The bovine herpesvirus-1 LR ORF2 is critical for this gene's ability to restore the high wild-type reactivation phenotype to a herpes simplex virus-1 LAT null mutant

Kevin R. Mott1, Nelson Osorio1, Ling Jin1, David J. Brick1, Julie Naito1, Jennifer Cooper1, Gail Henderson2, Melissa Inman2, Clinton Jones2, Steven L. Wechsler1 and Guey-Chuen Perng1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
During neuronal latency of herpes simplex virus (HSV)-1, the latency-associated transcript (LAT) is the only viral gene readily detectable. LAT is required for the high-level reactivation phenotype in animal models. LAT's anti-apoptotic activity was recently demonstrated by our group and it was proposed that LAT's anti-apoptotic function is involved in enhancing the reactivation phenotype. Recently, using chimeric virus CJLAT, it was shown that the reactivation phenotype of LAT- mutant dLAT2903 can be restored to wild-type levels by inserting the bovine herpes virus (BHV)-1 latency-related (LR) gene into the LAT locus of this HSV-1 LAT deletion mutant. Although transcription of the LR gene, like LAT, inhibits apoptosis, LR appears to be multifunctional. To investigate whether the LR gene's anti-apoptotic function was responsible for restoring the high-reactivation phenotype, a mutated BHV-1 LR gene was inserted into the LAT locus of HSV-1 generating the chimeric virus CJLATmut. This mutation consists of three stop codons inserted just after the ATG of the first LR open reading frame (ORF2). In plasmids and in a BHV-1 mutant, this mutation eliminated the LR gene's anti-apoptotic activity, strongly suggesting that ORF2 encodes a protein responsible for LR's anti-apoptotic activity. Reactivation of the CJLATmut virus, in both rabbits and mice, was significantly lower than in wild-type McKrae virus (P=0·0001 and P=0·0003, respectively) and CJLAT virus, containing wild-type LR in place of LAT (P<0·0001) and was similar to LAT- dLAT2903 (P=0·8 and P=0·7, respectively). Thus, disruption of BHV-1 LR ORF2 eliminated the high-reactivation phenotype.

K. R. Mott and N. Osorio contributed equally to this work.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Over 80 % of adults in the United States harbour latent herpes simplex virus type 1 (HSV-1). Following primary peripheral infection of the eye, HSV-1 travels along axons via antegrade transport to sensory neurons of the trigeminal ganglia (TG) where latency is established. The latent virus reactivates sporadically as a result of stress, UV exposure and other unknown stimuli. Reactivations can also occur in the absence of known stimuli, which we have operationally defined as spontaneous reactivation. Following reactivation, HSV-1 travels along axons in a retrograde direction to the initial infection site, where it can replicate and cause recurrent disease (Andreansky et al., 1998; Roizman & Whitley, 2001). Recurrent HSV-1 infection in the eye can cause corneal scarring leading to the loss of vision. As a result, HSV-1 is one of the leading infectious causes of corneal blindness in the developed world (Liesegang et al., 1989). The mechanism(s) by which the latent virus reactivates and causes disease are currently not completely understood.

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. 1C) 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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Fig. 1. Schematic detailed description of the HSV-1 LAT region. (A) HSV-1 wild-type LAT. The primary LAT transcript is approximately 8·3 kb. The open rectangle represents the promoter of LAT. The solid rectangle represents the very stable 2 kb LAT. The start of LAT transcription is indicated by the arrow at +1. Locations of the ICP0 and ICP34.5 transcripts antisense to the primary LAT are shown. (B) dLAT2903 LAT. dLAT2903 as a deletion from LAT nucleotides -161 to +1667 relative to the start of the primary transcript. The core of the LAT promoter is deleted and dLAT2903 does not make any LAT RNA. (C) LAT region of CJLAT. The complete LR gene of BHV-1, including the promoter, was inserted into the deletion in dLAT2903. The LR RNA is expressing from BHV-1 LR promoter. There are two potential ORFs in the LR gene. The shaded rectangle represents the LR ORF1, the solid rectangle represents the LR ORF2 and is the first LR ORF. (D) The beginning of CJLAT wt BHV-1 LR ORF2 transcript. The first ATG in the wild-type sequence is the first in-frame ATG for ORF2 and is underlined. (E) The beginning of CJLATmut BHV-1 LR ORF 2 transcript. A mutated oligonucleotide containing an unique EcoRI (underlined) restriction enzyme site (GAATTC) and three stop codons (boldface and underlined) are inserted at beginning of the ORF2 right after the first ATG and has been described in Inman et al. (2001a).

