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Identification of amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome

Journal article
Authors Isabella Muylaert
Zhiyuan Zhao
Torbjörn Andersson
Per Elias
Published in Journal of Biological Chemistry
Volume 287
Issue 40
Pages 33142-33152
ISSN 0021-9258
Publication year 2012
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 33142-33152
Language en
Links dx.doi.org/10.1074/jbc.M112.356782
Keywords DNA replication, Herpes simplex virus, helicase-primase, UL5, UL8, UL52, UL29, protein-protein interaction, confocal microscopy
Subject categories Chemistry

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.

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