is a frameshift alter ation in a homopolymeric run of C residues at codons 215 to 217. This resul ts in the predicted expression of a truncated, 289-residue molecule bearing 72 novel C-terminal residues derived from the +1 reading frame. Consistent with this, M16R expresses an ICP27-related molecule of the predicted size in the nuclei of infected cells. Transfection-based viral complementation a ssays confirmed that the truncated, frameshifted protein can partially subs titute for ICP27 in the context of viral infection. Surprisingly, its novel C-terminal residues are required for this activity. To see if the frameshi ft mutation is all that is required for M16R's viability, we re-engineered the M16R ICP27 allele and inserted it into a new viral background, creating the HSV-1 mutant M16exC. An additional mutant, exCd305, was constructed wh ich possesses the frameshift in the context of an ICP27 gene with the C ter minus deleted. We found that both M16exC and enCd305 are nonviable in Vero cells, suggesting that one or more extragenic mutations are also required f or the viability of M16R, Consistent with this interpretation, we isolated two viable derivatives of exCd305 which grow productively in Vero cells des pite being incapable of encoding the C-terminal portion of ICP27, Studies o f viral DNA synthesis in mutant-infected cells indicated that the truncated , frameshifted ICP27 protein can enhance viral DNA replication. In summary, our results demonstrate that the C-terminal portion of ICP27, conserved wi dely in herpesviruses and previously believed to be absolutely essential, i s dispensable for HSV-1 lytic replication in the presence of compensatory