Laboratoire de Virologie, UPRES EA 2387, CERVI1, Laboratoire de Génétique Moléculaire, Service de Biochimie Médicale2 and Service des Maladies Infectieuses et Tropicales3, Groupe Hospitalier Pitié-Salpêtrière, 83 Bld de lHôpital, 75651 Paris Cedex 13, France
Author for correspondence: Henri Agut. Fax +33 1 42 17 74 11. e-mail henri.agut{at}psl.ap-hop-paris.fr
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Abstract |
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Introduction |
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HHV-6 is the causative agent of exanthem subitum in young children (Yamanishi et al., 1988 ). Because of its tropism for T lymphocytes, HHV-6 is thought to act as a co-factor of human immunodeficiency virus (HIV) in AIDS progression, but this hypothesis is still being debated (Ablashi et al., 1998
; Garzino-Demo et al., 1996
; Lusso & Gallo, 1995
). HHV-6 has been also linked to several other diseases, such as multiple sclerosis, chronic fatigue syndrome and tumours (Ablashi et al., 2000
; Berti et al., 2000
; Ongradi et al., 1999
), but all of these results remain controversial (Dorrucci et al., 1999
; Taus et al., 2000
). There are now convincing studies that indicate that HHV-6 behaves as an opportunistic pathogen in liver, renal and bone marrow transplant recipients (Humar et al., 2000
; Ratnamohan et al., 1998
; Rogers et al., 2000
). Now, the precise frequency of such HHV-6-induced diseases has to be evaluated. Although the spectrum of HHV-6 pathogenicity still needs to be clarified, it has become clear that clinically serious HHV-6 infections should benefit from specific antiviral therapy, as pointed out in several reports (Cole et al., 1998
; Mookerjee & Vogelsang, 1997
).
In vitro, HHV-6 susceptibility to antiviral compounds was found to be broadly similar to that of HCMV. HHV-6 is sensitive to ganciclovir (GCV), a nucleoside analogue, cidofovir (CDV or HPMPC), a nucleoside phosphonate analogue, and foscarnet (PFA), a pyrophosphate analogue, which are the major antiviral agents used currently to treat HCMV infections (Biron et al., 1985 ; Crumpacker, 1996
; Freitas et al., 1985
). HHV-6 is relatively resistant to acyclovir (ACV). It is known that suboptimal, long-term therapy of HCMV infection leads to the emergence of drug-resistant HCMV. Numerous studies have shown that nucleotide mutations in two genes, the DNA polymerase (pol) (UL54) gene and the protein kinase (PK) (UL97) gene, are involved in this resistance (Baldanti et al., 1996
; Chou et al., 1995
; Erice, 1999
; Lurain et al., 1994
; Sullivan et al., 1993
). Pol is the final target of the three major antiviral agents and many of the mutations in the UL54 gene induce cross resistance to two or three drugs. The biological role of PK, which is encoded by the UL97 gene and which is also known as GCV kinase, is not understood completely, but PK has been shown to convert GCV and ACV into their monophosphate derivatives. Accordingly, mutations in the UL97 gene induce GCV resistance, but the HCMV strains carrying these mutations remain susceptible to HPMPC and PFA, unless they also carry UL54 gene mutations.
HHV-6 infection has been found to be frequently associated with HCMV infection and is probably underestimated in this context as it is not generally the target of specific diagnostic procedures. Therefore, it may be reasonably assumed that HHV-6 is exposed to antiHCMV drugs in the context of therapies driven both by virological HCMV markers and by clinical symptoms of the HCMV disease. This unrecognized exposure to drugs is susceptible to significantly inhibit HHV-6 replication, but, alternatively, may lead to the selection of drug-resistant HHV-6 strains. To date, there is no available data on HHV-6 resistance to GCV or on the possible molecular mechanisms that sustain this process. As with HCMV, HHV-6 pol, which is encoded by the U38 gene, is thought to be the common target of antiviral drugs. It has been shown that HHV-6 PK, which is encoded by the U69 gene and is homologous to the HCMV UL97 gene product, can phosphorylate GCV and confers GCV sensitivity to baculovirus grown in insect cells expressing HHV-6 PK (Ansari & Emery, 1999 ). However, there continue to be questions concerning the role of HHV-6 PK in HHV-6-infected human cells as well as the existence of specific U69 gene mutations that induce GCV resistance.
