Cytomegalovirus UL97 kinase catalytic domain mutations that confer multidrug resistance.

Human cytomegalovirus UL97 kinase mutations that commonly confer ganciclovir resistance cluster in different parts of the gene than those conferring resistance to maribavir, an experimental UL97 kinase inhibitor. The drug resistance, growth, and autophosphorylation phenotypes of several unusual UL97 mutations in the kinase catalytic domain were characterized. Mutations V466G and P521L, described in clinical specimens from ganciclovir-treated subjects, conferred a UL97 kinase knockout phenotype with no autophosphorylation, a severe growth defect, and high-level ganciclovir, cyclopropavir, and maribavir resistance, similar to mutations at the catalytic lysine residue K355. Mutations F342S and V356G, observed after propagation under cyclopropavir in vitro, showed much less growth attenuation and moderate- to high-level resistance to all three drugs while maintaining UL97 autophosphorylation competence and normal cytopathic effect in cell culture, a novel phenotype. F342S is located in the ATP-binding P-loop and is homologous to a c-Abl kinase mutation conferring resistance to imatinib. UL97 mutants with relatively preserved growth fitness and multidrug resistance are of greater concern in antiviral therapy than the severely growth-impaired UL97 knockout mutants. Current diagnostic genotyping assays are unlikely to detect F342S and V356G, and the frequency of their appearance in clinical specimens remains undefined.

Cyclopropavir susceptibility of cytomegalovirus DNA polymerase mutants selected after antiviral drug exposure.

Human cytomegalovirus (CMV) UL54 DNA polymerase (pol) mutants with known patterns of resistance to current antivirals ganciclovir (GCV), foscarnet (FOS), and cidofovir (CDV) were tested for cyclopropavir (CPV) susceptibility by a standardized reporter-based yield reduction assay. Exonuclease and A987G (region V) mutations at codons commonly associated with dual GCV-CDV resistance in clinical isolates paradoxically conferred increased CPV susceptibility. Various polymerase catalytic region mutations conferring FOS resistance with variable low-grade GCV and CDV cross-resistance also conferred CPV resistance, with 50% effective concentration (EC(50)) increases of 3- to 13-fold. CPV EC(50) values against several pol mutants were increased about 2-fold by adding UL97 mutation C592G. Propagation of a CMV exonuclease mutant under CPV selected for pol mutations less often than UL97 mutations. In 21 experiments, one instance each of mutations E756D and M844V, which were shown individually to confer 3- to 4-fold increases in CPV EC(50), was detected. Unlike GCV and CDV, exonuclease mutations are not a preferred mechanism of CPV resistance, but mutations in and near pol region III may confer CPV resistance by affecting its recognition as an incoming base for DNA polymerization.

Accelerated evolution of maribavir resistance in a cytomegalovirus exonuclease domain II mutant.

A human cytomegalovirus (CMV) UL54 pol exonuclease domain II mutation, D413A, found in a clinical specimen, conferred ganciclovir (GCV) and cidofovir resistance but not foscarnet resistance when incorporated into laboratory strain T2294. After several passages without drug, mutation was observed in five of eight plaques of T2294, and its plating efficiency under foscarnet was increased approximately 30-fold over that of a control strain. When T2294 was serially passed under maribavir (MBV), phenotypic changes and viral UL97 mutations were detected by passage 5, much earlier than previously reported for other CMV strains. By passage 15, mutations included two cases of H411Y, one each of H411L and H411N, three of T409M, five of V353A, and one of L397R. Five instances of codon 409 or 411 mutations evolved into double mutations including V353A. Marker transfer experiments showed H411N/Y/L to confer 9- to 70-fold-increased MBV resistance and combinations of H411L/Y and V353A to confer >150-fold-increased MBV resistance, but no GCV resistance. These findings are consistent with defective exonuclease activity of the pol D413A mutant T2294, leading to the accelerated evolution of UL97 mutations under MBV. This recapitulated the known resistance mutations V353A, L397R, and T409M; suggested their relative frequency; and identified new ones at codon 411. These UL97 mutations predict an MBV binding region overlapping the kinase ATP binding site and located upstream of known GCV resistance mutations. The existence of viable pol D413A mutants may facilitate the selection of additional drug resistance mutations in vivo and the study of these mutations in vitro.

Contrasting drug resistance phenotypes resulting from cytomegalovirus DNA polymerase mutations at the same exonuclease locus.

