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Journal of General Virology (2015), 96, 2794 –2805 DOI 10.1099/vir.0.000189 Mutation of UL24 impedes the dissemination of acute herpes simplex virus 1 infection from the cornea to neurons of trigeminal ganglia Pierre-Alexandre Rochette, Amélie Bourget,3 Carolina Sanabria-Solano, Soumia Lahmidi, Gabriel Ouellet Lavallée and Angela Pearson Correspondence Angela Pearson Université INRS, INRS-Institut Armand-Frappier, 531 boulevard des Prairies, Laval, Québec H7V 1B7, Canada [email protected] Received 16 December 2014 Accepted 17 May 2015 Herpes simplex virus 1 (human herpesvirus 1) initially infects epithelial cells of the mucosa and then goes on to infect sensory neurons leading ultimately to a latent infection in trigeminal ganglia (TG). UL24 is a core herpesvirus gene that has been identified as a determinant of pathogenesis in several Alphaherpesvirinae, although the underlying mechanisms are unknown. In a mouse model of ocular infection, a UL24-deficient virus exhibited a reduction in viral titres in tear films of 1 log10, whilst titres in TG are often below the level of detection. Moreover, the efficiency of reactivation from latency was also severely reduced. Herein, we investigated how UL24 contributed to acute infection of TG. Our results comparing the impact of UL24 on viral titres in eye tissue versus in tear films did not reveal a general defect in virus release from the cornea. We also found that the impairment of replication seen in mouse primary embryonic neurons with a UL24-deficient virus was not more severe than that observed in an epithelial cell line. Rather, in situ histological analyses revealed that infection with a UL24-deficient virus led to a significant reduction in the number of acutely infected neurons at 3 days post-infection (p.i.). Moreover, there was a significant reduction in the number of neurons positive for viral DNA at 2 days p.i. for the UL24-deficient virus as compared with that observed for WT or a rescue virus. Our results supported a model whereby UL24 functions in the dissemination of acute infection from the cornea to neurons in TG. INTRODUCTION Herpes simplex virus 1 (HSV-1; human herpesvirus 1) replicates in epithelial cells and then infects the ends of sensory neurons that form neuronal ganglions (Roizman et al., 2007). HSV-1 persists in neurons as a life-long latent infection and may reactivate, often following stress, resulting in recurrent infections (Roizman et al., 2007). In healthy individuals, symptoms typically vary from cold sores to viral keratitis and in rare cases infections can lead to a life-threatening encephalitis (Whitley & Roizman, 2001). In people who are immunocompromised, e.g. people living with AIDS, neonates or stem cell recipients, the symptoms are often severe (Fatahzadeh & Schwartz, 2007). Many questions remain regarding the spread of infection within the host, which has hindered the development of treatments to prevent recurrent infections. UL24 is highly conserved across the family Herpesviridae (Davison, 1993; Roizman et al., 2007). UL24 is involved in nucleolar modifications during HSV-1 infection and impacts 3Present address: Montreal Chest Institute, McGill University Health Centre, 3650 Saint-Urbain, Montreal, Québec H2X 2P4, Canada. 2794 the nuclear egress of capsids (Bertrand & Pearson, 2008; Bertrand et al., 2010; Lymberopoulos et al., 2011). UL24 has also been shown to induce a G2/M cell cycle block when expressed in the absence of infection (Nascimento et al., 2009). Following infection of an epithelial-derived cell line, a UL24-deficient HSV-1 strain produced five to 10 times fewer infectious virions and exhibited a reduced plaque size as well as a temperature-sensitive syncytial phenotype (Jacobson et al., 1989a; Sanders et al., 1982; Tognon et al., 1991). In vivo, HSV-1 UL24 has been shown to be important for pathogenesis (Jacobson et al., 1998). Mice infected with a UL24-null virus exhibited a reduction in viral titres in trigeminal ganglia (TG) of up to 4 log10, a 2 log10 reduction in viral DNA and a 1 log10 reduction in lat transcripts present in latently infected TG, a drastic reduction in the severity of disease, as well as a reduction in the frequency of reactivation from latency in explant assays (Jacobson et al., 1998; LeivaTorres et al., 2010). Reduced pathogenesis has also been observed for UL24 mutants of other alphaherpesviruses, such as herpes simplex virus 2 (HSV-2; human herpesvirus 2) (Blakeney et al., 2005), equid herpesvirus 1 (Kasem et al., 2010) and varicella-zoster virus (human herpesvirus 3) Downloaded from www.microbiologyresearch.org by 000189 G 2015 The Authors IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 Printed in Great Britain Role of UL24 in acute infection of TG in vivo (Ito et al., 2005). The exact mechanisms through which UL24 modulates pathogenesis are unknown. In this study, we tested the hypothesis that the large reduction in viral titres in mouse TG observed with a UL24-deficient virus is the result of a defect in the dissemination of infection from the cornea to neurons of TG. (a) KOS vUL24XRescue UL24X 34 ºC 