1 Department of Ophthalmology, University of California Irvine, UCIMC, 101 The City Drive, Orange, CA 92868-4380-02, USA
2 Department of Veterinary and Biomedical Sciences, University of Nebraska, Lincoln, NE 68583-0905, USA
Correspondence
Guey-Chuen Penrg
gperng{at}uci.edu
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ABSTRACT |
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K. R. Mott and N. Osorio contributed equally to this work.
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INTRODUCTION |
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The only gene that is actively transcribed during HSV-1 neuronal latency is the latency-associated transcript (LAT) (Rock et al., 1987b; Stevens et al., 1987
). A stable 2 kb LAT intron is spliced from the primary transcript (Farrell et al., 1991
) and is the major LAT expressed during neuronal latency (Dobson et al., 1989
; Spivack & Fraser, 1988
; Stevens, 1990
; Wechsler et al., 1988
, 1989
; Zwaagstra et al., 1989
).
LAT enhances the induced and spontaneous reactivation phenotypes in the rabbit ocular model (Hill et al., 1990; Perng et al., 1994
) and the induced reactivation phenotype in mice (Block et al., 1993
; Devi-Rao et al., 1994
; Leib et al., 1989
; Perng et al., 2001b
; Sawtell & Thompson, 1992
; Steiner et al., 1989
). The large 8·3 kb LAT overlaps and is antisense to ICP0, ICP34.5 and part of ICP4 (Rock et al., 1987b
; Stevens et al., 1987
). Thus, it has been proposed that LAT may block immediate-early gene expression via an antisense mechanism, which promotes latency (Chen et al., 1997
; Garber et al., 1997
). However, mutants expressing just the first 1·5 kb of the primary 8·3 kb LAT transcript (Bloom et al., 1996
; Perng et al., 1996
) have wild-type high-reactivation phenotypes. This 1·5 kb region does not overlap ICP0 or ICP4, and encompasses only the first 837 nucleotides of the stable 2 kb LAT (Perng et al., 1996
). Thus, in small animal models, the ability of LAT to enhance the reactivation phenotype does not require antisense repression of immediate-early gene expression or production of the stable 2 kb LAT. Interestingly, it was recently shown that the 5' end of LAT, a region not antisense to ICP0, can downregulate ICP0 expression in cis (Burton et al., 2003
). Thus, downregulation of ICP0 by LAT (by a mechanism unrelated to antisense) remains a potential mechanism by which LAT might regulate the latency-reactivation cycle.
LAT can reduce apoptosis in transient transfection assays in tissue culture and during the switch from acute to latent infection in rabbit TG (Inman et al., 2001b; Perng et al., 2000a
). The anti-apoptotic activity of LAT has been independently confirmed in tissue culture and in a mouse HSV-1 ocular model (Ahmed et al., 2002
). LAT's anti-apoptotic activity maps to sequences contained in the same 1·5 kb region that is capable of enhancing the spontaneous reactivation phenotype (Inman et al., 2001b
; Jin et al., 2003
). In addition, we recently reported that an alternative anti-apoptosis gene, the bovine herpes virus (BHV) type 1 latency related (LR) gene, could effectively replace HSV-1 LAT, producing a chimeric virus, CJLAT, with a high wild type-like reactivation phenotype (Perng et al., 2002
). Since the LR gene can inhibit apoptosis (Inman et al., 2001a
), these finding suggest that LAT's anti-apoptotic activity is important for efficient reactivation from latency.
The ability of LAT to reduce neuronal death during the establishment of latency (Perng et al., 2000a) may result in larger pools of latently infected neurons, which contribute to higher levels of reactivation from latency. This is consistent with reports suggesting that, in experimentally infected animals, more neurons become latently infected with LAT+ viruses than with LAT- viruses (Perng et al., 2000b
; Sawtell & Thompson, 1992
; Thompson & Sawtell, 1997
). It is important to note that LAT's anti-apoptotic activity may also play an important role in the maintenance of latency and in the reactivation process. In particular, many of the stimuli known to induce reactivation can also induce apoptosis, suggesting that LAT plays an important role in neuronal survival during reactivation from latency. The LAT region is also involved in virulence in infected animals because disruption of the genomic region encoding the 5' end of LAT alters the virulence phenotype in infected rabbits and mice (Perng et al., 1999a
, 2001a
).
