Establishment and maintenance of HSV latent infection is mediated through correct splicing of the LAT primary transcript

Abstract

To study the effect of the splicing of HSV-1 latency-associated transcript (LAT) on viral latency, we constructed two mutant viruses (FHλ+ and FHλ−) in which the 168-bp HpaI– HpaI fragment within the 2-kb LAT intron was replaced by a 447-bp bacteriophage lambda sequence. The lambda DNA was inserted in opposite orientations in FHλ+ and FHλ−. The mutation in FHλ+ disrupted the splicing of LAT primary transcript and altered both LAT exon and intron, whereas the mutation in FHλ− virus preserved the wild-type splice sites and the wild-type exon. Quantitative PCR analysis revealed that during latency there was a reduction in the number of viral genomes in mouse trigeminal ganglia infected with FHλ+ but not in those infected with FHλ−. The decrease in the latent genome numbers was not due to a defect in viral replication during the acute stage of infection. Furthermore, trigeminal ganglia from mice latently infected with FHλ+ displayed a slower reactivation kinetics compared to those infected with the parental strain. To elucidate the mechanism, we examined the antiapoptotic properties of these LAT constructs. A plasmid containing the pHλ+ construct was found to be less protective for cells against apoptosis than plasmid containing the wild-type or pHλ− constuct. These results suggest that the splicing of LAT primary transcript, and thus the correctly spliced exon product, play an important role in promoting the establishment and/or maintenance of viral latency.