37 ºC RESULTS Construction and characterization of a UL24X rescue virus To confirm that reinsertion of the WT UL24 gene within UL24X could correct the temperature-sensitive syncytial plaque phenotype (Jacobson et al., 1989a; Tognon et al., 1991), we infected Vero cells at low m.o.i. with KOS, UL24X or vUL24X-Rescue (Fig. 1a) for 48 h. UL24X produced syncytial plaques at 37 and 39 uC, whilst vUL24XRescue produced non-syncytial plaques similar to those of KOS at both temperatures. We next tested the rescue virus in a one-step viral replication assay using m.o.i. 5 (Fig. 1b). Although levels for vUL24X-Rescue were significantly lower than those observed with KOS, we found that viral yields were increased for vUL24X-Rescue as compared with UL24X, resulting in significantly higher viral titres for the rescue virus at both 12 and 24 h post-infection (p.i.). Importantly, we demonstrated that vUL24X-Rescue behaved similarly to KOS in vivo. CD-1 mice (8 weeks old) were infected with either 2|106 or 6|102 p.f.u. per eye of KOS, UL24X or vUL24X-Rescue (Fig. 2). Viral titres in tear films were determined for 1–3 days p.i. (Fig. 2a–c). We found that the mean ocular viral titre for vUL24XRescue at an inoculum of 2|106 p.f.u. per eye was higher than that for UL24X and similar to that for KOS. At 3 days p.i., vUL24X-Rescue produced viral titres in TG similar to those of KOS and values for both viruses were significantly higher than the viral titres produced by UL24X http://vir.sgmjournals.org 39 ºC (b) 108 Viral titre (p.f.u. ml–1) UL24X contains stop codons in all three reading frames to block translation at codon 44. This design ensures that the mutant retains WT levels of the viral thymidine kinase (TK), whose gene (UL23) is coded on the opposite strand and partially overlaps UL24. UL24X was originally constructed by homologous recombination of the appropriate transfer vector and the KOS-based strain tk LTRZ-1 (Davar et al., 1994). The latter has a lacZ gene inserted within UL23, which can be used to select for recombinants. A rescue of tk LTRZ-1 has been described demonstrating that this rescue virus has a WT phenotype (Jacobson et al., 1998). Nevertheless, we wished to generate a direct rescue of UL24X for use in our subsequent experiments. A UL24X rescue virus (vUL24-Rescue) was generated by homologous recombination of infectious DNA from UL24X co-transfected with the transfer vector pAG5, which contains UL22, UL23 and UL24 from KOS. 107 106 105 KOS 104 vUL24X-Rescue UL24X 103 6 12 18 Time (h p.i.) 24 Fig. 1. Characterization of a UL24X rescue virus in cell culture. (a) Comparison of plaque morphology for KOS, UL24X and vUL24X-Rescue on Vero cells at 2 days p.i. grown at the indicated temperatures. (b) One-step replication assay comparing KOS, UL24X and vUL24X-Rescue. Data represent mean¡SD ; *statistically significant differences between vUL24X-Rescue and UL24X. (Fig. 2d). Moreover, there was no statistical difference in titres obtained from mice infected with KOS or vUL24XRescue at 6|102 p.f.u. per eye. Furthermore, the severity of disease was restored to WT levels with vUL24X-Rescue at 2|106 and 6|102 p.f.u. per eye (Table 1). We found no statistical difference in the number of TG that reactivated ex vivo from vUL24XRescue-infected mice versus KOS-infected mice; thus the defect in reactivation observed with UL24X was corrected (data not shown). These data confirmed that in vivo defects observed for UL24X were attributable to the UL24 mutation. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 2795 (b) 7 10 107 106 106 (d) 7 10 107 106 106 cu 24 X e 2× 6× 10 6 10 2 10 2 6× S vU L2 UL e cu 6 2 10 10 24 UL cu es X e 2× 6× 10 6× 4X -R e cu vU L2 4X -R es KO 2 6 2× S 2× X 24 UL es L2 4X -R 10 6 10 10 6× e cu S KO vU e cu es 4X -R L2 6× 10 10 2× 10 2× S KO 2 100 2 100 6 101 6 101 vU 4X -R es 102 S 102 103 KO 103 104 10 104 105 2× 105 6 Viral titre (p.f.u. ml–1) (c) 4X -R es 10 6 2× S vU L2 UL KO cu 24 X e 2× 6× 6× 4X -R es L2 vU L2 4X -R es KO cu e S 2× 2× S KO 10 6 100 10 2 100 10 2 101 10 6 101 vU Viral titre (p.f.u. ml–1) 102 L2 102 103 KO 103 104 vU 104 105 2× 105 10 6 Viral titre (p.f.u. ml–1) (a) 10 6 Viral titre (p.f.u. ml–1) P.-A. Rochette and others Fig. 2. Rescue of UL24X mutation corrects in vivo defects observed in the mouse ocular infection model. (a–c) Viral titres in tear films for (a) 1, (b) 2 and (c) 3 days p.i. (d) Viral titres in homogenized TG at 3 days p.i. *Statistically significant difference between vUL24X-Rescue and UL24X. Dashed lines indicate the detection limit of the experiment. Importance of UL24 for viral replication in primary embryonic neurons is similar to that in epithelialderived cell lines One possible explanation for the disparity between the impact of UL24 mutations on viral titres in the corneal epithelia and in TG might be a specific requirement of UL24 for replication in neurons. To test this hypothesis, we compared replication of KOS, vUL24X-Rescue and UL24X in mouse primary embryonic neurons (Fig. 3a). Neurons were infected at m.o.i. 0.1, and total virus was harvested at 12, 24, 36, 48 and 72 h p.i. We found that the reduction 2796 in titres for UL24X in