BACKGROUND: Diverse mutations in the cytomegalovirus (CMV) DNA polymerase (pol) gene confer resistance to one or more of the antiviral drugs ganciclovir, foscarnet or cidofovir. The levels of resistance conferred by specific mutations are variable, ranging from insignificant resistance to triple-drug resistance. OBJECTIVES: Three pol mutations, I521T, P522A and P522L, detected in patients who received antiviral therapy for CMV infection, were studied by recombinant phenotyping to characterize their associated drug resistance. STUDY DESIGN: The individual mutations were transferred by homologous recombination into a reference CMV strain modified with a reporter gene and the drug concentrations required to reduce the reporter signal by 50% (IC50) were determined. RESULTS: The mutations I521T and P522A each conferred 3- to 4-fold increases in IC50 to both ganciclovir and cidofovir, while mutation P522L conferred no significant resistance to either drug. None of these mutations conferred foscarnet resistance. CONCLUSIONS: The resistance phenotypes of mutations I521T and P522A are as predicted from the known mutation P522S, but divergent results with P522L indicate that different amino acid substitutions at the same position may not have the same effect on drug resistance. New mutations must be individually validated for proper interpretation of genotypic resistance testing.

Development of new cytomegalovirus UL97 and DNA polymerase mutations conferring drug resistance after valganciclovir therapy in allogeneic stem cell recipients.

BACKGROUND: We report on two allogeneic stem cell transplant recipients who developed cytomegalovirus disease associated with new viral mutations that conferred antiviral drug resistance. METHODS: Blood specimens obtained during symptomatic disease were analyzed for mutations in the CMV UL97 and DNA polymerase genes and new mutations were assessed by recombinant phenotyping. RESULTS: Rising cytomegalovirus (CMV) antigenemia occurred after 4-5 months of preemptive valganciclovir therapy, followed by symptomatic CMV disease including fatal pneumonia in one case. In one case, a new viral UL97 mutation (deletion of codons 601-603) was found which conferred 15-fold increased ganciclovir resistance. In the other case, a known UL97 resistance mutation M460V and a new DNA polymerase (pol) mutation D413A were found. D413A conferred ganciclovir and cidofovir resistance. CONCLUSIONS: Known and newly discovered drug resistance mutations arising during preemptive therapy may complicate post-transplant CMV disease in stem cell recipients. Improved recombinant phenotyping methods enable the rapid quantitation of the resistance conferred by newly identified UL97 and pol mutations.

Maribavir sensitivity of cytomegalovirus isolates resistant to ganciclovir, cidofovir or foscarnet.

BACKGROUND: The cytomegalovirus (CMV) UL97 inhibitor drug maribavir (MBV) is undergoing clinical antiviral trials. OBJECTIVES: To assess the MBV sensitivity of CMV strains and isolates containing mutations that confer resistance to current antiviral drugs ganciclovir, cidofovir or foscarnet. STUDY DESIGN: Resistant clinical isolates and laboratory strains containing UL97 and or UL54 DNA polymerase mutations were tested for sensitivity to all four drugs by standard plaque reduction assay and a reporter-based yield reduction assay. Sensitive control strains were also tested. RESULTS: Eleven CMV strains or isolates resistant to GCV, four resistant to FOS and two resistant to CDV, were all sensitive to MBV. These viruses represent four UL97 mutations and three UL54 DNA polymerase mutations. The laboratory derived UL97 L397R mutant was highly MBV-resistant but remained sensitive to the other three drugs. CONCLUSIONS: No cross-resistance has been detected between viruses resistant to MBV and those resistant to one or more of the current CMV antiviral drugs, consistent with differences in their mechanisms of action.

A longitudinal analysis of T-cell epitope-coding regions of hepatitis C virus after liver transplantation.

BACKGROUND: Hepatitis C virus (HCV)-related liver failure is the single leading indication for orthotopic liver transplantation (OLT) worldwide. The mechanisms that underlie the observed differences in natural history of HCV recurrence remain poorly understood. We have previously demonstrated that differential T-cell responses correlate with histologic severity after OLT. We hypothesized that amino acid substitutions within critical T-cell epitopes could lead to increased severity of HCV disease. METHODS: We determined the peptide sequences from sequential serum-derived viral RNA by reverse transcription and direct polymerase chain reaction sequence analysis from 32 HCV genotype 1-infected patients with well-characterized outcomes after liver transplantation. Serum samples were analyzed for HCV sequence the day of OLT and at least one time point post-OLT. To construct evolutionary relationships among the different patient samples, phylogenetic analyses of core and NS3 sequences were performed using a matrix fed into a neighbor-joining tree algorithm. RESULTS: The phylogenetic analyses revealed remarkable conservation within a given individual and no significant differences when comparing patients with severe versus mild recurrence. Accordingly, the synonymous mutation rate was consistently greater than the nonsynonymous substitution rate. The nine epitopic regions analyzed were also preserved so that, with the exception of one patient with mild recurrence, none of the patients demonstrated a shift in viral peptide sequence. CONCLUSIONS: HCV core and NS3 viral peptide sequences are identical before and after OLT in most patients, suggesting that the prevalent sequence is preserved in most cases, and viral variants are competent to establish infection after OLT. Although these results do not support viral mutation as a dominant pathogenic mechanism after OLT, other viral regions need to be analyzed.

