Tissue-Specific Splicing of the Herpes Simplex Virus Type 1 Latency-Associated Transcript (LAT) Intron in LAT Transgenic Mice

Abstract

To study the regulation of herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) expression and processing in the absence of other cis and trans viral functions, a transgenic mouse containing the region encompassing the LAT promoter (LAP1) and the LAT 5 exon through the 2.0-kb intron was created. LAT expression was detectable by reverse transcriptase PCR (RT-PCR) in a number of tissues, including the dorsal root ganglia (DRG), trigeminal ganglia (TG), brain, skin, liver, and kidney. However, when the accumulation of the 2.0-kb LAT intron was analyzed at the cellular level by in situ hybridization, little or no detectable accumulation was observed in the brain, spinal cord, kidney, or foot, although the 2.0-kb LAT intron was detected at high levels (over 90% of neurons) in the DRG and TG. Northern blot analysis detected the stable 2.0-kb LAT intron only in the sensory ganglia. When relative amounts of the spliced and unspliced LAT within the brain, liver, kidney, spinal cord, TG, and DRG were analyzed by real-time RT-PCR, splicing of the 2.0-kb LAT intron was significantly more efficient in the sensory ganglia than in other tissues. Finally, infection of both transgenic mice and nontransgenic littermates with HSV-1 revealed no differences in lytic replication, establishment of latency, or reactivation, suggesting that expression of the LAT transgene in trans has no significant effect on those functions. Taken together, these data indicate that the regulation of expression and processing of LAT RNA within the mouse is highly cell-type specific and occurs in the absence of other viral cis-and trans-acting factors.Herpes simplex virus type 1 (HSV-1) latency has long been characterized by the accumulation of a single abundant RNA, the latency-associated transcript (LAT). From the 8.3-to 8.5-kb polyadenylated primary transcript, a very stable 2.0-kb intron, which can be readily detected in the nuclei of latently infected sensory neurons, is spliced (6,21,29,32). Further processing of the 2.0-kb intron can yield a 1.5-kb product in a subpopulation of neurons (20,31). In sensory ganglia, LAT expression seems to be tightly regulated; during the lytic infection, most neurons express either the LAT or lytic genes, not both (21). In addition, there is a sharp drop in the amount of LAT during reactivation (3, 30), with a decrease in transcriptional permissiveness of the LAT promoter occurring as early as 30 min postexplant (1). These observations, combined with genetic data describing LAT mutants as being defective in reactivation (2,10,13,16,24), have led to the model showing that the LAT RNA may play a role in transcriptional silencing of lytic genes during latency (2,7,15,36). Additionally, a cis element encompassing the promoter and enhancer (rcr) may regulate LAT expression to allow the establishment of latency and the occurrence of reactivation (1,14). Two central questions that have been difficult to address in vivo, however, are (i) how dependent the regulation of LAT transcription on other viral el...

Efficacy of Herpes Simplex Virus Vector Encoding the Human Preproenkephalin Gene for Treatment of Facial Pain in Mice

“…27 Analysis of vector DNA was performed with HSV-1 polymerase gene primers and probe (forward 5'-AGAGGGACATCCAGGACTTTGT-3'; reverse 5'-CAGGCGCTTGTTGGTGTAC-3'; probe 5'-ACCGCCGAACTGAGCA-3'), as described elsewhere. 28 Analysis of vector gene expression (cDNA) was performed using primers and probes specific to the HSV-1 latency-associated transcript (LAT)—a marker of HSV latency in neurons, as previously described, 29,30 and specific to the human preproenkephalin gene (forward 5'-ACGTGCAGCTACCGCCTAGT-3'; reverse 5'-GGCAGTTTACCTTCACATTCCATT-3'; probe 5'-CCGACATCAACTTCCTGGCTTGCG-3'), designed with the Primer Express 1.0 software (PE Applied Biosystems).…”

Herpes Simplex Virus Type 2 (HSV-2) Establishes Latent Infection in a Different Population of Ganglionic Neurons than HSV-1: Role of Latency-Associated Transcripts

“…Although there are limits to this type of data acquisition and analysis, this observation is consistent with the hypothesis that A5-and KH10-positive neurons are equally permissive for the accumulation of HSV-1 and HSV-2 LAT. Second, we examined the trigeminal ganglia of transgenic mice that contain the HSV-1 LAT-expressing region (8). In these mice, 7.7% (79/1,025) of the LAT-expressing neurons were A5 positive and 15.4% (154/1,000) were KH10 positive, which was very similar to the proportion of all ganglionic neurons that were A5 (8.4%; 86/1,026) and KH10 positive (14.9%; 150/1,007) in these tissues.…”

“…In those experiments requiring the use of an antiviral drug, acyclovir (1.2 mg/ml) was placed in the drinking water 40 h after viral inoculation. Uninfected trigeminal ganglia from LAT 3098 transgenic mice (8) were a generous gift of David Bloom (Gainesville, FL).…”

Characterization of a Novel Golgi Apparatus-Localized Latency Determinant Encoded by Human Cytomegalovirus

“…Similarly, the LATs of HSV are expressed during both the lytic and latent infections, but a role for LATs in viral replication has not been described. However, LATs are differentially transcribed and spliced depending on the context of infection (10,18). While pUL138 is synthesized in all infected cell types tested, the outcome of infection likely depends on the context-dependent balance of competing viral and cellular factors promoting virus replication or latency.…”