Effects of Charged Cluster Mutations on the Function of Herpes Simplex Virus Type 1 U L 34 Protein

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Abstract
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 U L 34 gene cannot undergo this event. U L 34 and U L 31, 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 U L 34 that are necessary for primary envelopment to occur, a library of 19 U L 34 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 U L 34-null virus. The remainder of the mutants complemented at or near wild-type U L 34 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 U L 34 protein in charged cluster mutants and ΔTM. In transfection-infection experiments, all of the functional U L 34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type U L 34. All of the noncomplementing U L 34 mutants mediated proper localization of U L 31. Charged clusters critical for U L 34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that U L 34 has at least one function in addition to mediating proper localization of U L 31 in infected cells and provide further support for the role of U L 34 in mediating proper localization of U L 31 in infected cells.