Effects of charged cluster mutations on the function of herpes simplex virus type 1 UL34 protein.
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 (DeltaTM) 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 DeltaTM. 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.
Bjerke
Susan L
SL
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242, USA.
Cowan
John M
JM
Kerr
Jelani K
JK
Reynolds
Ashley E
AE
Baines
Joel D
JD
Roller
Richard J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D019084:Fluorescent Antibody Technique, Indirect; D005816:Genetic Complementation Test; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D005810:Multigene Family; D009154:Mutation; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000235:genetics*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
D004279:DNA, Viral; C403420:UL34 protein, Human herpesvirus 1; D014764:Viral Proteins