A herpes simplex virus type 1 latency-associated transcript mutant reactivates with normal kinetics from latent infection

Abstract

The herpes simplex virus type 1 (HSV-1) latency-associated transcripts (LATs) accumulate in neuronal nuclei of latently infected ganglia. Explant reactivation kinetics of LAT deletion mutants in the mouse eye model have suggested a role for the LATs in the reactivation process. This report describes the construction and characterization of an HSV-1 strain HFEM mutant, TB1, disrupted within both copies of the LAT gene. TB1 contains a 440-base-pair segment of bacteriophage lambda DNA in place of a 168-base-pair deletion within the transcribed portion of the LAT gene. The 2.0-kilobase LAT was not produced after infection of tissue culture cells with TB1, but a 0.7to 0.8-kilobase RNA was expressed. TB1 did establish latent infection after corneal inoculation as efficiently as the parental virus, and its reactivation kinetics from explanted ganglia were similar to those of HFEM. During latent infection with TB1, HSV-1 transcripts were not detectable. Rescuant virus (TB1-R) contained intact LAT genes, synthesized full-length LAT transcripts during productive infection in tissue culture, and reactivated from ganglionic explants of latently infected mice with normal kinetics. Thus, any function these transcripts have in the reactivation process appears to include the region between the putative LAT promoter and the disruption in TB1-a region of approximately 1,600 nucleotides, 800 of which encode the LATs.

Selection of a nonconsensus branch point is influenced by an RNA stem-loop structure and is important to confer stability to the herpes simplex virus 2-kilobase latency-associated transcript

“…The TB1 mutation consists of a deletion and insertion of foreign DNA in the middle of the 2-kb LAT sequence. Surprisingly, no 2-kb LAT can be detected in TB1-infected cells or latently infected murine trigeminal ganglia (4).…”

“…However, the 2-kb LAT is replaced by smaller introns, the farthest 3Ј of which can be detected by Northern blot analysis (4). It was previously postulated that this RNA arose from a promoter activity lying within the lambda DNA insert (4).…”

“…CV-1 cells were maintained in Eagle's minimal essential medium plus 5% calf serum. HSV-1 mutant TB1 and HSV-1 F were prepared as previously described (4,9,10).…”

“…CV-1 cells were maintained in Eagle's minimal essential medium plus 5% calf serum. HSV-1 mutant TB1 and HSV-1 F were prepared as previously described (4,9,10).Plasmid construction. The 2.8-kb PstI-MluI restriction fragment encompassing the LAT gene of HSV-1 F was subcloned into the EcoRI and HindIII sites of pcDNA3 (Invitrogen) as described previously (58).…”

Experimental investigation of herpes simplex virus latency

“…This was first reported by Leib et al (150), who showed that fewer trigeminal ganglia explanted following latent infection with a mutant of the KOS strain of HSV-1 in which the latent phase promoter was specifically deleted were able to produce virus than were control ganglia latently infected with wt virus. Mutants of the 17syn ϩ strain of HSV-1 showed similar reduced and/or delayed recovery levels from latently infected murine dorsal root and trigeminal ganglia (15,56,275). Despite this, Izumi et al (119) and Devi-Rao et al (56) did not see such a delay or reduction in virus recovery from spinal ganglia latently infected with several KOS(M) derived LAT-negative mutants.…”