THE PRODUCT OF A 1.9-KB MESSENGER-RNA 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 herpe s 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). Th is mRNA is a member of a family of five unspliced 3'-coterminal messag es. 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 de scribed the isolation of AN-1, a deletion/ insertion mutant of the alk aline nuclease gene (S. K. Weller, R. M. Seghatoleslami, L. Shao, D. R owse, and E. P. Carmichael, 1990. J. Gen. Virol. 71, 2941-2952). The d eletion in AN-1 would be predicted to abolish gene products of both th e 2.3- and the 1.9-kb mRNAs. To investigate whether the putative trunc ated version of alkaline nuclease encoded by the 1.9-kb mRNA has enzym atic activity and plays a role in the viral life cycle, a viral mutant (AN-F1) was constructed which is predicted to abolish the gene produc t 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 poly clonal antiserum raised against a bacterially expressed full-length al kaline nuclease, we observed a 60-kDa protein in KOS- and AN-F1-infect ed 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 read ing 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 enzyma tically active, has low levels of activity, or possesses enzymatic act ivity which is not detected because of the low abundance of the polype ptide, AN-1 and AN-F1 are both severely restricted with respect to gro wth in Vero cells, as viral yields are 100- to 1000-fold lower than th ose of wild-type virus. We previously reported that the major defect i n AN-1 is in the ability of DNA-containing capsids which form in the n ucleus to mature into the cytoplasm (L. Shao, L. M. Rapp, and S. K. We ller, 1993. Virology 196, 146-162); AN-F1 exhibits the same defect. Th ese results indicate that although the 1.9-kb mRNA encodes a 60-kDa pr otein presumably from the UL12.5 open reading frame, this polypeptide cannot substitute for the full-length UL12 product. (C) 1996 Academic Press, Inc.