 
Since BHV-1 LR has multiple activities, it is possible that CJLAT virus (containing the LR gene in place of LAT) restored the high-reactivation phenotype due to an LR function other than anti-apoptosis. Therefore, as described here, we constructed a chimeric virus identical to CJLAT, except that the inserted LR gene contained the three stop codons described above that disrupt expression of LR ORF2.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus and cells.
The parental virus, HSV-1 McKrae, and all mutants were triple plaque purified and passed only one or two times in rabbit skin (RS) cells prior to use. The LAT mutant dLAT2903, and CJLAT, containing the wild-type BHV1 LR gene in place of the HSV-1 LAT, have been previously described (Perng et al., 1994, 2002). RS and CV-1 cells were grown in Eagle's minimal essential medium supplemented with 5 % foetal calf serum.

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 freeze–thawing 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Construction and genomic structure of CJLATmut
The construction and genomic structure of dLAT2903 and CJLAT were described previously (Perng et al., 1994, 2002). The LAT gene is located in the viral long repeats and thus there are two complete copies of LAT in the genome. In all the mutants, both LAT regions are identical and only one copy is shown for simplicity. A schematic representation of the HSV-1 wild-type LAT gene including the LAT promoter is shown in Fig. 1(A). A very stable and readily detected 2 kb LAT (solid rectangle) appears to be an intron derived by splicing of the primary LAT (Farrell et al., 1991). The LAT region of dLAT2903 (Fig. 1B) contains a deletion in both copies of LAT from -161 to +1667 relative to the start of the primary LAT transcript (indicated by ‘vir842975vir842975vir842975’). This deletion does not affect the ICP0 or ICP34.5 genes, which overlap LAT downstream of the deletion. dLAT2903 does not have key promoter elements, makes no LAT RNA, is a true LAT null mutant and has a low-reactivation phenotype (Perng et al., 1994). The LAT region of CJLAT (Fig. 1C) contains the complete BHV-1 LR gene, including the LR promoter, inserted into the deletion in dLAT2903 (Perng et al., 2002). There are two potential ORFs in the BHV-1 LR gene (Devireddy & Jones, 1998; Hossain et al., 1995) (Fig. 1C). There are also two reading frames in the LR gene that overlap ORF1 and ORF2 that lack an initiating ATG (data not shown).

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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Fig. 2. CJLATmut viral genome containing the mutated oligonucleotides. The mutated oligonucleotide containing a unique EcoRI facilitates the CJLATmut viral genome screening. The primers used for PCR were described in Methods. The PCR was performed on extracted viral DNA and the PCR products were run on a 2 % agarose gel. PCR products from CJLAT with wild-type BHV-1 LR yield a single band which migrated at 298 bp whether uncut or cut with EcoRI. PCR products from CJLATmut with mutated BHV-1 LR yield two bands which migrated at 105 and 185 bp after EcoRI digestion, while the uncut product yields one single band which migrated at about 298 bp. The original plasmids for CJLAT wild-type and CJLATmut were used as controls and size references. M is the 100 bp DNA marker. Bold arrow indicates the size of expected PCR product uncut, open arrow indicates the size of PCR products after digestion with EcoRI.

 
CJLATmut expresses the mutated BHV-1 LR transcript
RT-PCR was performed using primers to detect BHV-1 LR RNA or using primers to detect HSV-1 LAT RNA as described in Methods. Primers specific for GAPDH were used as an internal control as previously described (Perng et al., 1996). The RT-PCR products are shown in Fig. 3(A). PCR products generated from the original LR mutant plasmid DNA served as a size reference. Without EcoRI digestion the RT-PCR products from cells infected with CJLAT and CJLATmut viruses were of similar size. Following EcoRI digestion the RT-PCR product from cells infected with CJLATmut was cut into two pieces, similar to the marker CJLATmut plasmid. The RT-PCR product from CJLAT-infected cells was not cut by EcoRI. No RT-PCR products were seen with RNA isolated from mock-infected cells, cells infected with wild-type McKrae virus or dLAT2903, since the primers were specific for BHV-1 LR.



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Fig. 3. RT-PCR analysis of LR RNA expressed in CJLATmut. (A) CV-1 cells were infected with CJLATmut, CJLAT, dLAT2903 or wild-type McKrae viruses at an m.o.i. of 5 p.f.u. per cell. Total RNA was isolated at 24 h p.i. and RT-PCR was performed as described in Methods. The PCR products from original CJLATmut plasmid, uncut and cut with EcoRI, were used as size references. After EcoRI digestion, RT-PCR products from CJLATmut yielded two bands which migrated at 105 and 185 bp while RT-PCR products from CJLAT yielded one band which migrated at 298 bp. RT-PCR products from both the CJLATmut and CJLAT viruses yielded one band which migrated at around 298 bp without treatment with EcoRI. Since the primer set is very specific for the BHV-1 LR gene, no RT-PCR products were detected using RNA isolated from mock-, dLAT2903- or McKrae-infected cells. The primer set for HSV-1 LAT was applied as described in Methods. RT-PCR products (about 330 bp) could be detected with RNA isolated only from wild-type McKrae-infected cells but not from other virus-infected cells. No RT-PCR products were detected when reverse transcriptase was omitted during the cDNA reaction (-RT). Bold arrow indicates the size of PCR products uncut or cut with EcoRI. (B) Primers that amplify a segment of GAPDH transcript were used as an internal control. The size markers were a 100 bp DNA ladder. Regular arrow indicates the size of PCR products generated from the GAPDH primer set.

 
The RT-PCR products generated with RNA from McKrae-infected cells using HSV-1 LAT-specific primer were approximately 330 bp. Since these primers are within the deleted region of dLAT2903, no LAT-specific RT-PCR products were seen in dLAT2903-, CJLATmut- or CJLAT-infected cells. No RT-PCR products were detected in experiments performed without adding reverse transcriptase (-RT) as negative controls, which showed that no contaminating DNA was present in the RNA samples. GAPDH was also used as the internal control to show that all samples had similar amounts of RNA (Fig. 3B). These results confirm that CJLATmut expresses the proper LR RNA and that the mutations introduced into LR did not adversely affect LR RNA expression.

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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Fig. 4. Replication of CJLATmut in tissue culture. CV-1 cells were infected at an m.o.i. of 0·01 p.f.u. per cell. The infected monolayers were harvested by freeze–thawing at the indicated times, and the amounts of infectious virus were determined by plaque assays as described in Methods. wt, wild-type McKrae.

 
Replication of CJLATmut in rabbit and mouse eyes
Rabbits and mice were bilaterally ocularly infected with 2x105 p.f.u. or 1x106 p.f.u. per eye, respectively, of CJLATmut, CJLAT, dLAT2903 or wild-type McKrae viruses as described in Methods. Tear films were collected from 10 eyes per group at various times p.i., and the amount of virus in each tear film was determined by standard plaque assays on RS cells (Fig. 5). Replication of CJLATmut appeared similar to that of CJLAT, dLAT2903 and wild-type McKrae in both rabbit and mouse eyes at all times.



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Fig. 5. Replication of CJLATmut in eyes of rabbits and mice. Rabbits and mice were infected as described in Methods. Tears were collected from 10 eyes per group (one from each animal) on the days indicated and the amount of infectious virus was determined as described in Fig. 3. Panel (A) is from rabbits and panel (B) is from mice.

 
Rabbit and mouse survival
Rabbit and mouse survival (with death due to virus-induced encephalitis) was determined on day 21 p.i. There were no significant differences in rabbit survival (P>0·05) among the four viruses tested (Fig. 6A). In mice (Fig. 6B), CJLATmut, wild-type McKrae and dLAT2903 viruses had similar survival. Consistent with our previous study (Perng et al., 2002), CJLAT was more virulent than the other viruses in mice. This suggests that the increased virulence of CJLAT in mice compared to wild-type HSV-1 and dLAT2903 was due to a protein encoded by the LR gene.



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Fig. 6. Survival of rabbits and mice The numbers above each bar indicate the number of surviving animals on day 21 p.i. over the number of animals initially infected. P values were determined by chi-squared analysis. Bars: wt, wild-type McKrae; dLAT, dLAT2903; CJLAT, CJLAT; mut, CJLATmut. Panel (A) is from rabbits and panel (B) is from mice.

 
Spontaneous reactivation from rabbits
Spontaneous reactivation can be determined by analysing tears for the presence of infectious virus (i.e. virus shedding) in the rabbit ocular model. However, if severe corneal scarring develops in a large percentage of the eyes, as is the case with CJLAT (Perng et al., 2002) detection of recurrent virus is difficult and an alternative approach, such as increases in neutralizing antibody titres during latency (Perng et al., 1999b), must be used (Perng et al., 2000b). We previously showed that HSV-1 serum neutralizing antibody titres increase and are similar throughout the first 45 days p.i. in rabbits infected with HSV-1 viruses with high- and low spontaneous reactivation phenotypes. However, by day 59 p.i., rabbits latently infected with high-reactivation phenotype viruses develop neutralizing antibody titres that are significantly higher than that of rabbits latently infected with low-reactivation phenotype viruses (Perng et al., 1999b). This difference has been used to determine the efficiency of spontaneous reactivation with various HSV-1 mutants (Perng et al., 2000b, 2002). An increase in neutralizing antibody titres is also a sensitive measure of detecting dexamethasone-induced reactivation of calves latently infected with BHV-1 (Jones et al., 2000), demonstrating that an increase in virus-specific antibodies normally occurs as a result of reactivation of alphaherpesviruses from latency. This approach was used here.

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. 7A). 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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Fig. 7. Spontaneous reactivation and explant reactivation. (A) Individual sera from surviving rabbits shown in Fig. 6(A) were collected on day 59 p.i. Each datum point indicates the neutralizing antibody titre of one serum. (B) Thirteen surviving mice from each of the groups shown in Fig. 6(B) were selected randomly, and the TG were removed on day 30 p.i. and individually incubated in tissue culture media. An aliquot was removed from each culture daily for 18 days and inoculated onto RS cell monolayers to look for the presence of reactivated virus. The results are plotted as kinetics of the cumulative percentage of TG that had detectable reactivated virus. The P value was determined from survival curves using GraphPad Prism software. The P value between two arrows was obtained from CJLATmut and McKrae (P<0·0003). wt, wild-type McKrae; dLAT, dLAT2903; CJLAT, CJLAT; mut, CJLATmut.

 
Mouse explant cultivation reactivation
The mice that survived to the end of the experiment (Fig. 6B) were used to examine the reactivation phenotype of CJLATmut in mice (Fig. 7B). Since more mice survived in the CJLATmut-, dLAT2903- and wild-type McKrae-infected groups than in the CJLAT-infected group, 13 mice per group were randomly selected from each of the CJLATmut-, dLAT2903- and McKrae-infected groups so that the number of TG (i.e. 26) in each mouse group would be the same. Thus, any apparent differences in explant-induced reactivation among the groups should not be due to unequal statistical power. On day 31 p.i., the mice were euthanized, and individual TG was removed and cultured in tissue culture media. Aliquots of media were removed from each culture daily for up to 18 days and plated on indicator cells (RS cells) to look for the appearance of reactivated virus. Since the media from the explanted TG cultures were plated daily, the time at which reactivated virus first appeared in the explanted TG cultures could be determined. No significant difference in reactivation was detected between CJLATmut and dLAT2903 (P=0·7, Kaplan–Meyer survival curve). In contrast, reactivation of both CJLAT and McKrae viruses was significantly higher than either CJLATmut or dLAT2903 (P<0·0001 for CJLAT compared to either CJLATmut or dLAT2903; P=0·0003 for McKrae compared to CJLATmut; P<0·0001 for McKrae compared to dLAT2903). Thus, consistent with the rabbit results shown in Fig. 7(A), the anti-apoptosis protein encoded by LR ORF2 also appeared to be critical for LR's ability to support the high-reactivation phenotype in mice.

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, Kaplan–Meyer 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.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The latency-reactivation cycle can be divided into three separate, but partially overlapping phases at which LAT might function: (1) establishment of latency; (2) maintenance of latency; and (3) reactivation from latency. Some researchers have concluded that LAT is required for efficient establishment of latency because compared to mice infected with wild-type virus, mice infected with LAT- viruses contain less viral DNA in their TG (Sawtell, 1997; Sawtell & Thompson, 1992; Thompson & Sawtell, 1997). However, other studies reported no significant differences in the amount of viral DNA in TG of rabbits infected with LAT- compared to LAT+ viruses (Bloom et al., 1994; Perng et al., 1994). These discrepancies may be due to different animal strains, different virus strains or different animal models. We have shown that the effect of LAT on the HSV-1 latency-reactivation phenotype is mouse strain dependent (Perng et al., 2001b), whereas others have reported a gender bias (Han et al., 2001).

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.


   ACKNOWLEDGEMENTS
 
This work was supported by Public Health Service grants to G. C. P. (NIH EY13701), S. L. W. (NIH EY12823 and EY13191) and C. J. (NIH P20RR15635 and USDA 2000-02060 and 2003-02213). We thank Ada Yukht for her technical advice in mouse TG explantation and thank Susana Salina for her dedication in monitoring and recording the clinical symptoms of infected animals. We also thank Anita Avery for her excellent assistance with tissue culture and virus titrations.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 12 June 2003; accepted 10 July 2003.