The BHV-1 LR RNA, like LAT, is the only abundant viral transcript detected in latently infected neurons (Kutish et al., 1990; Rock et al., 1987a
, 1992
). A fraction of LR RNA is polyadenylated and alternatively spliced in TG, suggesting that this RNA is translated into more than one LR protein (Devireddy & Jones, 1998
; Hossain et al., 1995
). In addition to LR gene products inhibiting apoptosis (Ciacci-Zanella et al., 1999
), they also have the ability to inhibit S phase entry and an LR protein is associated with cyclin-dependent kinase 2 (cdk2)/cyclin complexes (Hossain et al., 1995
; Inman et al., 2002
). Similar to our original studies with HSV-1 (Perng et al., 2000a
), a BHV-1 LR mutant containing three stop codons inserted just downstream of the start of open reading frame 2 (ORF2) (the first ORF in LR; see Fig. 1
C) has higher levels of apoptosis in TG neurons during the transition from acute infection to latency (establishment of latency) compared to wild-type or marker-rescued virus (Lovato et al., 2003
). In transient transfection assays, plasmids expressing LR containing these alterations to ORF2 no longer inhibit apoptosis (Inman et al., 2001a
). These findings suggest that ORF2 encodes an anti-apoptosis protein.
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METHODS |
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Construction of CJLATmut.
CJLATmut was constructed exactly as previously described for CJLAT (Perng et al., 2002), except that the BHV-1 LR gene inserted into the HSV-1 LAT locus contained an EcoRI restriction site and three stop codons just downstream of the ORF2 ATG (see Fig. 1E
).
RT-PCR.
Subconfluent CV-1 cell monolayers were infected at a m.o.i. of 5 p.f.u. per cell, total RNA was isolated, and first-strand cDNA was synthesized using random hexamers as previously described (Inman et al., 2001a). RT-PCR for BHV-1 LR transcript was performed as previously described (Inman et al., 2001a
) using primers p4 and p5. These primers generate a 298 bp product specific for wild-type BHV-1 LR or a 290 bp product specific for the mutated BHV-1 LR. To detect HSV-1 LAT, aliquots of the cDNA product from above were amplified by PCR with primer WR43, 5'-AAGAAGGCATGTGTCCCACCCCGCCTGTGT-3' (HSV-1 genomic nucleotide 119730 to 119760) and primer WR 41, 5'-AAGGAGGGGGGGCGGTGCTTCTTAGAGACC-3' (120060 to 120030). These primers generate a 330 bp product specific for LAT nucleotides 929 to 1259. The conditions for cycling were: (i) one cycle of denaturation at 95 °C for 2·5 min; (ii) 30 cycles of denaturation at 94 °C for 1 min, annealing at 65 °C for 40 s, and extension at 71 °C for 2 min; and (iii) one cycle of extension at 72 °C for 10 min. GAPDH was used as an internal control as previously described (Perng et al., 1996
).
Rabbits.
Eight- to ten-week-old New Zealand White male rabbits (Irish Farms) were used. Rabbits were treated in accordance with ARVO (Association for Research in Vision and Ophthalmology), AALAC (American Association for Laboratory Animal Care) and National Institute of Health guidelines. Rabbits were bilaterally infected without scarification or anaesthesia by placing, as eye drops, 2x105 p.f.u. of virus into the conjunctiva cul-de-sac, closing the eyes, and rubbing the lid gently against the eye for 30 s as previously described (Perng et al., 1994). Analysis of virus replication in eyes was done as previously described (Perng et al., 1994
). Virulence (death due to encephalitis) was determined by survival at 21 days after infection as previously described (Perng et al., 2001a
).