neurons was of the same magnitude as the viral replication defect observed for UL24X in epithelial-derived cell lines (Jacobson et al., 1989a, 1998). Moreover, we obtained similar results using neurons derived from the human neuroblastoma cell line LA-N-5. Following differentiation by retinoic acid (RA), these cells develop into cholinergenic neurons. By day 8 post-RA treatment, w95 % of cells stained positive for the neuronal maturation marker Neurofilament M (Fig. 3b). Neurons were either mock-infected or infected with the WT virus KOS or UL24X at a m.o.i. 10. At 9 h p.i., cells were immunostained for the viral ssDNA-binding protein ICP8, a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 Journal of General Virology 96 Role of UL24 in acute infection of TG in vivo Table 1. Restoration of WT clinical signs with vUL24X-Rescue Disease score 0–4. Each row represents the clinical scores for one animal. Virus (p.f.u. per eye) Time (days p.i.) 1 2 3 4 5 6 7 8 9 10 KOS 26106 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + 0 + ++ 0 ++ ++ + ++ + + ++ +++ +++ + ++ ++ ++ ++ ++ ++ ++ +++ +++ + ++ +++ +++ +++ +++ +++ +++ ++++ +++ ++ ++ +++ +++ +++ +++ +++ +++ ++++ ++++ ++ +++ +++ +++ +++ +++ +++ +++ ++++ ++++ ++ +++ +++ +++ +++ +++ +++ +++ ++++ ++++ ++ vUL24X-Rescue 26106 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 + 0 0 0 ++ + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ +++ +++ ++ ++ ++ ++ ++ ++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ ++ +++ +++ +++ +++ +++ +++ ++++ +++ +++ ++ +++ +++ +++ +++ +++ +++ ++++ +++ +++ KOS 66102 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + + 0 0 0 0 0 0 0 0 + + 0 0 0 0 0 0 0 + + ++ 0 0 0 0 0 0 0 ++ ++ +++ 0 0 0 0 0 0 0 ++ ++ +++ 0 0 0 0 0 0 0 ++ ++ +++ 0 0 0 0 0 0 0 ++ ++ +++ 0 0 0 0 0 0 0 vUL24X-Rescue 66102 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 + 0 0 0 0 0 0 + 0 0 + 0 0 0 0 0 0 + 0 0 ++ 0 0 0 0 0 0 0 + + +++ 0 0 0 0 0 0 0 + + +++ 0 0 0 0 0 ++ 0 0 + ++ 0 0 0 0 0 ++ 0 0 + ++ 0 0 0 0 0 UL24X 26106 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 + 0 0 0 + 0 0 0 + 0 0 0 0 0 0 0 0 0 0 0 0 0 marker for viral replication compartments (VRCs). We observed similar large VRCs in both KOS- and UL24Xinfected LA-N-5 neurons (Fig. 3c). Next, differentiated LAN-5 cells were infected with KOS or UL24X at m.o.i. 10, http://vir.sgmjournals.org and titres determined at 6, 12, 18 and 24 h p.i. (Fig. 3d). In the absence of UL24, we observed a 10-fold reduction in viral titres as compared with KOS. Here too, reduction was of the same magnitude as the viral replication defect Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 2797 P.-A. Rochette and others Viral titre (p.f.u. ml–1) (a) 107 (b) Day 1 Day 5 Day 8 106 105 KOS 104 vUL24X-Rescue UL24X 104 102 12 24 36 48 72 Time (h p.i.) ICP8 Draq5 Merge (d) Viral titre (p.f.u. ml–1) (c) Mock KOS 108 107 106 105 KOS UL24X 104 103 6 12 18 Time (h p.i.) 24 UL24X Fig. 3. Replication of a UL24-deficient virus in neurons. (a) Multi-step replication assay in murine embryonic neurons. Data represent mean¡SD ; *statistically significant differences between vUL24X-Rescue and UL24X. (b) Differentiation of LA-N-5 neurons. Cells were immunostained for the neuronal maturation marker Neurofilament M (green). Nuclei were stained with Draq5 (blue). (c) Formation of VRCs. Infected neurons were immunostained for ICP8 (green) and nuclei stained with Draq5 (blue). Merged images are shown in the right-hand panels. (d) One-step replication assay in LA-N-5 neurons comparing UL24X with the WT virus KOS. Reduction of the ocular viral load in the absence of UL24 is not sufficient to explain the drastic reduction of viral titres in TG with 2|106 p.f.u. per eye of KOS, vUL24X-Rescue or UL24X. Eyes were harvested post-mortem 2 days p.i. and homogenized. We found that, similar to the reduction previously observed in tear films, viral titres were reduced 10fold in eye homogenates for mice infected with UL24X as compared with KOS and vUL24X-Rescue (data now shown). Thus, we did not observe a defect in virus release into tear films with UL24X and therefore concluded that the magnitude of the reduction of viral titres in tear films reflected the reduction of the viral load within cornea. Successful dissemination of HSV-1 infection from the eye to TG depends in part on the amount of virus present in the cornea (Chen et al., 2004; Leib et al., 1989; Sawtell, 1998). We first verified that reduction in viral titres detected in tear films of mice infected with UL24X reflected a defect in viral replication within the cornea and not a defect in virus release from corneal epithelial cells. Mice were infected We next tested if, under our conditions, the 10-fold reduction of the ocular viral load as observed for UL24X was sufficient to cause the up to 4 log10 reduction in viral titres in TG, possibly by a threshold effect. Thus, we attempted to infect mice with an amount of WT virus that would produce titres in tear films similar to those seen for UL24X under our standard infection conditions. observed for UL24X in epithelial-derived cell lines (Jacobson et al., 1989a, 1998). These data suggested that there was no inherent difference with respect to the importance of UL24 for viral replication in neurons as compared with epithelial-derived cells. 