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Short Communication |
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, MA 02115, USA
Correspondence
Donald M. Coen
Don_Coen{at}hms.harvard.edu
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ABSTRACT |
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MAIN TEXT |
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), including how well mutants resistant to an antiviral drug can reproduce relative to other genetic variants (fitness). The higher the fitness of drug-resistant mutants, the more likely that resistance to that drug will develop. Fitness can vary substantially depending on whether it is measured in cell culture or in animal hosts (in vivo). In vivo fitness can be especially important for the ability of resistant mutants to cause clinically relevant disease.
In the case of herpes simplex virus (HSV), the leading antiviral agent is acyclovir (ACV), a nucleoside analogue that is phosphorylated selectively to its monophosphate by the HSV thymidine kinase (TK). Cellular enzymes convert the monophosphate to a triphosphate, which is a selective inhibitor of HSV DNA polymerase (reviewed by Coen & Richman, 2007). Most ACV-resistant mutants exhibit little or no defect in fitness in cell culture; however, nearly all such mutants exhibit some reduction in fitness and pathogenicity in animal models of HSV infection (reviewed by Coen, 1994; Larder & Darby, 1984). These reductions are likely to be one of the factors contributing to the limited impact of ACV resistance in clinical settings (reviewed by Bacon et al., 2003).
Several newer anti-HSV agents target viral functions other than TK and DNA polymerase (reviewed by Coen & Schaffer, 2003; Visalli & van Zeijl, 2003). Among these are novel thiourea compounds that inhibit cleavage and packaging of viral genomes (van Zeijl et al., 2000). Further studies with one of these compounds, WAY-150138, suggest that it prevents complete assembly of the viral capsid (Newcomb & Brown, 2002). WAY-150138 and related compounds form an interesting inhibitor class containing some of the few antiherpes agents that do not target viral DNA replication. They have the advantage of targeting a process (encapsidation) and proteins that are specific to the virus. Thus, this class of drugs has promising features both for laboratory investigations and, potentially, for clinical use.
Mutants derived from HSV-1 strain Patton that were generated via serial passage in the presence of thiourea compounds are resistant to these compounds due to single amino acid substitutions in the UL6 protein (van Zeijl et al., 2000), the major component of the capsid portal complex through which viral DNA is presumably threaded during packaging (Newcomb et al., 2001; Patel et al., 1996; Sherman & Bachenheimer, 1988; Trus et al., 2004). One of the mutants, 138R/5, which harbours a glutamine to arginine mutation at position 621 (van Zeijl et al., 2000), exhibits more than 10-fold resistance to WAY-150138 (Pesola et al., 2005; van Zeijl et al., 2000). Based on experience with other anti-HSV agents, this degree of resistance would probably be clinically significant (Safrin et al., 1994). In the absence of WAY-150138, 138R/5 replicates with kinetics and to titres equivalent to those of the wild type in a 24 h yield assay performed at a low m.o.i. (van Zeijl et al., 2000). Whilst in vitro fitness, as observed by van Zeijl et al. (2000), is common with drug-resistant viral mutants, especially those that, like 138R/5, were isolated by passage in cell culture in the presence of drug, they are often found to be attenuated for replication or ability to cause disease in vivo.
