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.
Highlights
The herpes simplex virus replisome is composed of seven proteins encoded by the virus
Analyses of hybrid replisomes revealed species-specific interactions, and mutagenesis of UL8 identified amino acids required for DNA synthesis and UL52 primase interaction
Hybrid Replisomes Reveal Species-specific Interactions—To look for functionally important interactions within the herpes simplex virus type 1 (HSV-1) replisome, we settled for a comparison between HSV-1 strain 17 synϩ and an evolutionarily closely related virus equine herpesvirus type 1 (EHV-1) strain Ab4
Summary
The herpes simplex virus replisome is composed of seven proteins encoded by the virus. Results: Analyses of hybrid replisomes revealed species-specific interactions, and mutagenesis of UL8 identified amino acids required for DNA synthesis and UL52 primase interaction. 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. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8. An in depth search for interaction surfaces was carried out by systematic replacements of charged amino acids with alanines in UL8 and initially characterized in transient DNA replication assays. Our results suggest that the herpesvirus replisome operates as a functional unit requiring multiple species-specific interactions between the components of the replisome. Our results indicate that these interactions are likely to occur in evolutionarily conserved regions of the proteins
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