2798 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 Journal of General Virology 96 Role of UL24 in acute infection of TG in vivo We infected mice either with 2|106 p.f.u. per eye of UL24X or with quantities of KOS virus decreasing from 2|106 to 2|102 p.f.u. per eye (Fig. 4). As described previously, new infectious virus is first detected in the eye and tear films at 1 day p.i., whilst viral replication in neurons is first detected at 2 days p.i. (Summers et al., 2001). At 1 day p.i. (Fig. 4a), a time when we would expect the impact on TG titres to be the greatest, there was no significant difference between the viral titres in tear films for mice infected with KOS at 2|106, 2|105 and 2|104 p.f.u. per eye; for each condition, titres were *10-fold higher than for mice infected with UL24X at 2|106 p.f.u. per eye. At the other end of the spectrum, infection with 1|103 p.f.u. per eye or less led to viral titres in tear films on 1 day p.i. that were significantly below the levels produced upon infection (a) with 2|106 p.f.u. per eye of UL24X, with most viral titres falling below our detection threshold. Interestingly, although we were unable to detect infectious virus in tear films at 2 and 3 days p.i. in many of the mice that were infected with j1|103 p.f.u. per eye, in those where virus was detected, titres rose and surpassed levels seen with UL24X by day 3 p.i. (Fig. 4b, c). Furthermore, we found that a dose of 2|103 p.f.u. per eye of KOS was the only dose tested that resulted in tear films titres that were down to levels on day 1 p.i. similar to those seen for UL24X inoculated at 2|106 p.f.u. per eye and where infectious virus was detected in all mice tested. However, despite this similarity to UL24X, titres in TG at 3 days p.i. rose to levels seen for higher inocula of KOS, far surpassing titres obtained for UL24X (Fig. 4d). (b) 106 106 Viral titre (p.f.u. ml–1) 107 Viral titre (p.f.u. ml–1) 107 105 105 104 104 103 103 (c) 8 10 6 1× 10 2 KOS 2× 10 2 2× 10 3 6× 10 3 1× 10 4 UL24X 2× 10 5 2× 2× 10 10 8 10 6 10 1× 2 KOS 2× 10 2 10 2× 3 6× 10 10 1× 2× 10 10 2× 2× 10 2× 3 100 4 100 5 101 6 101 6 102 2× 102 UL24X (d) 106 106 Viral titre (p.f.u. ml–1) 107 Viral titre (p.f.u. ml–1) 107 105 105 104 104 103 103 UL24X KOS 8 1× 10 6 2× 10 2 2× 10 2 6× 10 3 1× 10 3 2× 10 4 10 2× 10 2× 10 2× 8 10 6 10 2× 1× 2 10 2 10 6× 2× 3 10 10 KOS 1× 2× 10 10 2× 2× 10 2× 3 100 4 100 5 101 6 101 5 102 6 102 UL24X Fig. 4. Reduction of the ocular viral load as observed in the absence of UL24 is not sufficient to explain the up to 4 log10 reduction of viral titres in TG. The inoculum (expressed in p.f.u. per eye of the indicated virus) used for each group is indicated at the bottom of each graph. (a–c) Virus in tear films was titrated at (a) 1, (b) 2 and (c) 3 days p.i. (d) Virus in homogenized TG was titrated at 3 days p.i. Dashed lines represent the detection limit of the experiment. *Statistically significant difference in viral titres obtained for the indicated group as compared with the group of mice infected with 26106 p.f.u. per eye of UL24X. http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 2799 P.-A. Rochette and others Thus, we concluded that the 1 log10 reduction in tear film titres with UL24X was not sufficient to explain the drastic reduction in viral titres in TG. Although the similarity in titres seen with the inoculum of 2|103 p.f.u. of KOS and 2|106 p.f.u. of UL24X was lost by 3 days p.i. (Fig. 4c), we would not expect virus levels in tear films to have an impact on TG titres of the same day. The reverse strategy of using an inoculum of UL24X (1|108 p.f.u. per eye) that was higher than that used for KOS did not produce higher viral titres in the eye or in TG. Although lowering the infectious dose of KOS to 1|103 p.f.u. per eye reduced the incidence of viral reactivation to levels similar to those observed for UL24X (Table 2), unlike UL24X, this reduction could be explained by a reduction in the likelihood of establishing a productive infection in the eye as demonstrated by the large number of mice infected with j1|103 p.f.u. per eye in which levels of virus in tear films were below the detection limit of the experiment. Similarly, we also noted a difference between UL24X and KOS with regard to the link between acute replication in the eye and the development of clinical signs. Lowering the infectious dose of KOS reduced the incidence of acute infection, but severe clinical signs were still evident in many animals; in contrast, for UL24X, acute replication in the eye was detected in all animals, but most animals exhibited no clinical signs and none exhibited severe clinical signs (Table 1). These results indicated that the *1 log10 defect in viral replication of UL24X observed in the eye could not account for the large reduction in viral titres in TG and in clinical signs, and suggested a specific role for UL24 in the acute infection of TG. UL24 is important to establish a productive infection in a high number of neurons at 3 days p.i. The low viral titres in TG observed