Mice.
Swiss Webster mice were used. Mice were ocularly infected with 1x106 p.f.u. per eye without corneal scarification as previously described (Perng et al., 2001b). Tear films were collected at various days after infection from one eye per animal. The amount of virus in each tear film was determined by standard plaque assays on RS cells. Virulence (death due to encephalitis) was determined by survival on day 21 after ocular infection as previously described (Perng et al., 2001b
).
Replication of CJLATmut in tissue culture.
CV-1 cell monolayers at approximately 70 to 80 % confluency were infected at 0·01 p.f.u. per cell, and all monolayers were refed with exactly the same amount of culture media. Viruses were harvested for titration at various times by two cycles of freezethawing of the monolayers with media (-80 °C to room temperature). Virus titres (p.f.u. ml-1) were determined by standard plaque assays on RS cells.
Neutralizing antibody assay.
Serum-neutralizing antibody titres were determined by standard plaque reduction assays as previously described (Perng et al., 1999b).
Mouse explant cultivation reactivation assay.
Mice were sacrificed at 30 days post-infection (p.i.) and individual TG (two per mouse) were cultured in tissue culture media. Aliquots of media were removed from each culture daily for up to 18 days and plated on RS cells to look for the presence of reactivated virus as previously described (Perng et al., 2001b).
Statistical analysis.
Statistical analyses were performed using GraphPad Prism version 3.02 for Windows. Results were considered statistically significant when the P value was <0·05.
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RESULTS |
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The sequence of wild-type BHV-1 LR ORF 2 in CJLAT virus is shown in Fig. 1(D). The alterations to the sequence of the start of the LR ORF2 in CJLATmut are shown in Fig. 1(E)
. ORF2 contains an inserted novel EcoRI restriction site and three stop codons (one in each potential reading frame).
To confirm that CJLATmut contained the mutated BHV-1 LR gene, PCR was performed using primers that flank the mutated region as described in Methods. The PCR product was then digested with EcoRI to differentiate CJLATmut from CJLAT (Fig. 2).
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Replication of CJLATmut in tissue culture
CV-1 cells were infected at an m.o.i. of 0·01 p.f.u. per cell with CJLATmut, CJLAT, dLAT2903 or wild-type McKrae viruses. Replication was similar for all four viruses (Fig. 4). Thus, insertion and expression of the mutated BHV-1 LR gene in place of the HSV-1 LAT gene did not appear to have an impact on virus replication in tissue culture. This also indicated that the ICP0 gene was functioning properly.
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Serum was collected on day 59 p.i. from each of the surviving rabbits that were shown in Fig. 6(A). Neutralizing antibody titres were determined for each serum, and the results were plotted as scattergrams (Fig. 7
A). CJLATmut- and dLAT2903-infected rabbits had similar antibody titres that were significantly lower than that of wild-type McKrae- or CJLAT-infected rabbits. This indicates that spontaneous reactivation of CJLATmut is similar to that of dLAT2903 and lower than that of wild-type McKrae or CJLAT viruses. This suggests that a protein encoded by LR ORF2 with anti-apoptotic function is associated with high-reactivation phenotype in rabbits. As we previous reported, CJLAT-infected rabbits had significantly higher neutralizing antibody titres than rabbits infected with wild-type McKrae virus (Perng et al., 2002
), suggesting that the BHV-1 LR gene is more efficient than the HSV-1 LAT gene in supporting spontaneous reactivation of HSV-1.
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As we previously reported (Perng et al., 2002) reactivation of CJLAT virus from explanted mouse TG appeared to occur slightly, but significantly more rapidly than for wild-type McKrae (Fig. 7B
; P=0·02, KaplanMeyer survival curve). This is consistent with CJLAT's higher neutralizing antibody titre in rabbits and suggests that CJLAT has a more efficient reactivation phenotype than wild-type HSV-1.
