The product of a 1.9-kb mRNA which overlaps the HSV-1 alkaline nuclease gene (UL12) cannot relieve the growth defects of a null mutant.
Alkaline nuclease, a relatively abundant viral phosphoprotein in herpes simplex virus type 1 (HSV-1)- or HSV-2-infected cells, is encoded by a 2.3-kb mRNA (R. H. Costa, K. G. Draper, L. Banks, K. L. Powell, G. Cohen, R. Eisenberg, and E. K. Wagner, 1983. J. Virol. 48, 591-603). This mRNA is a member of a family of five unspliced 3'-coterminal messages. Costa et al. proposed that another member of this family of mRNAs (1.9-kb) may encode an N-terminally truncated protein which shares its carboxy-terminus with the alkaline nuclease protein. We previously described the isolation of AN-1, a deletion/insertion mutant of the alkaline nuclease gene (S. K. Weller, R. M. Seghatoleslami, L. Shao, D. Rowse, and E. P. Carmichael, 1990. J. Gen. Virol. 71, 2941-2952). The deletion in AN-1 would be predicted to abolish gene products of both the 2.3- and the 1.9-kb mRNAs. To investigate whether the putative truncated version of alkaline nuclease encoded by the 1.9-kb mRNA has enzymatic activity and plays a role in the viral life cycle, a viral mutant (AN-F1) was constructed which is predicted to abolish the gene product of the 2.3-kb mRNA (full-length alkaline nuclease) but leave intact the putative product of the 1.9-kb mRNA. Using a highly sensitive polyclonal antiserum raised against a bacterially expressed full-length alkaline nuclease, we observed a 60-kDa protein in KOS- and AN-F1-infected cells but not in AN-1-infected cells. This suggests that the 60-kDa protein is likely to be expressed from the 1.9-kb mRNA; the open reading frame is now designated UL12.5. Despite the presence of the 60-kDa band, AN-F1 failed to exhibit any alkaline exonuclease activity. This result suggests that the truncated polypeptide (UL12.5) is not enzymatically active, has low levels of activity, or possesses enzymatic activity which is not detected because of the low abundance of the polypeptide. AN-1 and AN-F1 are both severely restricted with respect to growth in Vero cells, as viral yields are 100- to 1000-fold lower than those of wild-type virus. We previously reported that the major defect in AN-1 is in the ability of DNA-containing capsids which form in the nucleus to mature into the cytoplasm (L. Shao, L. M. Rapp, and S. K. Weller, 1993. Virology 196, 146-162); AN-F1 exhibits the same defect. These results indicate that although the 1.9-kb mRNA encodes a 60-kDa protein presumably from the UL12.5 open reading frame, this polypeptide cannot substitute for the full-length UL12 product.
Martinez
R
R
Department of Microbiology, University of Connecticut Health Center, Farmington 06030, USA.
Shao
L
L
Bronstein
J C
JC
Weber
P C
PC
Weller
S K
SK
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000096:biosynthesis / Q000378:metabolism; D016368:Frameshift Mutation; D005807:Genes, Overlapping; D005814:Genes, Viral; D005819:Genetic Markers; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D012260:Ribonucleases / Q000235:genetics* / Q000378:metabolism; D014709:Vero Cells; D014764:Viral Proteins / Q000378:metabolism
D004279:DNA, Viral; D005819:Genetic Markers; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D012260:Ribonucleases; C066964:viral alkaline nuclease