2 OPEN READING FRAMES (ORF1 AND ORF2) WITHIN THE 2.0-KILOBASE LATENCY-ASSOCIATED TRANSCRIPT OF HERPES-SIMPLEX VIRUS TYPE-1 ARE NOT ESSENTIAL FOR REACTIVATION FROM LATENCY

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) are dispensable for establishment and maintenance of latent in fection. However, the LATs have been implicated in reactivation of the virus from its latent state. Since the reported LAT deletion and/or i nsertion variants that are reactivation impaired contain deletions in the putative WT promoter, it is not known which LAT sequences are invo lved in reactivation. To examine the role of the 2.0-kb LAT in the pro cess of reactivation and the functional importance of the putative ope n reading frames (ORF1 and ORF2) contained within the 2.0-kb WT, we ha ve constructed an HSV-1 variant that contains a precise deletion and i nsertion within the WT-specific DNA sequences using site directed muta genesis. The HSV-1 variant FS1001K contains an 1,186-bp deletion start ing precisely from the 5' end of the 2.0-kb LAT and, for identificatio n, a XbaI restriction endonuclease site insertion. The FS1001K genome contains no other deletions and/or insertions as analyzed by a variety of restriction endonucleases. The deletion in FS1001K removes the ent ire 556-bp intron within the 2.0-kb WT, the first 229 nucleotides of O RF1, and the first 159 nucleotides of ORF2 without having an affect on the RL2 (ICP0) gene. Explant cocultivation reactivation assays indica ted that this deletion had a minimal effect on reactivation of the var iant FS1001K compared with the parental wild-type virus using a mouse eye model. As expected, Northern (RNA) blot analyses have shown that t he variant virus (FS1001K) does not produce the 2.0-kb WT or the 1.45 to 1.5-kb WT either in vitro or in vivo; however, FS1001K produces an intact RL2 transcript in tissue culture. These data suggest that the 2 .0-kb LAT putative ORF1 and ORF2 (or the first 1,186 bp of the 2.0-kb LAT) are dispensable for explant reactivation of latent HSV-1.