Nov 25, 1988 - HSV-1 latency in trigeminal ganglia, but suggest a role in reactivation ... any, that these transcripts play in one or more of the stages required for ...
The EMBO Journal vol.8 no.2 pp.505 - 511, 1989
Herpes simplex virus type 1 latency-associated transcripts are evidently not essential for latent infection
Israel Steiner, Jordan G.Spivack, Ronald P.Lirette, S.Moira Browni, Alasdair R.MacLean2, John H.Subak-Sharpe1 and Nigel W.Fraser The Wistar Institute, Philadelphia, PA 19104, USA, 'MRC Virology Unit, University of Glasgow, and Wellcome Unit of Molecular Parasitology, Department of Veterinary Parasitology, University of Glasgow, Church Street, Glasgow G 1I 5JR, UK Communicated by J.H.Subak-Sharpe
The herpes sinplex virus type 1 (HSV-1) transcripts that can be detected during latent infection by Northern blot analysis in human and experimental animal sensory ganglia are encoded by diploid genes. To investigate their role in latent infection we studied HSV-1 variant 1704, which has deleted most of the IRL copy of the coding region of these RNAs and has a 1.2-kb deletion that is immediately upstream of the coding region of the TRL copy. During primary infection, 1704 replicated in trigeminal ganglia with kinetics similar to the parent virus (17+) and established latent infection. However, while explant reactivation of latent HSV-1 from trigeminal ganglia was detected in 100% of 17+ infected mice within 7 days, the reactivation of 1704 was significantly delayed, and 31 days elapsed before eight out of nine mice became virus positive. The recognized HSV-1 latencyassociated RNAs were not detected during the latent state of 1704 by Northern blot analysis or in situ hybridization, which implies that the 1.2-kb deletion may contain the promoter or other important regulatory elements. The data indicate that detectable levels of these latencyassociated transcripts are not required for viral replication, establishment, or maintenance (>6 weeks) of HSV-1 latency in trigeminal ganglia, but suggest a role in reactivation. Key words: HSV- 1/herpes latency/latency-associated tran-
scripts/reactivation Introduction Herpes simplex virus type 1 (HSV-1) establishes latency in peripheral sensory ganglia and may reactivate to produce recurrent mucocutaneous lesions in the innervated dermatome (for review, see Hill, 1985). The molecular basis of latent herpetic infection within nervous tissue has been studied in ganglia obtained from both experimental animal models and human autopsy material (Rock and Fraser, 1983; Efstathiou et al., 1986; Stevens et al., 1987; Deatly et al., 1987; Spivack and Fraser, 1987, 1988a; Steiner et al., 1988). HSV-1 transcription during latency, as studied by in situ hybridization, is limited to the long repeat regions (Figure 1B). Three herpes-specific RNAs (latency-associated transcripts, LATs), 2.0, 1.5 and 1.45 kb, are detectable by ©IRL Press
Northern blot analysis, and map to a 3-kb location within the region positive by in situ hybridization (Spivack and Fraser, 1987). These HSV-1-specific RNAs map within the long internal (IRL) and terminal repeats (TRL), and are transcribed in the opposite direction to immediate early gene 1 (IEI, ICPO). These transcripts are present in reduced amounts in acutely infected mice (Spivack and Fraser, 1988a) and infected tissue culture cells (Spivack and Fraser, 1987), and do not belong to any of the recognized classes of HSV- 1 genes (Spivack and Fraser, 1988b). However, the role, if any, that these transcripts play in one or more of the stages required for latent infection, i.e. (i) transport from the site of initial infection to the nervous system, (ii) establishment of latent infection, (iii) maintenance of the latent state, and (iv) reactivation, is unknown, and is presently a main focus of research. HSV-l mutants can be used to investigate the function and importance of the LATs during acute infection, latent infection and reactivation. HSV-1 strain 17+ variant 1704 (MacLean and Brown, 1987) has a 3.8-kb deletion in the IRL and adjacent unique region, and a 1.2-kb deletion in the TRL (Figure 1). The exact end points of the deletions are at present being sequenced. The IRL/UL deletion removes between 1.2 kb and 2.4 kb of the 3.0-kb PstI-MluI fragment of BamHI b and extends upstream of the 5' end of the LATs. The deleted sequences in the TRL are contained within a 1.6-kb segment, nucleotide position 7057-8721, as assessed by positive hybridization using short synthetic oligonucleotides. The deletion does not extend into the 5' end of the LATs at nucleotide position 6906 but extends between 150 and 550 bases upstream of it (Perry and McGeoch, 1988; F.Junejo, personal communication). The proposed promoter region (TATA box, CAAT box and Spl binding sites) (Wechsler et al., 1988) starting at nucleotide position 7589 is removed by the deletion. The deletions in TRL and IRL do not extend into HpaI m within which the IE1 gene is fully contained (Perry and McGeoch, 1988). The deletions in 1704 do not significantly impair its ability to replicate in tissue culture (MacLean and Brown,