We decided to address these questions using two distinct approaches: (i) the selection and characterization of GCV-resistant HHV-6 isolates in vitro using prolonged exposure to GCV; and (ii) the characterization of HHV-6 isolates present in vivo in HCMV-infected patients treated with GCV for a long period of time. The results presented here converge to suggest that a U69 gene mutation induces the resistance of HHV-6 to GCV.
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Methods |
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Patient samples.
Heparinized blood samples were obtained from 21 AIDS patients with HCMV disease. Each patient was followed up at the Pitié-Salpêtrière Hospital in Paris. The 21 patients were treated with GCV either alone or in combination with PFA and CDV. In addition, one blood sample was obtained from one healthy untreated HHV-6-seropositive volunteer. PBMCs were separated from whole blood by centrifugation on FicollHypaque (Pharmacia), washed with PBS and stored at -80 °C until use.
Antiviral agents and monoclonal antibody (MAb).
GCV, the nucleoside analogue 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (Roche) was used for the selection of GCV-resistant HHV-6 mutants and susceptibility assays. Two other antiviral compounds, CDV/HPMPC, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)-cytosine (Pharmacia & Upjohn), and PFA (Astra), were evaluated for their activity against HHV-6 wild-type (wt) virus and the GCV-resistant mutant. MAb 7C7 (Argene Biosoft) was used for the detection of HHV-6 antigen expression by means of immunofluorescence assays (IFA) and flow cytometry in the follow-up of HHV-6 infection and susceptibility assays, respectively (Manichanh et al., 2000 ; Robert et al., 1998
).
Antiviral drug susceptibility assays.
Drug sensitivities were determined as described previously (Manichanh et al., 2000 ). Briefly, MT4 cells were infected at an m.o.i. of 0·01 TCID50 per cell during 1 h of incubation at 37 °C in the presence of 5% CO2. Cells were recovered by centrifugation, resuspended in culture medium and distributed at the concentration of 2x105 cells per well in a 24-well plate (Costar) in the presence of the appropriate concentrations of the drug. Two wells containing infected cells and two containing mock-infected cells without the drug were included as controls. At day 8 post-infection, virus antigen expression was analysed by flow cytometry using MAb 7C7. IC50 values were calculated as described previously (Manichanh et al., 2000
).
DNA extraction.
PBMCs or infected MT4 cells were resuspended in TE buffer (10 mM TrisHCl, pH 7·5, 1 mM EDTA, pH 8·0) containing 1% SDS and 200 µg/ml proteinase K. After 2 h of incubation at 56 °C, total DNA was extracted with an equal volume of TE-saturated phenolchloroformisoamylalcohol (25:24:1). DNA was then precipitated with 2 vols of ethanol in the presence of 0·3 M sodium acetate and the pellet was washed in 70% ethanol before resuspension in water. DNA extraction from ultracentrifuged culture supernatant was performed by resuspending the pellets in TE, incubating the mixture for 2 h at 56 °C followed by 10 min of incubation at 100 °C. The crude lysate was stored at -20 °C prior to DNA amplification.
PCR and nested-PCR for the amplification of HHV-6 DNA.