In the course of experiments investigating viral gene expression during reactivation from latency (Pesola et al., 2005), we observed preliminarily that 138R/5 (plaque-purified and kindly provided by Thomas R. Jones and Marja van Zeijl, Wyeth, Pearl River, NY, USA) exhibited considerable virulence in mice. We therefore followed up this observation to investigate whether this mutant was defective for in vivo fitness and virulence in a mouse model of HSV infection. In this model, virus is inoculated onto the scarified corneal epithelium, where it replicates productively and gains access to sensory nerve axon termini. From there, viral capsids travel to neuronal cell bodies in the trigeminal ganglion, where the virus can replicate productively and then establish latency, usually within 2 weeks (Stevens & Cook, 1971; Tenser & Dunstan, 1979). Virus can also infiltrate and replicate in the spinal cord and brain (reviewed by Enquist et al., 1998), which may result in fatal encephalitis. For the present studies, wild-type and 138R/5 viruses were inoculated bilaterally onto the corneas of 8-week-old male ICR mice (Harlan) as described previously (Pesola et al., 2005). On various days post-infection (p.i.), the progression of the acute infection was monitored by titration of virus from eye swabs, trigeminal ganglia and brains. These procedures were based on previously described methods (Coen et al., 1989; Leib et al., 1989), with some modifications detailed below. To collect eye swabs, mice were anaesthetized transiently in an induction chamber with isoflurane (3 % in oxygen; 2 ml min–1) using an anaesthesia machine (Colonial Medical). This method yields eye-swab titres indistinguishable from those collected without anaesthesia (M. F. Kramer & K. F. Bryant, personal communication). After wiping each eye with a separate cotton swab moistened in 1 ml cell-culture medium (containing 5 % newborn calf serum), swabs were returned to the medium and stored at –80 °C. To harvest tissue samples, mice were sacrificed and their brains and two trigeminal ganglia were removed, placed separately in tubes containing 1 ml culture medium and stored at –80 °C. These tissues were later thawed, homogenized and refrozen. On the day of titration, the homogenates were thawed, sonicated and (for brain only) clarified by centrifugation. The eye-swab media, ganglion homogenates and brain-homogenate supernatants were then titrated for infectious virus by standard plaque assays. Eye swabs, ganglia and brains from mock-infected mice were tested on several days p.i. and were always negative for infectious virus.
Over the first 5 days p.i., the titres of virus found in eye swabs (Fig. 1a) or ganglia (Fig. 1b) from mice infected with either wild-type or 138R/5 virus were very similar. The brains of four mice infected with each virus were harvested at 2, 3, 4, 5 and 7 days p.i. The virus titre in this tissue was indistinguishable between the two viruses (Fig. 1c). These data indicate that replication of the wild-type and drug-resistant viruses at the periphery (cornea), their transit to the ganglia and brain and their acute replication therein were equivalent.
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). In these studies, quantitative real-time PCR demonstrated no differences in the amount of viral DNA detected 48 h post-explant with or without ACV treatment. Because ACV is a specific inhibitor of viral DNA replication, the amount of DNA in ACV-treated ganglia is indicative of the DNA content of latently infected ganglia. Additionally, both wild-type virus and 138R/5 were able to reactivate from 100 % of infected mouse trigeminal ganglia upon explant for 48 h. Quantitative RT-PCR analyses showed that the expression of several HSV RNAs was similar in wild type- and 138R/5-infected ganglia after 48 h in the presence of ACV. Thus, these previous studies, together with the data reported here, revealed no differences in the in vivo fitness of the wild type and drug-resistant mutant.
We then asked whether the mutant virus retained virulence. In three independent experiments in which mice were infected in parallel with equivalent amounts of wild-type or 138R/5 virus, no reduction in mortality was observed for 138R/5-infected mice (Fig. 2). Thus, there was no indication that 138R/5 was attenuated for virulence in vivo [latently infected ganglia from mice infected with 138R/5, but not from those infected with Patton wild type, yield reactivated virus when explanted in the presence of WAY-150138 (Pesola et al., 2005), suggesting that the UL6 mutation did not revert during infection of mice]. In fact, in all three experiments, a higher lethality was observed consistently for 138R/5. In the two experiments with the largest sample numbers, these differences were statistically significant (Fig. 2a, b), and the difference was highly significant (P=0.0023) for the experiment in Fig. 2(a). It is notable that these differences were observed at two different doses of virus (confirmed by back titration of the inocula).
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; van Zeijl et al., 2000) and reveal that mutant 138R/5 is at least as fit and virulent as its wild-type parent in vivo. This is a surprising finding. There are only a few examples of drug-resistant virus mutants that maintain in vivo fitness and virulence in animal models (reviewed by Coen & Richman, 2007). Perhaps the best example is adamantane-resistant influenza (Bean et al., 1989; Oxford & Potter, 1973; Sweet et al., 1991). Of the many HSV drug-resistant mutants that have been tested for in vivo fitness and virulence, very few have been unimpaired (reviewed by Coen, 1994; Larder & Darby, 1984). One such exceptional mutant, KG111, which has partial TK activity, is indistinguishable from wild type in its ability to establish and reactivate from latency in the mouse ocular model (Coen et al., 1989) and in its virulence following intracranial inoculation (Pelosi et al., 1998a). It should be emphasized that, despite their decreased in vivo fitness and virulence, most ACV-resistant mutants of HSV replicate efficiently in cell culture. At least one other HSV-1 mutant, PAAr5, which is resistant to phosphonoacetic acid and ACV, replicates comparably to wild type in the peripheral nervous system (Jacobson et al., 1995) and in mouse brain following intracranial inoculation (Pelosi et al., 1998b). However, this mutant causes less lethality, due to the polymerase mutation (Pelosi et al., 1998b). In other words, like 138R/5, this mutant retains in vivo fitness, but unlike 138R/5, it does not retain virulence.