HIV protease mutations associated with amprenavir resistance during salvage therapy: importance of I54M.

OBJECTIVE: To examine the clinical significance of HIV protease mutations detected before and after therapy with amprenavir. STUDY DESIGN: Serial plasma HIV loads and protease gene mutations were monitored in 31 patients who received amprenavir, including 19 who had been exposed to other protease inhibitors (salvage therapy). Recombinant phenotyping was used to assess the significance of new mutations appearing after amprenavir therapy. RESULTS: After 6-8 months of amprenavir, 4 treatment-naïve and 5 salvage patients had an undetectable plasma HIV load, while 12 other salvage patients showed less than 10-fold HIV load reduction. HIV protease mutations associated with amprenavir resistance included I84V, I50V, I47V, V32I, and I54M. Among mutations newly detected after amprenavir treatment, I54M occurred in six cases, I54L in two cases, M46I in two cases, I47V in one case and I50V in one case. When compared with pretreatment plasma without the mutation, recombinant phenotyping showed that I54M increased the amprenavir resistance by at least 6-fold, resulting in up to 48-fold resistance over a drug-sensitive control. CONCLUSIONS: Protease I54M frequently appears after amprenavir therapy, and when combined with pre-existing mutations, leads to high-level amprenavir resistance and treatment failure.

Resistance to maribavir is associated with the exclusion of pUL27 from nucleoli during human cytomegalovirus infection.

Select mutations in the human cytomegalovirus (HCMV) gene UL27 confer low-grade resistance to the HCMV UL97 kinase inhibitor maribavir (MBV). It has been reported that the 608-amino acid UL27 gene product (pUL27) normally localizes to cell nuclei and nucleoli, whereas its truncation at codon 415, as found in a MBV-resistant mutant, results in cytoplasmic localization. We now show that in the context of full-length pUL27, diverse single amino acid substitutions associated with MBV resistance result in loss of its nucleolar localization when visualized after transient transfection, whereas substitutions representing normal interstrain polymorphism had no such effect. The same differences in localization were observed during a complete infection cycle with recombinant HCMV strains over-expressing full-length fluorescent pUL27 variants. Nested UL27 C-terminal truncation expression plasmids showed that amino acids 596-599 were required for the nucleolar localization of pUL27. These results indicate that the loss of a nucleolar function of pUL27 may contribute to MBV resistance, and that the nucleolar localization of pUL27 during HCMV infection depends not only on a carboxy-terminal domain but also on a property of pUL27 that is affected by MBV-resistant mutations, such as an interaction with component(s) of the nucleolus.

The unique antiviral activity of artesunate is broadly effective against human cytomegaloviruses including therapy-resistant mutants.

Current therapy options to treat infections with human cytomegalovirus face severe limitations leading to a continued search for novel drug candidates. Here, we describe novel characteristics of the strong antiviral potency of the drug artesunate. In vitro virus replication systems were applied to analyze a number of laboratory and clinically relevant strains of human cytomegalovirus. An inhibitory block at a very early stage of infection was demonstrated. Time-of-addition experiments indicated that the antiviral efficacy could be optimized when artesunate was applied as fractional doses consecutively added post-infection. Artesunate showed a clearly higher anti-cytomegaloviral activity than its parental drug artemisinin (approximately 10-fold) or other artesunate-related compounds. Mean IC(50) values of artesunate for a variety of standard therapy-resistant virus mutants were within a 2-fold range compared to wild-type virus. Furthermore, a synergistic effect was identified when artesunate was combined with the mechanistically distinct antiviral compound maribavir. These findings point to unique antiviral properties of artesunate which may offer an advantage over standard antiviral therapy particularly in cases of drug resistance.

Recombinant phenotyping of cytomegalovirus sequence variants detected after 200 or 100 days of valganciclovir prophylaxis.