PBMC DNA ranging from 100 ng to 1 µg was amplified during 40 cycles of PCR, each cycle consisting of denaturation at 92 °C for 1 min, primer annealing at 55 °C for 1 min and chain elongation with polymerase at 72 °C for times dependent on the size of the DNA fragment (1 min for 1000 bp). To improve the fidelity of DNA amplification, we used a mixture of Pfu (Promega) and Taq (Qiagen) polymerase at the ratio of 1:10. The U69 gene (1689 bp) from PBMC samples was amplified using a two-step PCR: first-round PCR was carried out using the primer pair GCVKA and GCVKB and second-round PCR consisted of three separate overlapping nested-PCRs using the three primer pairs GCVKA and GCVKB2, GCVKA1 and GCVKB1 and GCVKA2 and GCVKB, respectively (Table 1). Partial amplification of the U38 gene from PBMCs was performed using the primer pair POLA-MOR and POLB-MOR. When the starting DNA originated from HHV-6 propagated in cell culture, the entire U38 gene as well as the entire U69 gene were amplified using single-step PCR with the primer pairs POLA and POLB and GCVKA and GCVKB, respectively (Table 1
). Initial screening of HHV-6 DNA-positive PBMC samples as well as the follow-up of HHV-6 replication in vitro was carried out with a routine PCR diagnostic assay using the primer pair O10 and O15, as described previously (Collandre et al., 1991
).
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DNA sequencing and sequence analysis.
PCR products and cloned U69 DNA fragments were sequenced with the Prism Big Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). The corresponding primers used to sequence both the U69 and the U38 genes are described in Table 1. The average nucleotide sequence of each cloned U69 gene subfragment was established from the sequence of five to eight independent clones. The nucleotide and derived amino acid sequences were analysed using the following software: Sequencing Analysis, Sequence Navigator and AutoAssembler (Applied Biosystems).
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Results |
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Phenotypic analysis of GCVR1 HST
In the absence of GCV, GCVR1 HST exhibited replication dynamics similar to that of wt virus in terms of antigen expression and virus yield, as assessed by IFA and PCR, respectively (data not shown). GCVR1 HST was subsequently tested by flow cytometry-based susceptibility assay against three antiviral compounds, GCV, CDV and PFA. The results obtained from two independent experiments, each involving duplicate assays for each drug concentration, demonstrated a decrease in the sensitivity of GCVR1 HST to the three drugs tested as compared to wt virus (Table 2). Comparison of the IC50 values indicated that GCVR1 HST was 24-, 52- and 3-fold less sensitive to GCV, CDV and PFA, respectively, than wt HST. Although it had been selected in vitro under GCV pressure only, GCVR1 HST was cross-resistant to CDV and, to a much lower extent, PFA.
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Genetic analysis of GCVR1 HST
The U38 and U69 genes of GCVR1 HST were sequenced and compared to those of wt HST that had been serially propagated in parallel to GCVR1 HST selection. Only two mutations were detected (Figs 1 and 2
). A C
T nucleotide substitution at position 2882 of the U38 gene resulted in an A961V amino acid substitution in the pol gene. An A
G nucleotide substitution at position 952 of the U69 gene resulted in an M318V amino acid substitution in the PK gene. The sequences of wt HST fit perfectly with the HHV-6 variant B consensus sequences and none of the two mutations was located at any of the polymorphic sites detected among distinct GCV-sensitive HHV-6 strains. The consensus sequences of HHV-6 pol and PK were tentatively aligned with the homologous sequences of HCMV (data not shown) in order to predict the possible influence of the two mutations on enzyme function. The A961V mutation appeared to be far distant from the predicted catalytic domains of pol (the nearest putative catalytic domains of HHV-6 pol were domains VII and V at positions 771 and 803, respectively) and did not fit the position of HCMV mutations that had been involved in the resistance of HCMV to GCV (see Discussion). In contrast, the M318V mutation was located within the putative domain VI of HHV-6 variant B PK and affected the consensus sequence D314ISPMN, which is homologous to the HCMV D456ITPMN sequence. This sequence is believed to be a highly conserved region among protein kinases and, in the case of HCMV, has been shown to be the location of GCV-resistance mutations. Two distinct GCV-resistant HCMV strains, R6HS (Lurain et al., 1994
) and C9209 (Chou et al., 1995
), have been found to exhibit M460I and M460V amino acid substitutions in HCMV PK, respectively. These changes were both responsible for the GCV resistance phenotype and are homologous to the M318V change in HHV-6 PK. The results of the genetic analysis of GCVR1 HST thus strongly suggested that the M318V mutation of the HHV-6 PK was, at least in part, responsible for the phenotypic GCV resistance observed. However, a role of the A961V pol mutation for this phenotype, in the context of a single mutation-induced cross resistance to GCV, CDV and PFA, might also be considered.