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DISCUSSION |
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The BHV-1 LR gene, like HSV-1 LAT, is abundantly transcribed during latency (Kutish et al., 1990; Rock et al., 1987a
, 1992
) and inhibits apoptosis in transient transfection assays (Ciacci-Zanella et al., 1999
) and in the TG of cattle during the switch from acute to latent infection (Lovato et al., 2003
). We recently showed that the BHV-1 LR gene could substitute for the HSV-1 LAT gene and support the high-reactivation phenotype in rabbits and mice (Perng et al., 2002
). This was done by construction of a chimeric virus (CJLAT) in which the BHV-1 LR gene, including its promoter, was inserted into the LAT- virus dLAT2903 in the normal LAT location. The ability of the anti-apoptotic gene LR to restore the high-reactivation phenotype to an HSV-1 LAT- virus further supported the relationship between LAT's anti-apoptotic activity and the latency-reactivation cycle. However, because LR has other activities (Inman et al., 2002
; Jiang et al., 1998
), in addition to its anti-apoptotic activity, it was possible that an LR activity other than interfering with apoptosis was responsible for restoration of the high-reactivation phenotype. To begin to address these issues, we constructed and studied CJLATmut.
Despite CJLATmut virus replicating to similar levels as CJLAT, wild-type or dLAT2903 in vitro and in vivo, CJLATmut did not restore the reactivation phenotype compared to CJLAT or wild-type viruses. A similar level of LR RNA was detected, by semi-quantitative RT-PCR, in CJLATmut and CJLAT latently infected rabbit TG (data not shown). Considering the nature and complexity of LR RNA, it is possible that we may have detected additional alternative or aberrantly spliced LR RNA transcripts along with the expected transcript (Devireddy & Jones, 1998). However, this is unlikely since the primers used were specific for detection of LR ORF2 RNA (Inman et al., 2002
). Whether the primers used are capable of picking up other alternatively spliced LR RNAs is under investigation.
It has been demonstrated that the BHV1 LR gene encodes a protein both in vitro and in vivo (Hossain et al., 1995; Jiang et al., 1998
). In addition, a plasmid containing the same stop codon insertion mutation present in CJLATmut does not express ORF2 protein (Ciacci-Zanella et al., 1999
). Furthermore, the product of the LR ORF2 can inhibit programmed cell death (PCD) since a plasmid containing the same stop codons insertion mutation in ORF2 no longer protects cells from chemicals that induced PCD (Ciacci-Zanella et al., 1999
) and calves infected with a BHV-1 LR mutant containing the same stop codons insertion mutation have higher levels of apoptosis in TG (Lovato et al., 2003
). Since LR has multiple functions it is formally possible, but we think unlikely, that the LR mutation directly or indirectly interfered with a non-anti-apoptotic LR function and that this function was involved in the low-reactivation phenotype of CJLATmut virus.
The anti-apoptotic activity of HSV-1 LAT has been demonstrated by several groups both in vitro and in vivo (Ahmed et al., 2002; Inman et al., 2001b
; Perng et al., 2000a
). An HSV-1 LAT deletion mutant, dLAT2903, has a reduced reactivation phenotype and has higher levels of apoptosis in TG of infected rabbits (Perng et al., 1994
, 2000a
). Similarly, a different LAT- mutant was shown to increase apoptosis in TG of infected mice (Ahmed et al., 2002
). Another mouse study confirmed that LAT promotes neuronal survival but was unable to demonstrate that this was due to blocking of apoptotic events (Thompson & Sawtell, 2001
). In addition, the same phenomenon occurs with BHV-1 during the switch from acute to latent infection in the TG of infected calves. Significantly more apoptosis was seen with an LR mutant than with wild-type or marker rescued BHV-1 (Lovato et al., 2003
). Furthermore, it was recently found that the HSV-2 LAT also interferes with apoptosis (Stephen Straus, personal communication). Our mapping studies have shown a strong correlation between the ability of different LAT fragments to block apoptosis in transient transfection assays and the ability of the same LAT regions to support the high spontaneous reactivation phenotype in rabbits (Inman et al., 2001b
). Taken together, these findings suggest that an anti-apoptotic function plays a critical role in the latency-reactivation cycle of HSV-1 and BHV-1. Although we did not formally demonstrate that the LR mutation in CJLATmut prevented expression of the LR ORF2 protein or eliminated LR's anti-apoptotic activity, previous findings showed that the identical stop codon insertion mutant eliminated expression of the ORF2 protein and LR's anti-apoptotic activity in a plasmid and in a BHV-1 mutant. Thus, it is likely that this mutation had the same effect in CJALTmut, and it is also likely that the decreased reactivation phenotype of CJLATmut was due to loss of the anti-apoptotic activity of LR.