BACKGROUND: In a phase III controlled trial IMproved Protection Against Cytomegalovirus in Transplantation (IMPACT) comparing 200 with 100 days of valganciclovir prophylaxis in 318 cytomegalovirus D+/R- kidney transplant recipients, an equal number of patients (n=3 per arm) had known ganciclovir resistance mutations detected during viral breakthrough. In addition, many other viral sequence variants were observed that were of unknown significance for ganciclovir resistance. Recombinant phenotyping was performed to determine whether the previously uncharacterized genotypic changes affected ganciclovir susceptibility, especially in those receiving the longer duration of prophylaxis. METHODS: Sequences encoding individual amino acid substitutions in the UL97 kinase or UL54 DNA polymerase gene were transferred by recombination into a cloned cytomegalovirus laboratory strain, followed by reporter-based yield reduction phenotypic assay of the resulting virus for ganciclovir susceptibility. RESULTS: Twenty-six uncharacterized amino acid substitutions were detected, 2 in UL97 and 24 in UL54. All 10 substitutions in the 200-day arm and 9 of 17 substitutions in the 100-day arm (prioritized based on location and conservation) were selected for phenotyping; one substitution was detected in both subsets. Results were generated for nine of ten 200-day and eight of nine 100-day substitutions, with no substitution demonstrating a significant reduction in ganciclovir susceptibility. The two remaining amino acid substitutions, both in UL54, were not evaluated because of poor viral viability. CONCLUSION: Phenotypic evaluation of previously uncharacterized viral genotypes in the 200-day valganciclovir prophylaxis group showed no evidence of an increased incidence of genotypic ganciclovir resistance when compared with those in the 100-day prophylaxis group.

Cytomegalovirus UL97 kinase mutations that confer maribavir resistance.

The cytomegalovirus (CMV) UL97 kinase inhibitor maribavir (MBV) is undergoing clinical antiviral trials. Two clinical CMV isolates serially passaged in cell culture under MBV showed >20-fold increases in MBV resistance after the development of the UL97 mutation V353A in one of the isolates and of T409M in the other. Marker transfer studies confirmed that the V353A and T409M mutations conferred ~15-fold and ~80-fold increases, respectively, in MBV resistance without significantly affecting ganciclovir susceptibility. The 3 UL97 mutations now known to confer MBV resistance are located upstream of UL97 mutations linked to ganciclovir resistance, closer to kinase domains that are associated with adenosine triphosphate binding and phosphotransfer.

Growth and drug resistance phenotypes resulting from cytomegalovirus DNA polymerase region III mutations observed in clinical specimens.

Recombinant phenotyping of cytomegalovirus (CMV) pol region III mutations from clinical specimens showed that T813S and G841A each conferred foscarnet resistance and approximately threefold increased ganciclovir resistance; adding the UL97 mutation C592G increased ganciclovir resistance to approximately sixfold. Bacterial artificial chromosome CMV clones containing pol mutation L845P were nonviable unless repaired with the wild-type sequence.

Maribavir antagonizes the antiviral action of ganciclovir on human cytomegalovirus.

The cytomegalovirus (CMV) UL97 kinase inhibitor maribavir antagonized the anti-CMV effect of ganciclovir, increasing the ganciclovir 50% inhibitory concentration against a sensitive strain by up to 13-fold. Antiviral activities of foscarnet and cidofovir were unaffected by maribavir.

Effect of cell culture conditions on the anticytomegalovirus activity of maribavir.

The cytomegalovirus UL97 kinase inhibitor maribavir suppressed viral growth more effectively in lung fibroblasts than in skin fibroblasts, and some cellular kinase inhibitors enhanced its antiviral activity. These effects influence the phenotypic assay of drug susceptibility and suggest the possibility of therapeutically useful combinations of maribavir and cellular kinase inhibitors.

Phenotyping of cytomegalovirus drug resistance mutations by using recombinant viruses incorporating a reporter gene.

A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.

Mutations in the human cytomegalovirus UL27 gene that confer resistance to maribavir.

Previous drug selection experiments resulted in the isolation of a human cytomegalovirus (CMV) UL97 phosphotransferase mutant resistant to the benzimidazole compound maribavir (1263W94), reflecting the anti-UL97 effect of this drug. Three other CMV strains were plaque purified during these experiments. These strains showed lower-grade resistance to maribavir than the UL97 mutant. Extensive DNA sequence analyses showed no changes from the baseline strain AD169 in UL97, the genes involved in DNA replication, and most structural proteins. However, changes were identified in UL27 where each strain contained a different mutation (R233S, W362R, or a combination of A406V and a stop at codon 415). The mutation at codon 415 is predicted to truncate the expressed UL27 protein by 193 codons (32% of UL27) with a loss of nuclear localization. The expression of full-length UL27 as a green fluorescent fusion protein in uninfected fibroblasts resulted in nuclear and nucleolar fluorescence, whereas cytoplasmic localization was observed when codons 1 to 415 were similarly expressed. Viable UL27 deletion mutants were created by recombination and showed slight growth attenuation and maribavir resistance in cell culture. Marker transfer experiments confirmed that UL27 mutations conferred maribavir resistance. The UL27 sequence was well conserved in a sample of 16 diverse clinical isolates. Mutation in UL27, a betaherpesvirus-specific early gene of unknown biological function, may adapt the virus for growth in the absence of UL97 activity.

