Replication-competent herpes simplex virus 1 isolates selected from cells transfected with a bacterial artificial chromosome DNA lacking only the UL49 gene vary with respect to the defect in the UL41 gene encoding host shutoff RNase.
To generate a null U(L)49 gene mutant of herpes simplex virus 1 (HSV-1), we deleted from the viral DNA, encoded as a bacterial artificial chromosome (BAC), the U(L)49 open reading frame and, in a second step, restored it. Upon transfection into Vero cells, the BAC-DeltaU(L)49 DNA yielded foci of degenerated cells that could not be expanded and a few replication-competent clones. The replication-competent viral clones derived from independent transfections yielded viruses that expressed genes with some delay, produced smaller plaques, and gave lower yields than wild-type virus. A key finding is that the independently derived replication-competent viruses lacked the virion host shutoff (vhs) activity expressed by the RNase encoded by the U(L)41 gene. One mutant virus expressed no vhs protein, whereas two others, derived from independent transfections, produced truncated vhs proteins consistent with the spontaneous in-frame deletion. In contrast, cells infected with the virus recovered upon transfection of the BAC-U(L)49R DNA (R-U(L)49) accumulated a full-length vhs protein, indicating that in the parental BAC-DeltaU(L)49 DNA, the U(L)41 gene was intact. We conclude that expression of the vhs protein in the absence of U(L)49 protein is lethal, a conclusion bolstered by the evidence reported elsewhere that in transfected cells vhs requires both VP16 and VP22, the product of U(L)49, to be neutralized.
University of Chicago, Viral Oncology Laboratory, 910 East 58th St., Chicago, IL 60637, USA.
Maria Teresa
MT
Sciortino
Brunella
B
Taddeo
Maria
M
Giuffrè-Cuculletto
Maria Antonietta
MA
Medici
Antonio
A
Mastino
Bernard
B
Roizman
D036022:Capsid Proteins / Q000502:physiology; D002460:Cell Line; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics; D004279:DNA, Viral; D017353:Gene Deletion; D017405:Herpes Simplex Virus Protein Vmw65 / Q000502:physiology; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D012260:Ribonucleases; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*
D036022:Capsid Proteins; D004279:DNA, Viral; D017405:Herpes Simplex Virus Protein Vmw65; C081688:Ul41 protein, Human herpesvirus 1; C082563:VP19 protein, Human herpesvirus 1; D014764:Viral Proteins; C070722:virion host shutoff protein, Simplexvirus; D012260:Ribonucleases