HERPES-SIMPLEX VIRUS TYPE-1 ALKALINE NUCLEASE IS REQUIRED FOR EFFICIENT PROCESSING OF VIRAL-DNA REPLICATION INTERMEDIATES

Mutations in the alkaline nuclease gene of herpes simplex virus type 1 (HSV-1) (nuc mutations) induce almost wild-type levels of viral DNA; however, mutant viral yields are 0.1 to 1% of wild-type yields (L. Sha o, L. Rapp, and S. Weller, Virology 195:146-162, 1993; R. Martinez, L. Shao, J. C. Bronstein, P. C. Weber, and S. Weller, Virology 215:152-1 61, 1996). nuc mutants are defective in one or more stages of genome m aturation and appear to package DNA into aberrant or defective capsids which fail to egress from the nucleus of infected cells. In this stud y, we used pulsed-field gel electrophoresis to test the hypothesis tha t the defects in nuc mutants are due to the failure of the newly repli cated viral DNA to be processed properly during DNA replication and/or recombination. Replicative intermediates of HSV-1 DNA from both wild- type- and mutant-infected cells remain in the wells of pulsed-field ge ls, while free linear monomers are readily resolved. Digestion of this well DNA with restriction enzymes that cleave once in the viral genom e releases discrete monomer DNA. from wild-type virus-infected cells b ut not from nuc mutant-infected cells. We conclude that both wild-type and mutant DNAs exist in a complex, nonlinear form (possibly branched ) during replication, The fact that discrete monomer-length DNA cannot be released from nuc DNA by a single-cutting enzyme suggests that thi s DNA is more branched than DNA which accumulates in cells infected wi th wild-type virus. The well DNA from cells infected with wild-type an d nuc mutants contains XbaI fragments which result from genomic invers ions, indicating that alkaline nuclease is not required for mediating recombination events within HSV DNA, Furthermore, nuc mutants are able to carry out DNA replication-mediated homologous recombination events between inverted repeats on plasmids as evaluated by using a quantita tive transient recombination assay. Well DNA from both wild-type- and mutant-infected cells contains free U-L termini but not free U-S termi ni. Various models to explain the structure of replicating DNA are con sidered.