title: Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.
abstract: We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.
authors.affiliation: Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden. isabella.muylaert@medkem.gu.se
authors.forename: Isabella
authors.initials: I
authors.lastname: Muylaert
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Zhao
authors.forename: Torbjörn
authors.initials: T
authors.lastname: Andersson
authors.forename: Per
authors.initials: P
authors.lastname: Elias
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D009841:Oligonucleotides; D014764:Viral Proteins; D004247:DNA; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases; D000409:Alanine