In vivo genetic analysis of HHV-6 variant B among GCV-treated patients
In order to demonstrate unambiguously the causative role of the M318V PK mutation in GCV resistance, two distinct approaches, both of which were based on HHV-6 variant B replication in MT4 cells, were attempted: marker rescue experiments using the transfection of plasmids carrying the mutated gene and measurement of GCV phosphorylation rates with the hope of detecting a significant decrease of this activity in the presence of the mutation. To date, despite numerous attempts, both strategies have failed, in particular due to the difficulties of achieving a high multiplicity of infection and obtaining recombination events in MT4 cells (data not shown). We then turned to an alternative approach consisting of the study of the U69 gene among subjects exposed to GCV treatment for a long period of time and who were thought to suffer from a GCV-resistant HCMV infection. PBMC samples were obtained from 21 AIDS patients fulfilling these criteria and one untreated, healthy subject as a control. Five of the patients (24%) and the control subject were found to be HHV-6 variant B-positive by means of diagnostic PCR (Table 3). Due to the low virus load, genetic study of the U69 gene from these subjects required separate amplification of three overlapping U69 subfragments and their cloning prior to nucleotide sequencing. One AIDS patient, designated patient #4, exhibited the M318V mutation of PK, while the other five patients and the control subject did not. These findings were reproducibly obtained from two independent amplification runs from the same sample. Partial sequencing of the HHV-6 variant B pol gene indicated that the A961V mutation was absent in the PBMC sample from patient #4. Of note is the fact that, at the time of PBMC sampling, patient #4 had experienced a long-term exposure to GCV and exhibited clinical symptoms of ongoing HCMV retinitis despite the prolonged GCV therapy. These circumstances suggest the selection of the M318V mutation in vivo, in parallel to prolonged inefficient GCV therapy. Isolation of the corresponding HHV-6 variant B strain from PBMC culture was not possible and, thus, did not permit us to test its susceptibility to GCV. A second sample from patient #4 was obtained a few weeks later. The patient was beginning to recover from retinitis and was found to be HHV-6-negative by PCR. Thus, we could not confirm the persistence of the M318V mutation.
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Discussion |
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HHV-6 resistance to antiviral compounds is still an emerging question. Although this virus has been found to be the cause of severe opportunistic infections among immunocompromised patients, there are, to date, only a few reports about the treatment of HHV-6 infection by GCV, PFA and CDV, either alone or in combination (Cole et al., 1998 ; Mookerjee & Vogelsang, 1997
; Rieux et al., 1998
). However, it is clear, in particular from our results, that HHV-6 is very often exposed to GCV in vivo through the prophylaxis and treatment of HCMV infections. Our findings indicate that this unrecognized exposure may select HHV-6 mutations distinct from the usual polymorphism profile of the virus. These mutations, in particular the reported M318V amino acid substitution, are likely to confer a replication advantage to HHV-6 mutants under GCV pressure. Due to the structural similarity of GCV and ACV, prolonged exposure to high concentrations of ACV, as in the situation of HCMV infection prophylaxis, might also be considered in terms of partial selective pressure against both HCMV and HHV-6 PKs. Although the IC50 of ACV against both viruses is high, we cannot rule out the hypothesis that partially effective ACV concentrations may select PK changes, thus inducing a higher resistance profile to GCV. It should be analysed to what extent this unrecognized exposure of HHV-6 to antiherpetic compounds may induce the emergence of drug resistance and compromise the success of further treatments against this virus.