1987). We have studied the behavior of HSV-1 17+ and the variant 1704 in the trigeminal ganglia of mice during acute infection, latent infection and explant reactivation. 1704 replicated in the trigeminal ganglia of infected mice to the same extent as 17+ and established latency in almost all of the infected animals. However, following trigeminal ganglia explant, the reactivation time for 1704 was significantly prolonged relative to 17+. The HSV-1 LATs which were present during latent infection with 17+, could not be detected with 1704 either by Northern blot analysis or by in situ hybridization. Therefore, these data indicate that: (i) the deletion in 1704 may contain the promoter or important regulatory elements for the HSV-1 LATs, (ii) if these RNAs are essential for the establishment, maintenance and reactivation of latency, levels below detection by 505
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Northern blot analysis or in situ hybridization are sufficient, and (iii) the deleted genetic information in 1704 modifies the reactivation process.
Results
day 10, reached a peak on day 11 and the last death was day 15. The survival rate for 1704-infected mice (38/48, 79%) was greater than for 17+-infected mice (15/40, 38%). Thus, the information deleted from 1704 may affect HSV-1 virulence in the mouse.
Acute viral replication in trigeminal ganglia and survival rates Following corneal inoculation, the parental virus 17+, and the variant 1704, replicated in mouse trigeminal ganglia (Figure 2). Virus titers of 5.6 a 3.1 x 103 and 3.5 + 2.8 x 102 p.f.u. per ganglion were observed with 1704 and 17+, respectively, on the second day post-infection. The peak of replication for 1704 was on the third day and for 17+ on the fourth day, and the titers then declined until day 9. No virus was detectable in trigeminal ganglia after the ninth day post-infection. Thus, there was no significant impairment in the ability of 1704 to replicate in mouse trigeminal ganglia or in the ability of the mouse to clear the acute infection caused by the virus. In the group infected with strain 17+, the first death occurred on day 6 postinfection, peaked on day 8 and the last death was observed on day 11. With mice infected with 1704 the first death was
Explant reactivation of latent 17+ and 1704 Reactivation of latent HSV-1 was assayed for 28-44 days post-infection by incubating explanted ganglia with monolayers of susceptible cells (CV-1), and inspecting the monolayers daily for cytopathic effects. Every 5-7 days the explanted ganglia were transferred to fresh cells. No infectious virus was detectable in latently infected ganglia at explant, as measured by virus titer count in ganglia homogenates. In all mice infected with strain 17+ (5 mice, 9/10 ganglia) reactivation was detectable between 6 and 7 days post-explant (Figure 3). Similarly, in the trigeminal ganglia of mice infected with strain F reactivation was present in all animals (4) between days 8 and 9 post-explant (data not shown). The reactivation of latent 1704 from trigeminal ganglia was significantly delayed. Reactivated 1704 was first detected on day 10, and was present in 14 out of 18 ganglia (8/9 mice) by the 31st day. Since 17+ as
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Fig. 2. HSV-1 titers in mouse trigeminal ganglia during acute infection. Each point represents the geometric mean titer from eight individually titered ganglia at the indicated time (days) post-infection, in two different experiments. Titers are plotted on a logarithmic scale as plaque forming units/trigeminal ganglia (PFU/TG). The standard deviations for all time points was ' o
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Fig 5. In situ hybridization of HSV-1 latently infected mice trigeminal ganglia. The probe used was 35S-labeled nick-translated HSV-1 Pst-Mlul DNA (Figure 1). Positive hybridization over nuclei of neurons from trigeminal ganglia of mice latently infected with HSV-1 strain 17+ (panel A, 20 h). No hybridization signal was present in trigeminal ganglia tissue from mice latently infected with 1704 (panel B, 48 h) or when over exposed (panel C, 4 days).
