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. 2012 Aug;86(16):8848-58.
doi: 10.1128/JVI.00652-12. Epub 2012 Jun 13.

Influence of herpes simplex virus 1 latency-associated transcripts on the establishment and maintenance of latency in the ROSA26R reporter mouse model

M P Nicoll  1 , J T Proença, V Connor, S Efstathiou
Affiliations

Influence of herpes simplex virus 1 latency-associated transcripts on the establishment and maintenance of latency in the ROSA26R reporter mouse model

M P Nicoll et al. J Virol. 2012 Aug.

Abstract

Herpes simplex virus 1 (HSV-1) can establish life-long latent infection in sensory neurons, from which periodic reactivation can occur. During latency, viral gene expression is largely restricted to the latency-associated transcripts (LATs). While not essential for any phase of latency, to date the LATs have been shown to increase the efficiency of both establishment and reactivation of latency in small-animal models. We sought to investigate the role of LAT expression in the frequency of latency establishment within the ROSA26R reporter mouse model utilizing Cre recombinase-encoding recombinant viruses harboring deletions of the core LAT promoter (LAP) region. HSV-1 LAT expression was observed to influence the number of latently infected neurons in trigeminal but not dorsal root ganglia. Furthermore, the relative frequencies of latency establishment of LAT-positive and LAT-negative viruses are influenced by the inoculum dose following infection of the mouse whisker pads. Finally, analysis of the infected cell population at two latent time points revealed a relative loss of latently infected cells in the absence of LAT expression. We conclude that the HSV-1 LATs facilitate the long-term stability of the latent cell population within the infected host and that interpretation of LAT establishment phenotypes is influenced by infection methodology.

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Figures

Fig 1
Fig 1
Generation of Cre reporter viruses bearing deletions of the core LAT promoter. (a) Genomic structures of HSV CMVCre, HSV CMVCreΔLAT, HSV CMVCreΔLAT-GFP, and HSV CMVCreREV. All four viruses harbor an HCMV MIEP-Cre recombinase expression cassette within the nonessential HSV-1 US5 locus. (b) Genomic structures as analyzed by Southern blot hybridization. Restriction digest with HpaI demonstrates all predicted restriction fragments. Deletion of the core LAT promoter within HSV CMVCreΔLAT and HSV CMVCreΔLAT-GFP as well as its rescue within HSV CMVCreREV was confirmed by PstI restriction digest. The 3.3-kb HpaI fragment encoded within pPSTD1 was utilized as a radiolabeled probe. (c) LAT expression was quantified by qRT-PCR from total RNA extracted from TG latently infected with all four viruses, utilizing primers for major LAT and cyclophilin RNA. Histograms represent the mean (± SEM) numbers of major LAT RNA copies per 103 copies of cyclophilin RNA from triplicate PCRs.
Fig 2
Fig 2
Cre reporter virus growth characterization. (a) Low (0.01) MOI. In vitro growth curves of recombinant viruses and wild-type strain SC16 from duplicate experiments performed in BHK cells are shown. (b and c) In vivo replication of recombinant viruses in the ears (b) and pooled CII, CIII, and CIV DRG (c) 3, 5, and 7 d.p.i. following inoculation of BALB/c mice on the left ear with 2 × 106 PFU. Data points represent mean (± SEM) viral titers from five mice at each time point. (d) Virus latent DNA loads. qPCR was performed on DNA extracted from pooled CII, CIII, and CIV DRG from five latently infected mice, utilizing primers for the ICP0 promoter and cellular APRT. Values represent mean (± SEM) numbers of HSV genome copies per 104 copies of APRT from triplicate PCRs. (e) Explant reactivation competence of HSV recombinants. CII, CIII, and CIV ganglia from latently infected mice were cultured for 5 days and assayed for reactivating virus. Each point represents viral titers detected per mouse, and the bars represent the averages of these data. (f to h) In vivo replication of recombinant viruses in both whisker pads (f), TG pairs (g), and brain (h) 3, 4, and 6 d.p.i. following inoculation of BALB/c mice on both whisker pads. Data points represent mean (± SEM) viral titers from five mice at each time point. (i and j) In vivo replication of recombinant viruses in both whisker pads (i) and TG pairs (j) 3, 4, and 5 d.p.i. following inoculation of ROSA26R mice on both whisker pads with 106 PFU. Data points represent mean (± SEM) viral titers from five mice at each time point. No mortality was observed in any of the above-described experiments.
Fig 3
Fig 3
LAT promoter-negative HSV-1 establishes latency at a higher frequency than the wild type in the TG. (a and b) Detection of Cre-marked cells following infection of ROSA26R mice with HSV CMVCre, HSV CMVCreΔLAT, HSV CMVCreΔLAT-GFP, and HSV CMVCreREV in the DRG (a) and TG (b). Histograms represent the mean (± SEM) numbers of positive cells per ganglion detected at the specified time points postinfection. P values of 0.005 and 0.058 are represented by ** and *, respectively. (c and d) Light micrographs of latently infected DRG (c) and TG (d, panels i and ii). Bars, 1 mm. A single HSV CMVCre-infected mouse was euthanized following whisker pad infection when pathology reached a predetermined threshold of severity. No other mortality was recorded.
Fig 4
Fig 4
Explant reactivation kinetics are unimpaired in the absence of LAT expression following latency in the TG. (a) ROSA26R mice were inoculated with 106 PFU of each recombinant virus per whisker pad. At 40 d.p.i., TG were dissected (n = 10 per recombinant), cut into five pieces, and plated onto MRC-5 cell monolayers, with pieces from one whole TG per dish. Plates were scored positive for reactivation upon observation of CPE within monolayers. (b) PCR with primers flanking the LAT region was designed to identify each recombinant. (c) Reactivating virus identity was successfully confirmed by PCR. The identities of viruses reactivating 5 and 7 days postexplant are shown. No mortality was recorded within this experiment.
Fig 5
Fig 5
In the absence of LAT expression, the frequency of latency establishment is enhanced at “low” doses but diminishes at increasing doses. (a) R26R mice were infected on both whisker pads at a range of doses (105 to 6 × 107 PFU) and TG dissected ∼30 d.p.i. for marked-cell quantification. Histograms represent the mean (± SEM) number of positive cells per ganglion at each dose. P values of <0.00005, <0.002, and 0.01 are represented by ***, **, and *, respectively. (b) R26R mice were infected on the left ear at 105 or 107 PFU doses. CII, III, and IV DRG were dissected 30 d.p.i. for marked-cell quantification. Histograms represent the mean (± SEM) number of positive cells per ganglion at each dose.
Fig 6
Fig 6
Maintenance of the latent cell reservoir is unstable in the absence of LAT expression. (a) R26R mice were infected on both whisker pads with 106 PFU and TG dissected at latency (29 to 33 d.p.i.) and “late” latency (110 to 140 d.p.i.) for marked-cell quantification. Histograms represent the mean number of marked cells lost per TG per day ± SEM. “LAT-pos” denotes both HSV CMVCre and HSV CMVCreREV data. “LAT-neg” denotes both HSV CMVCreΔLAT and HSV CMVCreΔLAT-GFP data. (b) Light micrographs of HSV-1 antigen-positive cells as detected via whole-ganglion immunohistochemistry. An example of acute infection within the TG at 4 d.p.i. is displayed in panel i. Panel ii displays antigen-positive cells detected in 20-μm-thick sections of latently infected TG at 44 d.p.i. Panel iii displays examples of antigen-positive cells detected at 44 d.p.i. to 69 d.p.i. All bars represent 100 μm.
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