The mechanism of HHV-6 resistance to GCV remains to be understood. The function of HHV-6 PK needs to be clarified in this context. A role in the first step of GCV phosphorylation is strongly suggested by the GCV-induced cytotoxic effect in insect cells expressing HHV-6 PK (Ansari & Emery, 1999 ). This mimics the effect observed with eukaryotic cells expressing herpes simplex virus (HSV) thymidine kinase and which are exposed to GCV, a well-known strategy for suicide gene therapy. The mutation M318V is believed to alter the phosphorylation activity of HHV-6 PK. This would consequently reduce the level of GCV monophosphate within the cell, leading to the decrease of final concentrations of GCV triphosphate active on HHV-6 pol. As mentioned previously, the proof of this deficient phosphorylating activity cannot be obtained under the current standard experimental conditions of HHV-6 infection and, therefore, requires other strategies.
However, another mechanism might be considered for HHV-6 resistance to GCV. As in the case of HCMV, mutations of the pol gene can induce GCV resistance, in particular by lowering the affinity of GCV triphosphate to the binding site of the enzyme and/or reducing the efficiency of the catalytic polymerization step involving this triphosphate analogue. In this case, the GCV-resistance pol gene mutations may also induce resistance to compounds that do not require the first step of phosphorylation into the monophosphate moiety to be active, e.g. CDV and PFA. GCVR1 HST does exhibit this pattern of cross resistance and, in this context, the role of the single pol A961V change must be discussed. This mutation is far distant from the predicted nucleotide binding and catalytic sites of HHV-6 pol, namely domains IV, II, VI, III, I and V located at positions 346, 551, 629, 662, 736 and 803, respectively (Teo et al., 1991 ). In particular, it is far distant from the domains that are homologous to those carrying GCV-resistance mutations in HCMV strains, domains IV and V (Lurain et al., 1992
; Sullivan et al., 1993
). Nevertheless, any amino acid change may alter the general conformation of the protein and, therefore, enzyme functions. As an example, two mutations of the HSV pol gene located at the positions 1007 and 1028 of the amino acid sequence, i.e. beyond the last conserved enzymatic domain, are able to induce resistance to HPMPA, a nucleotide analogue closely related to CDV (Andrei et al., 2000
). However, in this case, the HPMPA-resistant mutants were not resistant to either ACV or PFA, suggesting that the mechanism of resistance was rather specific for HPMPA and did not affect the sensitivity to either nucleoside or pyrophosphate analogues. It is, therefore, tempting to assume that the A961V change of HHV-6 pol is responsible for its high resistance to CDV and moderate resistance to PFA. Its contribution to GCV resistance cannot be completely ruled out, in particular, because sequential processes may be considered for the emergence of GCV resistance: the U69 mutations generating low level resistance to the drug might be followed by the appearance of the U38 mutations inducing a higher level of resistance. This would be similar to the sequential emergence of the UL97 and UL54 mutations observed with GCV-resistant HCMV (Baldanti et al., 1996
). However, when a retrospective analysis was carried out on the samples reserved from the serial intermediary passages under GCV, no temporal dissociation was observed in the emergence of either mutation in the GCV-resistant HHV-6 population. In any case, no mutation of HCMV analogous to A961V has been reported for GCV-resistant viruses and a unique role of A961V for the GCV-resistant phenotype, excluding any contribution of the M318V PK change, is unlikely. The question is why this mutation has been selected in vitro without any use of CDV during HHV-6 propagation and not in vivo in the HHV-6 variant B pol gene from patient #4. The GCV selection pressure in vitro broadly differs from that exerted in vivo due to the consistency of the drug concentration used, the monomorphic nature of cells sustaining virus replication and the absence of many other interfering factors specific for a human organism. In vitro, the selection of GCV-resistant HHV-6, which, in our hands, resulted from a long-standing procedure, may require some compensatory mutations in the pol gene that, by chance, may induce CDV resistance. This illustrates the fact, well known for other herpesviruses, that the isolation and characterization of drug-resistant HHV-6 mutants may be helpful to elucidate both the mechanisms of selective antiviral activity and the dynamics of genetic evolution under selective pressure.
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Acknowledgments |
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Received 14 March 2001;
accepted 11 July 2001.