with UL24X could theoretically result from there being the same number of infected neurons as seen with KOS, but a lower viral yield, or due to fewer acutely infected neurons with the same or a lower viral yield than for KOS. We next tested Table 2. Frequency of ex vivo viral reactivation from latently infected TG comparing different inocula of KOS and UL24X Virus (p.f.u. per eye) 6 KOS 2610 KOS 26104 KOS 16103 KOS 66102 KOS 26102 UL24X 26106 UL24X 16108 Mock Reactivated/total TG (%) 16/16 (100)* 10/10 (100)* 2/6 (33) 2/6 (33) 2/28 (7) 5/18 (27) 4/10 (40) 0/8 (0) *Statistically different from UL24X with Fisher’s exact test. 2800 the hypothesis that the defect was at the level of establishment of lytic infection in neurons. TG were harvested from mice infected with KOS, UL24X or vUL24X-Rescue at 3 days p.i., sectioned, stained using an antibody against HSV-1, and visualized by immunohistofluorescence using a stereoscope. We found that for UL24X, the size of the infected area in TG, when one could be detected, was typically smaller than that observed for KOS or vUL24XRescue (Fig. 5a). Furthermore, for both KOS and vUL24X-Rescue, we often observed multiple large foci of infection per TG, which was in contrast to our observations for UL24X where we typically detected either none or a single centre of infection. Moreover, all TG from KOSand vUL24X-Rescue-infected mice stained positive for viral lytic antigens, whilst for UL24X, lytic replication was detected in only half of TG from infected mice. For quantification purposes, sections were then analysed by confocal microscopy. Foci of infection consisted primarily of large pseudomonopolar neurons as identified by their characteristic morphology (Krastev et al., 2008; Mochizuki et al., 1995). For each virus, infected neurons were quantified from individual TG harvested from at least three infected mice. We found that the mean number of infected neurons per TG section for KOS and vUL24X-Rescue was *7 and *6, respectively, with individual values ranging from 1 to 16 (Fig. 5b). In contrast, in the absence of UL24, there was a mean 30-fold reduction in the number of infected neurons detected per section as compared with that observed for both KOS and vUL24X-Rescue. Absence of UL24 leads to a reduced number of TG neurons staining positive for HSV-1 DNA at 2 days p.i. At 3 days p.i., the total number of neurons expressing late viral proteins was the cumulative result of newly infected cells from the periphery, as well as possible interneuronal dissemination of the infection within TG. Moreover, detection of infected neurons by immunohistofluorescence using our anti-HSV-1 antibody depended on the expression of late viral genes. Therefore, using this technique, we could miss neurons where the genome gained access to the nucleus and began to be replicated, but there was little expression of late proteins. In order to have a better idea of the number of neurons that were infected prior to extensive spread of the virus, we used whole-mount in situ hybridization (WmISH) to quantify the number of neurons with HSV DNA at 2 days p.i. (Fig. 5c, d). At this time point, we could expect that on average the virus could have replicated once in epithelial cells and then gone on to infect neurons of TG. Thus, infected neurons would mostly represent cells infected from the periphery. We found that in the absence of UL24, the size of the infected area in TG, when one could be detected, was typically smaller than that observed for KOS or vUL24X-Rescue (Fig. 5c). For each virus, infected neurons were quantified from individual TG harvested from at least three infected mice (Fig. 5d). Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 Journal of General Virology 96 Role of UL24 in acute infection of TG in vivo (a) Autofluo HSV-1 Merge (c) KOS vUL24X-Rescue UL24XG KOS vUL24X-Rescue UL24XG (b) (d) 6 15 No. of infected neurons 10 5 4 2 X vU vU L2 UL 24 e cu es 4X -R X 24 es 4X -R L2 UL cu e S KO S 0 0 KO No. of infected neurons 20 Fig. 5. UL24 is important for dissemination of the acute infection to a high number of neurons. (a) Sections of TG from mice infected with KOS, UL24X or vUL24X-Rescue at 3 days p.i. were analysed by immunohistofluorescence for HSV-1. Lefthand panels show the tissue autofluorescence (green), centre panels show HSV-1-infected cells (red) and right-hand panels show merged images. (b) The number of positive neurons in (a) was quantified. Each point represents the mean number of HSV-1-positive neurons per section for a given TG. Results presented were pooled from two independent experiments. *Statistically significant difference as compared with the values obtained for UL24X. (c) TG from mice infected with KOS, UL24X or vUL24X-Rescue were harvested at 2 days p.i., hybridized against HSV-1 DNA, immunochemically stained, sectioned and analysed by phase-contrast microscopy. Boxes in the upper panels indicate areas with neurons harbouring HSV-1 DNA and are enlarged in the corresponding lower panels. Arrowheads indicate other infected neurons outside the boxed area. Bar, 0.5 mm. (d) The number of positive neurons in (c) was quantified as described in (b). For