ACV exhibits selectivity for two viral enzymes, TK and DNA polymerase, and mutations affecting either of these proteins can confer ACV resistance. Resistance-conferring TK mutations need only eliminate or significantly reduce TK activity. There is good evidence that these mutations reduce in vivo fitness and virulence, due to a greater requirement for thymidine phosphorylation for virus replication in neurons than in cultured cells (Chen et al., 1998). Polymerase mutations must necessarily be functionally conservative, because this enzyme is essential for HSV replication. How these mutations reduce in vivo fitness and virulence is unclear, but it has been speculated that the mutant polymerases have decreased affinities for deoxynucleoside triphosphates that are in lower concentrations in neurons (Field & Coen, 1986).
The probable mechanism of action of WAY-150138 and, therefore, the modes for resistance, are different. There is evidence suggesting that WAY-150138 may prevent assembly of intact capsids by interfering with the association of the portal and scaffolding proteins (Newcomb et al., 2003). If WAY-150138 interferes directly with this association, the finding that 138R/5 maintains fitness and virulence would be especially notable, because mutations that are able to avoid the effects of WAY-150138 while maintaining essential protein–protein interactions are expected to be rare. However, it may be that WAY-150138 acts allosterically, and mutations that affect its binding may not affect protein–protein interactions. Nevertheless, other mutations that affect capsid proteins, including one that merely alters kinetics of expression, and have little or no effect on in vitro fitness can diminish in vivo fitness (Desai et al., 1998; Tran et al., 2002).
A second surprising finding of the present study is that 138R/5 exhibited a modest, but significant, increase in virulence compared with its wild-type parent. The replication of 138R/5 in ganglia and brain gave no indication of increased fitness in that environment and thus no obvious suspect for the increased virulence. Further investigations of specific regions of the central nervous system or types of cell in which 138R/5 replicates may uncover differences in neurotropism or neuroinvasion that could account for this increased virulence.
It is not yet known whether the increased virulence is due to the UL6 mutation or to coincident, unidentified mutations. Regardless, it may be relevant that UL6 sequences can precipitate an inflammatory response in the cornea in mice (Zhao et al., 1998). It may be that some kind of increased inflammatory response is involved in the increased-virulence phenotype observed in our studies. Uncovering the basis for increased virulence could be valuable in ongoing efforts to adapt herpesvirus vectors for gene therapy.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 December 2006;
accepted 25 January 2007.
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) or 138R/5 (
). Horizontal bars designate arithmetic means and dotted lines denote the limit of detection for the assay when no plaques were observed. Symbols on the abscissa were taken as zero when calculating mean values. Eye swabs on day 0 were collected 5 h p.i. To analyse these data statistically, each observation, x, of p.f.u. was transformed into x' by the function x'=log10(x+1) in order to equalize within-group variances, approximate a normally distributed variable and avoid the undefined logarithm of zero. Two-way ANOVA was performed on the x' values by using Prism 4 (GraphPad Software). For ganglion and brain titres, virus was not a significant source of variation (P>0.8). For eye swabs, ANOVA detected an interaction between virus and days p.i. (P<0.01) and Bonferroni post-tests indicated a significant difference between viruses on days 1 and 4 p.i. (P<0.001). However, titres for both viruses were very similar on all other days p.i. In a repeat experiment, no significant differences were found on days 1–4 p.i. and the relative positions of the mean p.f.u. for wild-type and 138R/5 viruses on days 1 and 4 p.i. were reversed from those in (a) (data not shown). Thus, we do not consider these distinctions to be biologically relevant.