Effects of Charged Cluster Mutations on the Function of Herpes Simplex Virus Type 1 UL34 Protein

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American Society for Microbiology

RESUMO

Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the UL34 gene cannot undergo this event. UL34 and UL31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of UL34 that are necessary for primary envelopment to occur, a library of 19 UL34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (ΔTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a UL34-null virus. The remainder of the mutants complemented at or near wild-type UL34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the UL34 protein in charged cluster mutants and ΔTM. In transfection-infection experiments, all of the functional UL34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type UL34. All of the noncomplementing UL34 mutants mediated proper localization of UL31. Charged clusters critical for UL34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that UL34 has at least one function in addition to mediating proper localization of UL31 in infected cells and provide further support for the role of UL34 in mediating proper localization of UL31 in infected cells.

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