Existence of Transdominant and Potentiating Mutants of UL9, the Herpes Simplex Virus Type 1 Origin-Binding Protein, Suggests that Levels of UL9 Protein May Be Regulated during Infection

Abstract

UL9 is a multifunctional protein required for herpes simplex virus type 1 (HSV-1) replication in vivo. UL9 is a member of the superfamily II helicases and exhibits helicase and origin-binding activities. We have previously shown that mutations in the conserved helicase motifs of UL9 can have either a transdominant or potentiating effect on the plaque-forming ability of infectious DNA from wild-type virus (A. J. Malik and S. K. Weller, J. Virol. 70:7859-7866, 1996). In this paper, the mechanisms of transdominance and potentiation are explored. We show that the motif V mutant protein containing a G to A substitution at residue 354 is unstable when expressed by transfection and is either processed to a 38-kDa N-terminal fragment or degraded completely. The overexpression of the MV mutant protein is able to influence the steady-state protein levels of wild-type UL9 and to override the inhibitory effects of wild-type UL9. Potentiation correlates with the ability of the UL9 variants containing the G354A mutation to be processed or degraded to the 38-kDa form. We propose that the MV mutant protein is able to interact with full-length UL9 and that this interaction results in a decrease in the steady-state levels of UL9, which in turn leads to enhanced viral infection. Furthermore, we demonstrate that inhibition of HSV-1 infection can be obtained by overexpression of full-length UL9, the C-terminal third of the protein containing the origin-binding domain, or the N-terminal two-thirds of UL9 containing the conserved helicase motifs and the putative dimerization domain. Our results suggest that transdominance can be mediated by overexpression, origin-binding activity, and dimerization, whereas potentiation is most likely caused by the ability of the UL9 MV mutant to influence the steady-state levels of wild-type UL9. Taken together, the results presented in this paper suggest that the regulation of steady-state levels of UL9 may play an important role in controlling viral infection.The UL9 gene is required for herpes simplex virus type 1 (HSV-1) replication in vivo (6, 9). The UL9 protein is a dimer in solution and exhibits helicase, ATPase, and origin-binding activities (8, 13). UL9 is believed to play a key role in the initiation of HSV-1 replication by binding the HSV-1 origin of replication via its C-terminal domain and unwinding it in the presence of ATP and ICP8, the HSV-1 single-stranded DNA binding protein. It is likely that UL9 plays an important role in the assembly of the viral replisome (10, 20, 26, 41) through its interactions with other viral replication proteins (7,28,29).UL9 is a member of the superfamily II helicases (14). The conserved helicase motifs that are characteristic of this superfamily are positioned within the N-terminal domain of the protein (14). Genetic studies have previously shown that conserved residues within the helicase motifs are essential for HSV-1 replication in vivo; most engineered motif mutants fail to complement the growth of hr94, a UL9 null virus (24,27). Furthermore, biochemi...

DNA Binding Activity of the Herpes Simplex Virus Type 1 Origin Binding Protein, UL9, Can Be Modulated by Sequences in the N Terminus: Correlation between Transdominance and DNA Binding

“…Several mutants were shown to potentiate viral infection or to reverse the inhibition seen with wildtype plasmid. For instance, cotransfection with the motif V mutant (G354A, UL9-MV) consistently resulted in 150 to 200 plaques in this assay (18). We were intrigued by the fact that we were not able to detect full-length UL9 in cells transfected with this mutant and instead detected an N-terminal 38-kDa protein fragment.…”

“…Previously described UL9 mutants were classified as transdominant or potentiating according to a plaque reduction assay in which Vero cells were cotransfected with the HSV-1 infectious DNA and plasmid DNA capable of expressing wildtype or mutant versions of UL9 (18). The number of plaques observed with only HSV-1 infectious DNA was normalized to 100% (17), and plasmids which resulted in 10% or fewer plaques were considered to be transdominant.…”

“…The pCDNA3-UL9-WT plasmid expressing wild-type UL9 under the control of a cytomegalovirus promoter was reported previously (18). The UL9 mutant plasmids which were used in this study and the PCR primers used for generating these plasmids are listed in Tables 1 and 2, respectively.…”

“…The pCDNA3-UL9-1-359ϩ3FS plasmid was generated using UL9-start-BamHI as the downstream primer and oligo II as the upstream primer. Plasmids UL9-1-450-WT, UL9-1-354MV, UL9-MV (renamed UL9-MV-FS in this paper), and UL9-1-321-WT were generated previously (18). The pCDNA3-UL9-WT plasmid was also used as a template in PCR for the generation of plasmids UL9-C, UL9-494-851, UL9-394-851, and UL9-304-851.…”

“…Subconfluent monolayers of Vero cells in 60-mm 2 dishes were transfected with 2 g of plasmid DNA containing wild-type or mutant versions of the UL9 gene with Lipofectamine Plus reagent (Invitrogen, Calif.) according to the manufacturer's instructions. After 24 h, concentrated cell lysates were prepared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer as described previously (18).…”