Construction and Characterization of HSV-1 Mutants Encoding Truncated VP16 in the Presence and Absence of Functional VHS

Abstract

VP16 is an integral element in the HSV-1 strategy to overturn normal cellular functions and use the host machinery for the production of new virus. VP16 enhances the expression of viral immediate-early genes in the nucleus of the infected cell. By binding to the virion host shutoff protein (vhs), VP16 is able to downregulate its activity and prevent destruction of the viral transcripts. Finally, VP16 has an essential but specifically undetermined role in virus assembly. While a null mutation in VP16 correlates to a lethal defect in virus assembly, recent work has revealed that the C-terminal acidic activation domain of VP16 is not essential to virus survival. Viral mutants containing 422 and 379 amino acid tail-deleted derivatives of VP16 are viable. We have constructed a viable viral mutant which encodes a 369 amino acid VP16, revealing that the N-terminal 369 amino acids of VP16 retain a function which is essential to virus survival. The viral mutants encoding truncated VP16 derivatives displayed a characteristic preference for growth on a cell line which supplied full length VP16 in 𝘵𝘳𝘢𝘯𝘴. Furthermore, the preference for growth on the complementing cell line became more apparent as VP16 was progressively truncated from the C-terminus. To determine if full length VP16 was aiding these virtues in attenuating vhs activity or in performing a structural role in the presence of vhs, we constructed double mutant viruses encoding the truncated VP16s and containing an inactivating mutation in the vhs locus (ΔSma). The elimination of vhs activity and/or vhs-VP16 binding alleviated the preference for growth on the complementing cell line in these viruses. Thus, while the acidic activation domain of VP16 is not essential for viral replication in tissue culture, it clearly confers a growth advantage to the virus. The viral mutants constructed here will prove useful in understanding the significance of the interaction between VP16 and vhs.