Analysis of reactivated 1704 DNA To confirm that the genome of 1704 remained unchanged during the latent infection, 32P-labeled DNA was prepared in vivo from virus stocks of HSV-1 strain 17+, variant 1704 and plaque-purified 1704 reactivated from the trigeminal ganglia of several latently infected mice, and subjected to restriction enzyme analysis. The BamHI and HpaI profiles are shown in Figure 6. The profiles of the four plaquepurified reactivated 1704 DNA samples are not distinguishable from the pattern of the original 1704 stock and differ from that of the parental HSV-1 strain 17+, as previously described (MacLean and Brown, 1987). To further confirm this, Southern blots were carried out on DNA isolated from the above viruses, digested with BamHI or HpaI and hybridized with nick-translated HSV-1 strain 17+ BamHI restriction fragment b (Figures 1 and 7). The data demonstrate that the deletions in the 1704 isolates were not altered from the input 1704. The profiles of 17+ and 1704 were the same as previously described (MacLean and Brown, 1987). Thus, the 1704 deletions have not changed during acute infection, latency or reactivation.
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Discussion The mechanism(s) employed by human neurotropic herpes viruses (HSV-1, HSV-2 and varicella-zoster) to establish latent infection in peripheral sensory ganglia, to maintain the latent state for the lifetime of the individual and to reactivate once or many times, years following the primary infection, are unknown. Experimental animal models have yielded some information about the molecular state of HSV-l DNA in latently infected nervous tissue (Rock and Fraser, 1983; Efstathiou et al., 1986), and viral transcripts expressed during latency. These models have been found to be a valid paradigm for HSV- 1 transcription during latency in human peripheral nervous system tissue. The LAT pattern of viral transcription, identified in latently infected ganglia of the mouse initially infected via the cornea or via the foot pad (Spivack and Fraser, 1987; Stevens et al., 1987), reflects the situation in human trigeminal ganglia (Steiner et al., 1988). Several roles have been postulated for these latency508
Fig. 6. Autoradiographs of in vivo 32P-labeled HSV-1 DNA digested with BamHI (panel A) and HpaI (panel B). Lanes 1-4, plaquepurified reactivated virus from trigeminal ganglia of mice latently infected with deletion variant 1704; lane 5, variant 1704; lane 6,
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associated RNAs. Since they are transcribed in the opposite direction to the immediate early (alpha) gene IEI (ICPO), but overlap only its 3' end (Spivack and Fraser, 1987), it was suggested by Stevens et al. (1987) that they could be
trans-acting inhibitory messages (Green et al., 1986; Chang
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interrupt the latent state and initiate reactivation. However, since spontaneous reactivation has not been observed during Reactivated 1704 Re te 4704 latent infection in the mouse eye and foot pad experimental :0 and in vivo reactivation is variable, reactivation is A B models, E ) in explanted ganglia. The present study elucidates examined b a d d the role the LATs may play during the first, second and VAMP e' partially also the fourth stages of latent infection. e The genes encoding the LATs are diploid. HSV-1 variant 1- - - fft "a b' 1704 contains two deletions. The 3.8-kb deletion within m BamHI b covers the location of the LATs apart from their 3' end. The 1.2-kb deletion within BamHI e is located within the HpaI o fragment, between nts 7057 and 8721 and does not extend into the 5' end of the LATs at 6906. The deletion within the repeat region of BamHI e is completely contained within the region deleted in BamHI b. Neither deletion AW s > _tv overlaps with ICPO (Figure 1) or affects ICPO mRNA synthesis (Figure 4B). The observation that the LATs are not detectable during latent 1704 infection (sensitivity
