Deletion mutants of the herpes simplex virus type 1 UL8 protein: effect on DNA synthesis and ability to interact with and influence the intracellular localization of the UL5 and UL52 proteins.
The herpes simplex virus type 1 (HSV-1) helicase-primase, an essential component of the viral DNA replication machinery, is a trimeric complex of the virus-coded UL5, UL8, and UL52 proteins. An assembly of the UL5 and UL52 subunits retains both enzymic activities, and the UL8 protein has been implicated in modulating these functions, facilitating efficient nuclear uptake of the complex and interacting with other viral DNA replication proteins. To further our understanding of UL8, we have constructed plasmids expressing mutant proteins, truncated at their N- or C-termini or lacking amino acids internally, under the control of the human cytomegalovirus major immediate-early promoter. Deletion of 23 amino acids from the N-terminus or 33 from the C-terminus abolished the ability of UL8 to support DNA replication in transient transfection assays. None of the UL8 mutants tested exhibited a strong dominant negative phenotype in the presence of the wild-type product, although some inhibition of replication was observed with mutants lacking 165 N-terminal or 497 C-terminal amino acids. The ability of the UL8 mutants to facilitate efficient nuclear localization of UL52 in the presence of coexpressed UL5 was examined by immunofluorescence. Selected mutants were also expressed by recombinant baculoviruses and tested for interaction with UL5 and UL52 in immunoprecipitation assays. The replicative ability of the mutants was found to correlate with their ability to localize UL52 to the nucleus, but not their interaction with UL5 and UL52. This property precluded the identification of any region of UL8 important for its presumed nuclear functions.
Barnard
E C
EC
MRC Virology Unit, Institute of Virology, Church Street, Glasgow, G11 5JR, United Kingdom.
Brown
G
G
Stow
N D
ND
D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D006224:Cricetinae; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics*; D017353:Gene Deletion; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011817:Rabbits; D014764:Viral Proteins; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases