1 Division of Virology, Department of Pathology, Cambridge University, Tennis Court Road, Cambridge CB2 1QP, UK
2 GlaxoSmithKline, UK Virology Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK
Correspondence
Rob Fenton
rob.j.fenton{at}gsk.com
Finn Grey
greyf{at}ohsu.edu
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ABSTRACT |
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Present address: Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, L220, Portland, Oregon 97239, USA.
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INTRODUCTION |
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It is generally accepted that TK-deficient viruses have low neurovirulence (Field & Darby, 1980; Field & Wildy, 1978
; Tenser et al., 1981
; Sibrack et al., 1982b
) and are unable to reactivate from latency (Tenser & Dunstan, 1979
; Coen et al., 1989
; Efstathiou et al., 1989
; Jacobson et al., 1993
). Periodically, however, reports have emerged that describe individual TK-deficient isolates which appear to challenge these principles, i.e. they can be recovered, albeit somewhat inefficiently, from latently infected animals (Sakuma et al., 1988
; Erlich et al., 1989
; Kost et al., 1993
; Sasadeusz et al., 1997
; Horsburgh et al., 1998
). A possible explanation is that these isolates induce low levels of TK, which accounts for their neurovirulence, either because they are heterogeneous and contain a small proportion of wild-type virus or because the mutant gene permits expression of limited TK activity (Hwang et al., 1994
). A more intriguing possibility is that, in some cases, other gene products have the potential to complement TK function and influence the ability of HSV to replicate in the nervous system and to reactivate from latency (Horsburgh et al., 1998
).
In a study of ACV-resistant clinical isolates, collected in Europe between 1980 and 1995 (Harris et al., 2003), we confirmed that viruses with mutations that disrupt the TK region are generally attenuated with respect to their ability to replicate in the nervous system, with no evidence of zosteriform spread in infected animals. Furthermore, consistent with previous observations (Horsburgh et al., 1998
; Hwang et al., 1994
; Sasadeusz et al., 1997
), it was shown that variants with the potential to express minimal levels of TK can be recovered, albeit inefficiently, from cultured ganglia. These include isolates with single G insertions in the G-string (Fig. 1
), in which translational frame-shifts (+1) during protein synthesis are thought to result in the expression of a low level of wild-type polypeptide (Hwang et al., 1994
), and those with amino acid substitutions, where the mutated polypeptides retain minimal activity.
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METHODS |
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BuBHK cells are derived from baby hamster kidney cells (BHK) and are devoid of cellular TK activity. They were generated by passage in the presence of 5-bromo-2'-deoxyuridine and were grown under the conditions stated above.
All cells were grown, and assays performed, at 37 °C in 5 % CO2.
HSV clinical isolates and reference laboratory strains.
The ACV-resistant, TK-deficient HSV-1 clinical isolate strain 4 was obtained from an immunocompromised patient who failed to respond to ACV treatment. The isolate was plaque-purified twice by limiting dilution in Vero cells. Two clones, C4a and C4b, were selected for further analysis. Sequence analysis (Harris et al., 2003) had shown that the clones carried identical TK genes, each with a GG insertion in the G-string motif. One of these clones, C4b, was used in the studies described in this paper.
The recombinant virus, SC16 GG, was constructed by inserting two G residues (GG) into the G-string motif of the SC16 TK gene, which was cloned into the HindIII and XbaI sites of the plasmid pcDNA3 (Invitrogen) using the Exsite Mutagenesis kit (Promega). The integrity of recombinant plasmids was confirmed by appropriate restriction enzyme digestion and by sequencing. Plasmids were co-transfected into Vero cells with infectious SC16 DNA and overlaid with medium containing 2·5 mM ACV. Resistant plaques carrying the GG insertion in TK were plaque-purified twice and the presence of the mutation was then confirmed by sequencing.
To construct the TK-disrupted recombinant virus C4bgfp, the plasmid pAR29 (a gift from C. Preston, MRC Institute of Virology, Glasgow, UK) was used. This plasmid comprises the BamHI P fragment of HSV-1 (nt 45 05548 634) containing the human cytomegalovirus (HCMV) immediate-early promoter driving expression of the green fluorescent protein (GFP) gene inserted in the SacI site of the TK gene (nt 47 359). GFP is transcribed anti-sense in relation to the TK gene. Recombinant virus was generated by co-transfection into Vero cells of 10 µg clone C4b virus-infected cell DNA with 3 µg of linearized plasmid DNA by the CaCl2DMSO boost method (Stow & Wilkie, 1976). Progeny virus was recovered by limiting dilution and selection of GFP-positive recombinant viruses. The expected genomic structure was verified by Southern hybridization analysis (data not shown).
HSV-1 strain SC16 (Hill et al., 1975) was used as the reference type 1 strain. The deletion mutant, DM21, derived from it (Efstathiou et al., 1989
), which carries an 816 bp deletion in TK, was used as the control TK-negative strain.
The characteristics of these viruses are outlined in Table 1.
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Enzyme assays.
A 50 µl sample of protein extract was added to 200 µl of reaction mixture containing 6 mM ATP, 2 mM disodium orthophosphate, 18 mM sodium orthophosphate, 6 mM MgCl2 and 0·4 µCi [14C]thymidine (sp. act. 4060 mCi per mmol; 1·482·22 GBq per mmol), and incubated at 37 °C. Samples were taken at 10 and 20 min and heated at 100 °C for 2 min to stop the reaction. Samples were spun in a Heraeus Biofuge at 13 000 r.p.m. for 2 min, then duplicates of 50 µl samples were spotted onto DEAE filter paper and allowed to dry. Filter papers were washed three times for 20 min with 1 mM ammonium formate. The incorporation of [14C]thymidine was then quantified by liquid scintillation counting.
Genotype analysis.
Sequencing was performed by the cycle sequencing of PCR-generated products from infected cell DNA using 12 overlapping sense and anti-sense internal primers to encompass the entire TK open reading frame. Sequence reactions were carried out using the ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit and electrophoresis on an ABI 377 sequencer, according to the manufacturer's instructions. The resulting sequence was analysed using Lasergene software (DNASTAR).
In vivo analysis.
The left ear pinnae of anaesthetized 4-week-old BALB/c mice were infected by inoculating 10 µl of virus suspension (containing 5x105 p.f.u.) onto an area scarified previously with a 27 gauge needle. Mice were sacrificed at days 3 and 5 p.i. and the inoculated ear and dorsal root ganglia C2, C3 and C4 were removed and homogenized in 500 µl GMEM supplemented with 10 % FCS, 100 units penicillin G ml-1, 100 µg streptomycin ml-1 and 0·01 mM glutamine. For latency reactivation assays, mice were sacrificed at least 30 days p.i. and the dorsal root ganglia C2, C3 and C4 were removed into GMEM supplemented with 10 % FCS, 100 units penicillin G ml-1, 100 µg streptomycin ml-1 and 0·01 mM glutamine. Ganglia were incubated at 37 °C in a 5 % CO2 humidified atmosphere for 72 h before being homogenized and assayed for infectious virus on Vero cells.
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RESULTS |
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In vivo characteristics of clone C4b
To characterize further the phenotype of clone C4b, a detailed in vivo analysis was carried out. The in vivo characteristics of clone C4b were compared with SC16 and the TK deletion mutant DM21. Mice were infected with 5x105 p.f.u. of virus via scarification of the ear pinnae. At days 3 and 5 p.i., the ears and ganglia of infected mice were taken and assayed for the presence of infectious virus. Infected ganglia were also explanted 34 days p.i. to examine virus reactivation from latency (Fig. 3a). Virus titres in the ear at days 3 and 5 indicated efficient replication of all three viruses, although there may be a slight attenuation of DM21 at day 3 and both clone C4b and DM21 at day 5 compared with SC16. The attenuation of DM21 is far more pronounced in the ganglia of infected mice at both days 3 and 5. Clone C4b is also attenuated in its ability to replicate in the ganglia at both days when compared with SC16. However, clone C4b was able to replicate with greater efficiency than DM21 on both days and was able to reactivate from a latent state with an efficiency equivalent to that of SC16. In contrast, no infectious virus was detected in explanted ganglia from mice infected with DM21.
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To address the question of whether the TK gene of clone C4b and its low level of TK activity is playing a role in its unusual in vivo phenotype, we disrupted the TK gene by insertion of the GFP reporter gene. The in vivo characteristics of the recombinant virus, C4bgfp, were then compared to SC16 and clone C4b. The results showed that this modified virus lost its ability to replicate in the peripheral nervous system of mice and its ability to reactivate from latency (Fig. 3c). Furthermore, in situ data showed a reduced level of latency establishment of the recombinant clone C4bgfp relative to clone C4b and SC16 (Table 2
).
Can translational frame-shifting explain the low level of TK activity in clone C4b?
One possible explanation for the low level of TK activity induced by clone C4b was that frame-shifting in the G-string during translation could result in the synthesis of a small quantity of functional product. Data to support this mechanism had been obtained in the case of a single G insertion mutant (Hwang et al., 1994), although, in this case, the frame-shift invoked was +1 (Fig. 1
). Clone C4b has a double G insertion and so the frame-shift required in this case to move translation back to the correct reading frame would be +2. Alternatively, a -1 frame-shift would also shift translation back to the correct reading frame but with the insertion of an additional glycine residue. If this is the explanation, then it is likely that any virus having a -2 (or +1) frame-shift in the G-string would exhibit a similar high level of neurovirulence and that virus recovered from the nervous system of animals would be genotypically and phenotypically identical to the infecting virus. SC16 GG was, therefore, constructed. SC16 GG contains in the SC16 genetic background a double G insertion in the G-string and, therefore, has a run of nine G residues. However, this virus was shown to be TK negative in all assays (data not shown) and reactivated at low levels from only 2 of 10 explanted ganglia cultures (2·3x102 and 1x102 p.f.u., respectively).
As translational frame-shifting could not explain the low level of TK activity or the ability of the virus to replicate in the peripheral nervous system, further phenotypic analyses of the virus recovered from the peripheral nervous system of mice infected with clone C4b were undertaken.
TK phenotype of virus recovered from the nervous system
Virus samples were recovered from animals infected with clone C4b from local sensory ganglia during the acute phase of the infection and following reactivation from latency. Virus stocks were grown on Vero cells from the original ganglionic homogenates derived from individual mice. These were tested first for their ability to express TK activity (Fig. 4). All were found to be positive for TK expression. Viruses isolated from acutely infected ganglia expressed variable levels of TK activity, with one clonal isolate expressing levels as high as SC16 (Fig. 4a
). All viruses recovered from latently infected ganglia expressed levels similar to those expressed by SC16 (Fig. 4b
). Therefore, it appears that passage through the peripheral nervous system exposes the virus to positive selection for TK expression.
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DISCUSSION |
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The first task was to establish whether clone C4b induced any measurable TK activity. Although levels were extremely low (<1 % of the level of the wild-type virus SC16), there appeared to be consistent TK activity. The nature of the genetic lesion in clone C4b (a double G insertion inducing a -2 frame-shift) appeared to preclude any induction of TK activity by authentic translation of the gene since many functionally critical regions are disrupted by the frame-shift (Brown et al., 1995). Amino acids with direct involvement in the active site of the enzyme [e.g. residues 161192 involved in nucleoside binding and other conserved residues in the active site, such as Arg222 and Glu225 and Asp162, which is believed to be responsible for Mg2+ co-ordination (Brown et al., 1995
)] would be lost following the frame-shift.
Two alternative explanations were considered for the low level of TK expression. Hwang et al. (1994) produced evidence that a compensating +1 translational frame-shift could result in the induction of a small amount of wild-type polypeptide from a TK gene with a single G insertion in the G-string, and so frame-shifting had to be considered as a possible explanation. An alternative explanation was suggested by the work of Horsburgh et al. (1998)
who showed that other gene products could compensate for the loss of TK function in interactions of the virus with the nervous system. Furthermore, work with the drug ganciclovir in HCMV, which has no TK gene, has demonstrated that phosphorylation of this nucleoside analogue is carried out by the UL97 gene product, a product believed to be a protein kinase (Biron et al., 1986
; Littler et al., 1992
). The HCMV UL97 gene is conserved across the herpesviruses and is homologous to the UL13 product of HSV. Therefore, another gene product compensating for the loss of TK was considered also.
A simple way of assessing whether the TK gene had a role in neurovirulence was to introduce an irreversible lesion into the TK gene of clone C4b. This was achieved by introducing an HCMV GFP gene cassette into the TK open reading frame. The resulting virus exhibited no detectable expression of TK activity (Fig. 2c). Furthermore, it resembled the deletion mutant DM21 in its profound attenuation in the peripheral nervous system of mice during acute infection and its inability to reactivate from latency (Fig. 3c
). The data demonstrated the necessity for the uninterrupted TK gene for neurovirulence and appeared to eliminate a role for a compensatory gene function. However, although the data appear to argue against compensatory functions, they do not provide any direct evidence for frame-shifting.
It was argued that if frame-shifting explained the neurovirulence of clone C4b, it might be expected that other viruses with mutations in the G-string that induce -2 (or +1) frame-shifts would exhibit similar neurovirulence. However, SC16 GG (-2 frame-shift) showed diminished neurovirulence. Consistent with this are the observations described in the previous paper on clone C3c, which also exhibits a +1 frame-shift and which appears to lack neurovirulence (Harris et al., 2003).
Another prediction was that if frame-shifting explained the neurovirulence of clone C4b, virus recovered from the nervous system during acute infection and following reactivation from latency would have the phenotype and genotype of the input virus. This was not the case. Expression of TK was observed with viruses recovered from the nervous system of animals infected with clone C4b, intermediate levels with those isolated during the acute phase and almost wild-type levels with viruses recovered from latency. Consistent with these phenotypic observations, virus recovered from latently infected ganglia had acquired an additional mutation in the G-string (a third G residue), ensuring that the TK gene was now translated in frame although the protein would be expected to have an additional glycine residue.
A single G insertion in the G-string appears to be the most commonly observed TK mutation (Sasadeusz et al., 1997; Harris et al., 2003
). If this mutation is capable of reverting clone C4b to wild-type TK functionality, it may not be surprising that it emerges during replication of the virus in the nervous system, where there would be positive selection pressure for functional TK activity. However, it is still unclear why this phenomenon is so marked with clone C4b and is not seen with SC16 GG or clone C3c described in the previous paper (Harris et al., 2003
). There may yet be a significant role for genes other than TK in regulating the genetic stability of the virus. Evidence to support this comes from sequence data on reactivated SC16 GG. The TK region of two of the cloned isolates from explanted ganglia infected with SC16 GG was sequenced. One of these contained the same GG insertion as the input virus. The other, however, had gained an extra G residue, the same secondary mutation seen in reactivated isolates of clone C4b. This suggests that SC16 GG is able to revert to a wild-type phenotype by the same mechanism as clone C4b but at a slower rate. As genetic instability of C4b has been shown to lead to mixed populations of the TK-positive and -negative viruses in vivo, it is likely that the same instability occurs in vitro and accounts for the low level TK activity detected. The lower levels of TK activity detected before passage in mice reflect the lack of selection of TK-positive viruses in tissue culture. In contrast, during replication of the virus in vivo, strong selective pressure would favour the replication of the TK-positive variant of C4b.
In summary, the low level of expression of TK by clone C4b and its considerable neurovirulence result from inherent genetic instability at the TK locus, resulting in significant phenotypic reversion to a virus with wild-type TK activity by the addition of a third G residue (Fig. 5). The phenotypic and genotypic data regarding SC16 GG suggests that this may be due to factors other than the specific GG insertion in the TK gene of clone C4b.
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NOTE ADDED IN PROOF |
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ACKNOWLEDGEMENTS |
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Received 7 October 2002;
accepted 21 February 2003.