Replication, recombination and packaging of amplicon DNA in cells infected with the herpes simplex virus type 1 alkaline nuclease null mutant ambUL12.
The alkaline nuclease (AN) encoded by gene UL12 of herpes simplex virus type 1 (HSV-1) is essential for efficient virus replication but its role during the lytic cycle remains incompletely understood. Inactivation of the UL12 gene results in reductions in viral DNA synthesis, DNA packaging, egress of DNA-containing capsids from the nucleus and ability of progeny virions to initiate new cycles of infection. Mechanistically, AN has been implicated in resolving branched structures in HSV-1 replicative intermediates prior to encapsidation, and promoting DNA strand-exchange. In this study, amplicons (bacterial plasmids containing functional copies of a virus replication origin and packaging signal) were used to analyse further the defects of the UL12 null mutant ambUL12. When ambUL12 was used as a helper virus both replication and packaging of the transfected amplicon were reduced in comparison with cells infected with wild-type (wt) HSV-1, and to extents similar to those previously observed for genomic ambUL12 DNA. By using amplicons differing at a specific restriction endonuclease site it was demonstrated that replicating molecules exhibit high frequency intermolecular recombination in both wt- and mutant-infected cells. Surprisingly, in the absence of the UL12 product, amplicons lacking a functional encapsidation signal were packaged. Moreover, these packaged molecules could be serially propagated indicating that they had been incorporated into functional virions. This difference in packaging specificity between wt HSV-1 and ambUL12 might indicate that replicative intermediates accumulating in the absence of AN contain an increased incidence of structures that can serve for the initiation of DNA packaging.
Porter
Iain M
IM
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
Stow
Nigel D
ND
MRC Virology Unit, Institute of Virology, Church Street, Glasgow G11 5JR, UK.
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D004261:DNA Replication; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D012260:Ribonucleases / Q000502:physiology*; D019065:Virus Assembly; D014779:Virus Replication
D012260:Ribonucleases; C066964:viral alkaline nuclease