all the viruses tested, the number of cells for which HSV1 DNA was detected at 2 days p.i. was much lower than the number of cells expressing viral proteins at 3 days p.i. We found that the mean number of HSV-DNA-positive neurons per TG section for KOS and vUL24X-Rescue was *1.5 and *3, respectively, with individual values ranging from 0.19 to 5. In stark contrast, in the absence of UL24, there was a mean 50- and 100-fold reduction in the http://vir.sgmjournals.org number of infected neurons detected per section as compared with that observed for both KOS and vUL24XRescue, respectively, with a mean value of 0.03 and individual values ranging from 0.018 to 0.18. Owing to the limited sensitivity of the in situ approach, we also analysed the relative amount of viral DNA in TG by quantitative real-time (qRT)-PCR. TG were harvested at 1.5 days p.i. before the expected production of new infectious viral Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 2801 P.-A. Rochette and others particles (Summers et al., 2001), total DNA isolated and viral DNA quantified by qRT-PCR (Fig. 6). As expected (Sawtell, 1997), there was a large drop in the relative amount of viral DNA in TG for KOS and vUL24X-Rescue when the inoculum was reduced from 2|106 to 2|103 p.f.u. per eye. Nevertheless, even at the lower inoculum – one that led to titres in tear films similar to those obtained with 2|106 p.f.u. per eye of UL24X – there was still more viral DNA detected in TG from mice infected with the WT or rescue virus than when mice were infected with 2|106 p.f.u. per eye of UL24X (five and 3.5 times more, respectively). Thus, in the absence of UL24, reduced spread of acute infection from the cornea to neurons of TG appeared to be a major factor leading to the drastic reduction in viral titres seen in TG at 3 days p.i. DISCUSSION Reduced viral titres in the eye cannot explain the drastic reduction of viral titres in TG seen with UL24X It is known that a decrease in the viral load within the cornea can impact the amount of virus reaching TG (Chen et al., 8 Relative quantification of viral DNA 7 6 5 4 3 2 1 6 10 3 24 es c L2 4 XR UL ue 2× 2× 10 3 2× 10 KO S vU vU L2 4 XR es c KO S ue 2× 2× 10 10 6 6 0 Fig. 6. Reduced amounts of viral DNA in TG for UL24-deficient virus even upon normalization of viral titres in the eye. Relative amounts of viral DNA from TG harvested at 1.5 days p.i. were determined by qRT-PCR. The results for the KOS 26103 p.f.u. samples were defined as 1. Data represent mean¡SEM for two mice per condition. 2802 2004; Leib et al., 1989; Sawtell, 1998); however, we identified inocula for KOS where titres at 1 day p.i. were below those for UL24X, but nevertheless resulted in TG titres that far surpassed those for UL24X. Thus, our results do not support a model whereby a reduction of viral load in tear films is the cause of the drastic reduction in viral titres observed in TG for the UL24 mutant. Rather, our results point to UL24 playing a direct role in neurovirulence. A UL24 mutant replicates similarly in epitheliaderived cells, in murine embryonic neurons and in LA-N-5-derived neurons HSV-1 infection of neurons depends on a select subset of genes required to compensate for the lack of robust nucleotide metabolism in these non-dividing cells, e.g. the viral ribonucleotide reductase gene (Goldstein & Weller, 1988; Jacobson et al., 1989b; Perkins et al., 2002). Infection of primary mouse embryonic neurons can reveal neuronal replication defects of HSV-1 strains harbouring critical mutations in such neurovirulence genes (Perkins et al., 2002). We found that infection of such primary neurons in the absence of UL24 led to a decrease in viral titres as compared with KOS, but this decrease of *1 log10 was no greater than what is observed in an epithelial cell line. Similar results were obtained after infection of neurons derived from a human neuroblastoma cell line (LA-N-5). Thus, whilst UL24 does seem to play a role in the efficiency of viral replication in a variety of cell types, our results do not support a model whereby UL24 is critical for productive infection in neurons. It has recently been shown that infection of neurons at the level of the cell stroma favours an acute infection; in contrast, infecting neurons through their axonal termini favours a latent infection (Hafezi et al., 2012). This effect is possibly due to loss of specific transcription factors from the tegument of the incoming virion as it makes its way along the axon to the nucleus, thus shifting the balance between viral lytic gene expression and latency (Hafezi et al., 2012; Roizman & Sears, 1987). In our experiments, we did not restrict infection of neurons to a specific substructure of the cell. Thus, we cannot rule out the possibility of a UL24-specific function related to infection of neurons via their axonal termini. Although such a function could theoretically account for the discrepancy between the in vivo observations and our results obtained for UL24X infections in neuronal cultures, UL24 of HSV-1 has never been detected in extracellular virions (Loret et al., 2008; Pearson & Coen, 2002) and thus is unlikely to play a direct role in retrograde transport to the neuronal body following entry. Nevertheless, we cannot exclude the possibility that absence of UL24 during cellular infection has an impact on tegument composition, which could ultimately impact subsequent transport within neuronal axons. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 Journal of General Virology 96 Role of UL24 in acute infection of TG in vivo UL24 affects the spread of infection from the cornea to neurons of TG Whilst the five- to 10-fold reduction in the efficiency of viral replication of UL24X in neurons, as observed in cell culture, undoubtedly contributes in part to the drop in viral titres in TG, it cannot by itself account for the severity of the defect in neurovirulence seen with UL24X. Rather, the huge drop in TG viral titres appears to be due to a significant reduction both in the number of neurons and in the number of TG where acute infection takes hold. To identify infected neurons where viral replication may not have progressed to the late phase of gene expression, we also analysed TG at 2 days p.i. using WmISH targeting the HSV1 genome. Of note, we could not distinguish between incoming and newly synthesized DNA in these experiments, so we cannot formally rule out the possibility that the UL24 mutant entered and released its genome into neurons as efficiently as the WT virus, but then failed to replicate its genome. Nevertheless, this strategy enabled us to identify infected neurons prior to a major contribution of any intragangliolar spread of the infection. We observed a significant decrease in the number of neurons harbouring HSV-1 DNA in the absence of UL24. In all our experiments aimed at detecting HSV-1 proteins or DNA in situ, we never detected infected neurons in more than half of TG isolated from UL24X-infected mice. Importantly, we also found that levels of viral DNA in TG at 1.5 days p.i. were lower for UL24X even when inoculation doses of KOS or vUL24Xrescue were reduced 1000-fold. Our results point to UL24 being a novel player in the dissemination of HSV-1 infection from the cornea to TG neurons. Further studies will be required to uncover the precise mechanism underlying the role of UL24 in the neuropathogenesis of HSV-1. METHODS Viruses and cells. Virus was propagated and titrated on Vero cells as described previously (Lymberopoulos & Pearson, 2007). Primary embryonic neurons were harvested from E16-18 CD-1 mice (Jacomy et al., 2006). LA-N-5 cells were maintained and differentiated with RA (Hill & Robertson, 1998). The HSV-1 strains KOS and UL24XG (Jacobson et al., 1998) were originally provided by D. M. Coen (Harvard Medical School). vUL24X-Rescue was generated by homologous recombination essentially as described previously by cotransfecting UL24X-infectious DNA with the linearized transfer vector pAG5 containing the Bam HI fragment of KOS from nt 45 001 to 48 575 (Griffiths & Coen, 2003) into Vero cells using Lipofectamine (Life technologies). Screening for recombinant virus was done by PCR designed to detect the 24 bp insertion within the UL24 ORF of UL24X (59-TACGAAGCCATACGCGC-39 and 59-GTCAACAGCGTGCCGC-39). The UL24 ORF of vUL24X-Rescue was sequenced to ensure the absence of undesired mutations. Viral yield in LA-N-5 neurons was assessed in a one-step replication assay. LA-N-5 cells (4|104 cells per well) were seeded in duplicate in six-well Cell+ plates and differentiated into neurons. Neurons were infected at m.o.i. 10 in 2 ml of preheated complete RPMI 1640 medium containing 1 % FBS. Viral replication assays in Vero cells were described previously (Lymberopoulos & Pearson, 2007). Statistical analysis was performed using Student’s t-test, a50.05. Immunofluorescence on cells in culture. LA-N-5 cells were dif- ferentiated on #1 coverslips pre-treated with 0.1 % cold water fish skin gelatin (Sigma-Aldrich). Neurons were infected at m.o.i. 10 for 9 h in 200 ml preheated RPMI 1640 medium containing 1 % FBS. Cells were fixed with PBS containing 4 % (w/v) paraformaldehyde, permeabilized with chilled methanol for 5 min at 220 uC, washed and immunostained. Coverslips were mounted using ProlongGold antifade reagent (Life Technologies). The following antibodies were used: mouse monoclonal anti-ICP8 (Abcam), rabbit polyclonal anti-Neurofilament M (Millipore) and Alexa Fluor 488-conjugated goat polyclonal antibodies directed against rabbit or mouse IgG. Nuclei were stained using Draq5 (Biostatus). Confocal microscopy was carried out at the INRS-Institut Armand-Frappier imaging facility using a Bio-Rad Radiance 2000 confocal system with an argon/krypton laser at 488 and 568 nm (diode 638 nm) mounted on a Nikon E800 microscope using a |60 objective, with an aperture of 1.4, and |1.6 software magnification in Lasersharp software (Bio-Rad). Images were prepared using Adobe Photoshop. Murine model of ocular infection. Animal experiments were conducted at the INRS-Centre for Experimental Biology in accordance with institutional good animal care practices. Ocular infection of CD-1 mice deeply anaesthetized with a mixture of ketamine and xylazine, analyses of acute replication in the eye and TG, and ex vivo reactivation