ABSTRACT
The UL4 gene of herpes simplex virus type 1 is predicted to encode a 21.5 kDa protein of 199 amino acids. Although UL4 is dispensable for growth in cell culture, its function is not known. In the present study, the promoter of UL4 was examined and found to contain a cAMP-response element which bound the transcription factor CREB, and was strongly activated by cAMP. A recombinant virus, termed UL4HS, was constructed with a nonsense linker inserted into the UL4 open reading frame, to make a truncated UL4 protein of 60 amino acids. In addition, a marker-rescued virus, UL4HSMR, was constructed. Western immunoblot analysis revealed a 23 kDa band in extracts of wild-type and marker-rescued virus infected cells which was missing for UL4HS. Only modest differences were observed in the abilities of wild-type and UL4-mutant viruses to grow in Vero cells or in contact-inhibited mouse C3H/10T1/2 cells. In addition, there were only modest differences between the ability of UL4HS to replicate in murine corneas and trigeminal ganglia relative to wild-type viruses, and reactivation of UL4HS from latency was unaffected. Taken together, these data demonstrate that UL4 is dispensable for latency and pathogenesis in mice.
Subject(s)
Herpesvirus 1, Human/physiology , Viral Proteins/genetics , Virus Latency , Amino Acid Sequence , Animals , Cells, Cultured , Chlorocebus aethiops , Cyclic AMP/metabolism , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/growth & development , Humans , Kinetics , Mice , Mice, Inbred C3H , Molecular Sequence Data , Mutagenesis , PC12 Cells , Promoter Regions, Genetic , Rats , Recombination, Genetic , Vero Cells , Viral Proteins/physiology , Virus Activation , Virus ReplicationABSTRACT
The promoters of the latency-associated transcripts (LATs) of herpes simplex virus type 1 (HSV-1) strains KOS and McKrae were compared to examine their influence upon the reactivation phenotypes of these two strains. Unlike strain KOS, McKrae is readily reactivable using in vivo reactivation models. We found greater than 96% sequence conservation between KOS and McKrae in the LATs promoter region, and both promoters showed equivalent basal and inducible activities. An inter-strain recombinant (termed MK13) was constructed in which the LATs promoter of HSV-1 McKrae was recombined into the background of HSV-1 strain KOS. In a murine u.v. light-induced reactivation model, virus shedding was detected by eye swabbing in two of 44 (5%) mice infected with KOS, 20 of 42 (48%) mice infected with McKrae and none of 45 (0%) mice infected with MK13. These data show that the LATs promoters of these viruses are structurally and functionally similar and that transfer of the LATs promoter from McKrae into KOS is insufficient to confer a reactivatable phenotype.