Transcription at Early Stages of Herpes Simplex Virus 1 Infection and during Reactivation

The program of gene expression exhibited by herpes simplex virus during productive infection of cultured cells is well established; however, less is known about the regulatory controls governing reactivation from latency in neurons. One difficulty in examining gene regulation during reactivation lies in distinguishing between events occurring in initial reactivating cells versus events occurring in secondarily infected cells. Thus, two inhibitors were employed to block production of infectious virus: acyclovir, which inhibits viral DNA synthesis, and WAY-150138, which permits viral DNA synthesis but inhibits viral DNA encapsidation. Latently infected murine ganglia were explanted in the presence of either inhibitor, and then amounts of RNA, DNA, or infectious virus were quantified. In ganglia explanted for 48 h, the levels of five immediate-early and early RNAs did not exhibit meaningful differences between acyclovir and WAY-150138 treatments when analyzed by in situ hybridization or quantitative reverse transcription-PCR. However, comparative increases in viral DNA and RNA content in untreated ganglia suggested that virus was produced before 48 h postexplant. This was confirmed by the detection of infectious virus as early as 14 h postexplant. Together, these results suggest that high levels of viral gene expression at 48 h postexplant are due largely to the production of infectious virus and subsequent spread through the tissue. These results lead to a reinterpretation of previous results indicating a role for DNA replication in immediate-early and early viral gene expression; however, it remains possible that viral gene expression is regulated differently in neurons than in cultured cells.

Section: Discussionmentioning

confidence: 69%

“…In addition, it has been reported that during explant reactivation from murine TG, E and leaky late transcripts are detected before IE transcripts (43). On the other hand, a recent investigation of this question with rabbit TG found no deviation from the canonical regulatory order (28).…”

mentioning

confidence: 82%

Herpes Simplex Virus 1 Immediate-Early and Early Gene Expression during Reactivation from Latency under Conditions That Prevent Infectious Virus Production

Pesola¹,

Zhu

Knipe

et al. 2005

“…Several groups have studied HSV gene transcription and translation in explanted ganglia as a model of reactivation (4,6,10,18,24,25,33,34). In some cases it was concluded that the transcriptional program during reactivation differed from that seen during lytic infection or that viral DNA replication was required for immediate-early gene expression.…”

Section: Discussionmentioning

confidence: 99%

Comparison of Herpes Simplex Virus Reactivation in Ganglia In Vivo and in Explants Demonstrates Quantitative and Qualitative Differences

Sawtell

Thompson

2004

The in vivo ganglionic environment directs the latent herpes simplex virus transcriptional program. Since stress-driven perturbations in sensory neurons are thought to play a critical role in the transition from latency to reactivation, a primary concern in the selection of a valid model of the molecular interactions leading to reactivation is the faithful recapitulation of these environments. In this study reactivation of latently infected ganglia excised and cultured in vitro (explanted) is compared to reactivation occurring in latently infected ganglia in vivo following hyperthermic stress. Three notable points emerged. (i) Neurons in explanted ganglia exhibited marked morphological changes within 2 to 3 h postexplant. DNA fragmentation in neuronal nuclei was detected at 3 h, and atypical expression of cell cycle-and stress-regulated proteins such as geminin, cdk2, cdk4, and cytochrome c became apparent at 2 to 48 h. These changes were associated with axotomy and explant and not with the initiation or progression of reactivation and were not observed in ganglia following in vivo hyperthermic stress. (ii) Despite these differences, during the first 22 h primary reactivation events were restricted to a very small number of neurons in vivo and in explanted ganglia. This suggests that at any given time only a few latently infected neurons are competent to reactivate or that the probability of reactivation occurring in any particular neuron is very low. Importantly, the marked changes detected in explanted ganglia were not correlated with increased reactivation, demonstrating that these changes were not associated with the reactivation process per se. (iii) Secondary spread of virus was evident in explanted ganglia within 36 h, an event not observed in vivo. We conclude that explant reactivation may provide an ancillary system for selected studies of the early events in reactivation. However, clear signs of neuronal degeneration within 2 to 3 h postexplant indicate that these ganglia are undergoing major physiological changes not associated with the reactivation process. This ongoing neurodegeneration could alter even the early virus-host interactions in reactivation, and thus caution in the extrapolation of results obtained in explants to the in vivo interactions initiating reactivation is warranted.

“…Since the gene encoding the ICP0 protein of HSV-1 is contained entirely within the region of the HSV-1 genome encoding LATs (reviewed in reference 3), it might be transcribed during latency, as recently reported by Chen et al for latently infected TG neurons (10). ICP0 is the first viral protein produced during the productive cycle and is thus classically considered a relevant indicator of early-stage reactivation (18,59,65). It nonspecifically transactivates both viral and cellular promoters (18,25,42) and plays a crucial role in the initiation of productive infection following low-multiplicity infections of cells in culture (7,8,17,63).…”

mentioning

confidence: 99%

Herpes Simplex Virus Type 1 Latently Infected Neurons Differentially Express Latency-Associated and ICP0 Transcripts

Maillet

Naas

Crépin

et al. 2006

Herpes simplex virus type 1 (HSV-1) is a human neurotropic virus, which establishes lifelong latent infection. During the acute phase of infection, viral replication is characterized by the expression of immediate early (IE), early, and late genes in an ordered cascade that leads to the release of infectious particles that can attach to nerve endings. Following axonal transport to neuronal cell bodies, HSV-1 can either replicate or persist in a latent state in the infected host (62).During latency infectious virus cannot be detected in neuronal tissues (62). The viral genome is circularized, complexed with histones, and maintained in a repressed state with the exception of the latency-associated transcripts (LATs) that are encoded within the repeats flanking the unique long region of the HSV-1 genome (12, 33, 60). The LATs (2 kb and 1.5 kb in size) are stable unpolyadenylated introns that are retained within the nuclei of latently infected neurons (reviewed in references 33 and 56) and are spliced from a primary 8.3-kb transcript (19). Their unusual stability has been related to modifications in the consensus sequences of splicing (84), resulting in an unstable 6.3-kb spliced exon (13, 85) and a stable 2-kb LAT intron that is expressed during both acute and latent infections (73,84). During latent neuronal infection, the 2-kb LAT may undergo subsequent splicing that results in a 1.5-kb LAT (1,46,70). The functions of LATs are not yet clearly understood, but mutational analyses and transfection assays of LATs strongly suggest that they have a key role in establishment of latency and/or viral reactivation (29,31,53,64,71,77), besides their antiapoptotic, anti-interferon, and proneuronal survival properties (24,32,52,54,55,78).Several studies failed to detect lytic viral gene expression during HSV-1 latency (20,33). However, some studies reported the detection of transcripts from genes encoding the immediate-early viral protein ICP4 and the early viral protein thymidine kinase (TK) in latently infected trigeminal ganglion (TG) neurons (9, 34). This observation was initially interpreted as a possible result of spontaneous reactivation (33). Since the gene encoding the ICP0 protein of HSV-1 is contained entirely within the region of the HSV-1 genome encoding LATs (reviewed in reference 3), it might be transcribed during latency, as recently reported by Chen et al. for latently infected TG neurons (10). ICP0 is the first viral protein produced during the productive cycle and is thus classically considered a relevant indicator of early-stage reactivation (18,59,65). It nonspecifically transactivates both viral and cellular promoters (18,25,42) and plays a crucial role in the initiation of productive infection following low-multiplicity infections of cells in culture (7,8,17,63). ICP0 is able to initiate both viral gene expression from quiescent genomes in cells in culture and viral reactivation from latently infected sensory neurons in vivo (6, 26, 27,