following latency were conducted essentially as described previously (Coen et al., 1989; Leib et al., 1989; Leiva-Torres et al., 2010). Backtiters of inocula were verified following infection of the mice. Viral titres were determined for at least three mice per condition. Data points below the level of detection of the experiment were treated as the limit values for calculation of means. Statistical analysis was performed using Student’s t-test, a50.05. For the reactivation assays, the statistical analysis was performed using Fisher’s exact test. Disease scoring. Clinical disease was assessed as described pre- viously (Leiva-Torres et al., 2010). Five animals per group were followed daily for the indicated periods of time. Histological sectioning. TG were harvested 3 days p.i. and processed for histological sectioning. Each TG was entirely processed into 8 mm sections on a Microm HM525 cryostat using replaceable blades (Edge-Rite, Thermo Scientific). All sections were serially placed on slides and overlaid with PBS. Immunohistofluorescence. Immediately following tissue section- ing, samples were permeabilized, dried briefly and incubated overnight at 4 uC in a humid chamber with a rabbit polyclonal anti-HSV-1 (Abcam) antibody and Hoechst (Life technologies) for nuclear staining. The next day, slides were washed three times with PBS containing 5 % newborn calf serum (NCS), incubated for 1.5 h at room temperature with a goat polyclonal anti-rabbit IgG conjugated to Alexa Fluor 568 and washed, and coverslips were mounted using ProlongGold. Antibodies were diluted in PBS containing 5 % NCS. Analysis of stained histological sections by fluorescence microscopy. Low-magnification imaging of whole TG sections Viral replication assays. Viral yield in primary mouse embryonic was performed using a stereoscope fluorescence microscopy system (Nikon SMZ800; X-Cite 120 series) and a Q-Imaging camera (01-RET-2000R-F-M-12-C) at |6.3 optical magnification. neurons was assessed in a multi-step replication assay at m.o.i. 0.1. Primary neurons (2|105 cells cm22) were seeded in duplicate in sixwell Cell+ plates (Sarstedt) that were pretreated with poly-D -lysine. Quantification of the number of infected neurons per section was performed using a high-magnification epifluorescence microscope (Nikon Eclipse TE2000-U) at |40 optical magnification. Neurons http://vir.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Mon, 19 Jun 2017 00:46:57 2803 P.-A. Rochette and others were identified qualitatively by the large size of the nucleus, their large cell body size and their inherent autofluorescence that emits in the green spectrum. Neurons were identified as infected by positive antiHSV-1 immunohistofluorescence staining. For each ganglion analysed, the number of infected TG neurons was determined for each section produced. Results are presented as the total number of infected neurons per number of sections obtained for a given TG. Statistical analysis was performed using Student’s t-test, a50.05. Chen, S. H., Pearson, A., Coen, D. M. & Chen, S. H. (2004). Failure of Probe preparation for WmISH. Samples of 3 mg of EcoR1b plasmid Cohrs, R. J., Randall, J., Smith, J., Gilden, D. H., Dabrowski, C., van Der Keyl, H. & Tal-Singer, R. (2000). Analysis of individual human (Goldin et al., 1981) containing a 20 kb fragment of HSV-1 DNA were used in conjunction with a DIG-High-Prime DNA labelling kit (Roche) using a 1 : 2 ratio of DIG-labelled dUTP to dTTP. WmISH analysis of TG sections. Detection of cells that stained positive for HSV-1 DNA in histological sections was performed by WmISH as described previously (Escalante & Loomis, 1995). Quantification of the number of infected neurons per section was performed using a high-magnification phase-contrast microscope (Nikon Eclipse TE2000-U). For each ganglion analysed, the number of infected TG neurons was determined for each section produced. Results are presented as the mean number of infected neurons per section of TG. Statistical analysis was performed using Student’s t-test, a50.05. qRT-PCR. TG harvested from each mouse were pooled and placed on dry ice. DNA was extracted using a DNeasy Blood and Tissue kit (Qiagen) (Cohrs et al., 2000). Reactions were performed in 10 ml using iTaq Universal SYBR Green Master Mix (Bio-Rad), 100 ng of ganglionic DNA, 500 nM each primer. For TK: 59-CCCAACGGCGACCTGTATAAC; 59-CCGGAGGTAAGTTGCAGCAG. MT-1 and MT-2 primers for mouse adipsin were described previously (Kramer & Coen, 1995). qRT-PCR was performed using the Rotor-Gene RG3000 (Corbett Research). The relative amount of viral DNA normalized against adipsin was determined using the 22DDCt method (Livak & Schmittgen, 2001). All samples were run in triplicate. thymidine kinase-negative herpes simplex virus to reactivate from latency following efficient establishment. J Virol 78, 520–523. Coen, D. M., Kosz-Vnenchak, M., Jacobson, J. G., Leib, D. A., Bogard, C. L., Schaffer, P. A., Tyler, K. L. & Knipe, D. M. (1989). Thymidine kinase-negative herpes simplex virus mutants establish latency in mouse trigeminal ganglia but do not reactivate. 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