Fine mapping of the latency-related gene of herpes simplex virus type 1: alternative splicing produces distinct latency-related RNAs containing open reading frames

Abstract

The latency-related (LR) gene of herpes simplex virus type 1 (HSV-1) is transcriptionally active during HSV-1 latency, producing at least two LR-RNAs. The LR gene partially overlaps the immediate-early gene ICPO and is transcribed in the opposite direction from ICPO, producing LR-RNAs that are complementary (antisense) to ICPO mRNA. The LR gene is thought to be involved in HSV-1 latency. We report here the fine mapping and partial sequence analysis of this HSV-1 LR gene. 32P-labeled genomic DNA restriction fragments and synthetic oligonucleotides were used as probes for in situ hybridizations and Northern (RNA) blot hybridizations of RNA from trigeminal ganglia of rabbits latently infected with HSV-1. The two most abundant LR-RNAs appeared to share their 5' and 3' ends and to be produced by alternative splicing. These LR-RNAs were approximately 2 and 1.3 to 1.5 kilobases in length and were designated LR-RNA 1 and LR-RNA 2, respectively. Their 5' ends started approximately 1,210 nucleotides downstream from the 3' end of the ICPO mRNA. Their 3' ends overlapped ICPO by nearly 1,000 nucleotides. LR-RNA 1 appeared to have at least one intron removed, while LR-RNA 2 appeared to have at least two introns removed. The LR-RNAs contained two potential long open reading frames, suggesting the possibility that one or more of the LR-RNAs may be a functional mRNA.

Experimental investigation of herpes simplex virus latency

“…While the fact that secondary splicing of introns is not a well-understood or described phenomenon has led some to speculate that an independently expressed small species is generated from a "second" LAT promoter, the recent demonstration that it can be isolated in lariat form is clearly consistent with a resplicing mechanism for its formation. Finally, while some laboratories have reported that the lower-abundance smaller species is actually made up of two partially contiguous transcripts differing by 50 bases (52,179,304,305), the report by Wu et al demonstrates that these two species are actually the same RNA differing in their physical configuration (321).…”

“…Although not strictly necessary for investigation of the genetic requirements for reactivation by using mutant viruses or for general characterization of LATs (96,205,207,208,304,305), the ability to efficiently induce reactivation is of great value for analyzing the nature of the changes seen in neural tissue during reactivation and for analyzing the function of critical portions of the viral genome on reactivation itself (16,17,109,294). Despite these advantages, efficient establishment of latency and reactivation is confined to HSV-1 in rabbits and is highly strain dependent (87,112).…”

“…Much of our picture of how latent phase transcripts are expressed and how they may function in reactivation is based upon the detailed characterization of these transcripts by Northern blot, S1, and RNase resistance analysis of RNA-DNA and RNA-RNA hybrids (52,57,60,137,178,179,270,271,282,286,299,300,304,305,331,332). For this review, all our mapping data have been correlated with the sequence of the 17syn ϩ strain, and minor differences in sequence between strains noted in the primary references have no impact upon the general picture outlined below.…”

A Herpes Simplex Virus Type 1 Latency-Associated Transcript Mutant with Increased Virulence and Reduced Spontaneous Reactivation

“…The construction and properties of dLAT2903 and its marker-rescued virus dLAT2903R, dLAT371 and its marker-rescued virus dLAT371R, and LAT3.3A (previously designated LAT1.5a) have been described previously (14,17,18). The LAT transcript (24,25). A very stable and easily detected 2-kb LAT (solid rectangle) appears to be an intron derived by splicing of the primary LAT (6).…”

“…LAT is initially transcribed as an 8.3-kb RNA (4,26). This primary LAT transcript gives rise to a family of LAT RNAs, including the very stable 2-kb LAT (20,24,25), which appears to be an intron produced by splicing (6). LAT transcription-negative mutants have been shown to reactivate poorly by explant or induced reactivation in the mouse (9,10,21), by induced reactivation in the rabbit (1,23), and by spontaneous reactivation in the rabbit (14,17).…”