The exonuclease activity of HSV-1 UL12 is required for in vivo function.
The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline pH-dependent deoxyribonuclease termed alkaline nuclease. A recombinant UL12 knockout mutant, AN-1, is severely compromised for growth, and analysis of this mutant suggests that UL12 plays a role in processing complex DNA replication intermediates (R. Martinez, R. T. Sarisky, P. C. Weber, and S. K. Weller, (1996) J. Virol. 70, 2075-2085). This processing step may be required for the generation of capsids that are competent for egress from the nucleus to the cytoplasm. In this report, we address the question of whether the AN-1 growth phenotype is due to the loss of UL12 catalytic activity. We constructed two point mutations in a highly conserved region (motif II) of UL12 and purified wild-type and mutant enzymes from a baculovirus expression system. Both mutant proteins are stable, soluble, and competent for correct nuclear localization, suggesting that they have retained an intact global conformation. Neither mutant protein, however, exhibits exonuclease activity. In order to examine the in vivo effects of these mutations, we determined whether expression of mutant proteins from amplicon plasmids could complement AN-1. While the wild-type plasmid complements the growth of the null mutant, neither UL12 mutant can do so. Loss of exonuclease activity therefore correlates with loss of in vivo function.
Goldstein
J N
JN
Department of Microbiology, University of Connecticut Health Center, Farmington 06030-3205, USA.
Weller
S K
SK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D003673:Defective Viruses / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D017353:Gene Deletion; D005814:Genes, Viral; D005816:Genetic Complementation Test; D018259:Herpesvirus 1, Human / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012260:Ribonucleases / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D012260:Ribonucleases; C066964:viral alkaline nuclease