The first 1·5 kb of the primary 8·3 kb LAT does not appear to encode a protein that is well conserved among three HSV-1 LAT genes each capable of supporting the high-reactivation phenotype (Drolet et al., 1998). This same region is capable of both fully supporting the high-reactivation phenotype and inhibiting apoptosis (Inman et al., 2001b
, Jin et al., 2003
). This suggests that these LAT activities are not due to a LAT-encoded protein. This is in contrast to LR's anti-apoptotic activity which appears to be due to the protein encoded by ORF2. Several possible mechanisms exist by which LAT could interfere with apoptosis without expressing a protein. For example: (1) LAT RNA could directly interact and interfere with one or more apoptosis factors; (2) LAT RNA could induce a cell protein that interferes with apoptosis; (3) one or more small portions of LAT could interfere with apoptosis via an RNA interference (RNAi) mechanism; (4) LAT RNA could associate with ribosomes (Ahmed & Fraser, 2001
; Goldenberg et al., 1997
) and alter expression of one or more apoptosis-related proteins; or (5) LAT could alter splicing (Ahmed & Fraser, 2001
) of one or more apoptosis-related transcripts.
LAT can interfere with the caspase 8 apoptotic pathway (Ahmed et al., 2002). More recently, we showed that a plasmid expressing LAT can block apoptosis induced by either caspase 8 or caspase 9 (Henderson et al., 2002
; Jin et al., 2003
). Thus, LAT appears capable of interfering with both major apoptotic pathways. This further supports the importance of LAT's anti-apoptotic activity. There are two major apoptotic pathways; the death receptor-mediated pathway (Fas or tumour necrosis factor receptor, for example) and the mitochondrial pathway (Krueger et al., 2001; Schmitz et al., 2000
; Wang, 2001
). The death receptor-mediated pathway activates caspase 8 leading to caspase 3 activation. Activation of the mitochondrial pathway results in release of several proapoptotic molecules, cytochrome c and Smac/DIABLO for example (Wang, 2001
). Caspase 3 activation leads to the morphological hallmarks of apoptosis.
There have been several reports of proteins encoded by LAT (Doerig et al., 1991; Thomas et al., 1999
, 2002
). However, all of these putative proteins are encoded by ORFs located completely downstream of the first 1·5 kb of LAT, a region capable of interfering with apoptosis and supporting the high-reactivation phenotype. Thus, although one or more of these proteins may play an important role in the HSV-1 life cycle, they cannot be essential elements of LAT's ability to enhance the reactivation phenotype in rabbits or mice.
The phase of the latency-reactivation cycle during which LAT exerts its main influence on reactivation remains unclear. LAT's anti-apoptotic activity may result in more neurons becoming latently infected and in the long-term survival of these latently infected neurons. LAT may also play a direct or indirect role in the reactivation process. Since stimuli that induce reactivation are likely to also induce apoptosis, the continued presence of LAT RNA may be important in survival of neurons following induction stimuli. It is also possible that reactivation is triggered by an interaction between LAT and one or more factors in the apoptotic pathway.
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ACKNOWLEDGEMENTS |
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Received 12 June 2003;
accepted 10 July 2003.