Helicase-primase complex of herpes simplex virus type 1: a mutation in the UL52 subunit abolishes primase activity.
The UL52 gene product of herpes simplex virus type 1 (HSV-1) comprises one subunit of a 3-protein helicase-primase complex that is essential for replication of viral DNA. The functions of the individual subunits of the complex are not known with certainty, although it is clear that the UL8 subunit is not required for either helicase or primase activity. Examination of the predicted amino acid sequence of the UL5 gene reveals the existence of conserved helicase motifs; it seems likely, therefore, that UL5 is responsible for the helicase activity of the complex. We have undertaken mutational analysis of UL52 in an attempt to understand the functional contribution of this protein to the helicase-primase complex. Amino acid substitution mutations were introduced into five regions of the UL52 gene that are highly conserved among HSV-1 and the related herpesviruses equine herpesvirus 1, human cytomegalovirus, Epstein-Barr virus, and varicella-zoster virus. Of seven mutants analyzed by an in vivo replication assay, three mutants, in three different conserved regions of the protein, failed to support DNA replication. Within one of the conserved regions is a 6-amino-acid motif (IL)(VIM)(LF)DhD (where h is a hydrophobic residue), which is also conserved in mouse, yeast, and T7 primases. Mutagenesis of the first aspartate residue of the motif, located at position 628 of the UL52 protein, abolished the ability of the complex to support replication of an origin-containing plasmid in vivo and to synthesize oligoribonucleotide primers in vitro. The ATPase and helicase activities were unaffected, as was the ability of the mutant enzyme to support displacement synthesis on a preformed fork substrate. These results provide experimental support for the idea that UL52 is responsible for the primase activity of the HSV helicase-primase complex.
Klinedinst
D K
DK
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Challberg
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004279:DNA, Viral / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014764:Viral Proteins; D014779:Virus Replication / Q000235:genetics / Q000502:physiology
D004279:DNA, Viral; D014764:Viral Proteins; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases