Role of herpes simplex virus type 1 UL46 and UL47 in alpha TIF-mediated transcriptional induction: characterization of three viral deletion mutants.
The transcriptional induction of the alpha or immediate-early gene class of herpes simplex virus type 1 effected by the alpha trans-induction factor (alpha TIF, ICP25, VP16, Vmw65) requires an alpha-specific cis-acting site. Increased transcription does not result from the direct, independent binding of alpha TIF, but rather from an alpha TIF-dependent formation of a protein-DNA complex containing, in addition to alpha TIF, at least one host cell factor. One of the host factors is a POU domain protein which recognizes an octamer element in the alpha-specific consensus. There is evidence that alpha TIF may drive the formation of multiple protein-DNA complexes containing a POU protein and additional host factors. Previously, the gene products of UL46 and UL47 have been implicated in modulating the alpha TIF-dependent transcriptional induction of alpha genes. Our current studies have extended these analyses from a transient-expression system to a series of viral deletion mutants. In these studies we demonstrate that neither UL46- nor UL47-encoded gene product, either separately or in combination, is required for viral growth in cell culture. The absence of UL47 reduces by up to 80% the ability of the virus to induce an alpha-regulated thymidine kinase reporter gene resident in 143TK- cells. Autoradiograms of [35S]methionine pulse-labeled infected cell proteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, show that deleting UL46 and/or UL47 has no discernable effect on the synthesis of alpha TIF or alpha TIF-containing proteins. Subsequent Western immunoblot analysis, with rabbit anti-alpha TIF antibodies made to an alpha TIF-Staphylococcus aureus protein A fusion, demonstrated that the accumulation and steady-state levels of alpha TIF or alpha TIF-containing proteins was indistinguishable from that of the thymidine kinase-negative isogenic parental virus, R delta 305.
Zhang
Y
Y
Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pennsylvania 15261.
Sirko
D A
DA
McKnight
J L
JL
D000818:Animals; D015139:Blotting, Southern; D002460:Cell Line; D002472:Cell Transformation, Viral; D002872:Chromosome Deletion; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D017405:Herpes Simplex Virus Protein Vmw65; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D015183:Restriction Mapping; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics*; D013937:Thymidine Kinase / Q000235:genetics; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D017405:Herpes Simplex Virus Protein Vmw65; D015534:Trans-Activators; D014764:Viral Proteins; D013937:Thymidine Kinase