Analysis of sequence configurations of the ISDR, PKR-binding domain, and V3 region as predictors of response to induction interferon-alpha and ribavirin therapy in chronic hepatitis C infection.

Interferon (IFN) and ribavirin combination therapy for chronic hepatitis C virus (HCV) infection yields a sustained response rate of only approximately 40%. Previous studies have linked IFN responsiveness to viral sequence variation in parts of the E2 and NS5A genes, but this remains controversial. We studied pretreatment sera from 28 subjects (23 with HCV genotype 1a) who received high-dose IFN induction followed by IFN-ribavirin combination therapy. Serum HCV sequences were amplified and compared from 14 responders with undetectable HCV RNA 24 weeks after therapy and 11 nonresponders (excluding three who dropped out of the study). Analysis included the E2 PKR eIF-2alpha phosphorylation homology domain (PePHD, codons 659-670), where the sequence was well conserved, and codons 2001-2420 of NS5A. In NS5A, the proposed PKR binding domain (codons 2209-2274), containing the putative IFN sensitivity determining region (ISDR, codons 2209-2248), showed too little variation among subjects to differentiate responders and nonresponders. NS5A codons 2356-2385 (which includes the V3 region) exhibited more variation. Here, six of 12 genotype 1a responders showed four or more amino acid changes from the prototype HCV-1 sequence, as compared with one of eight nonresponders, but this fell short of statistical significance (P = 0.16). NS5A sequences from posttreatment sera were examined in six nonresponders to look for selection of treatment-resistant viral subpopulations, but no consistent change was detected. In conclusion, our results indicate that the sequences of the ISDR, the PKR-binding domain, and the PePHD are unlikely to have predictive value for IFN treatment success in those infected with HCV genotype 1a. However, the finding of greater variability among treatment responders in the carboxy end of NS5A suggests that the V3 region merits further investigation.

Tumor necrosis factor genetic polymorphisms and response to antiviral therapy in patients with chronic hepatitis C.

OBJECTIVE: Hepatitis C virus (HCV) is the major causal agent of non-A, non-B hepatitis and the leading indication for liver transplantation worldwide. The emerging field of immunogenetics has confirmed the significant role of heritability in host immune responses to infectious pathogens. Both the major and non-major histocompatibility complex genes are increasingly identified as candidate genes hypothesized to influence the susceptibility to, or the course of, a particular disease. We hypothesized that polymorphisms within the major histocompatibility complex class III region that encode for tumor necrosis factors (TNF)-alpha and TNF-beta might be predictive of response to antiviral therapy in patients with chronic hepatitis C. METHODS: A total of 155 subjects, including 110 HCV-seropositive individuals undergoing antiviral therapy and 45 ethnically similar HCV-negative controls, were studied. The HCV-positive patients had undergone antiviral treatment with either interferon monotherapy (n = 73) or in combination with ribavirin (n = 37) and were categorized as either nonresponders, sustained responders, or relapsers. Sixty (55%) patients had genotype 1 (1a or 1b). Genomic DNA was extracted, followed by polymerase chain reaction amplification and sequencing for two promoter TNF-alpha variants (at positions -238 and -308), as well as restriction fragment length analysis for four polymorphic loci within the TNF-beta gene (NcoI, TNFc, aa13, aa26). RESULTS: Although there was a trend toward higher frequency of the A allele in the TNF 238 promoter among HCV-infected patients (12% vs 4%), there were no significant differences in the distribution of the genotypic polymorphisms between patients and controls. Patients with the TNF 238 A allele had higher pretreatment viral loads as compared with patients homozygous for the wild type allele (7.2 x 10(6) +/- 4.2 x 10(6) copies/ml vs 3.8 x 10(6) +/- 0.34 x 10(6) copies/ml, p = 0.03). However, there was no association between TNF genetic markers, including multiple haplotypic combinations, and response to therapy. In addition, there was no correlation with these polymorphic loci and histological severity of liver disease. CONCLUSIONS: Although previous work has suggested potential roles for TNF in the pathogenesis of HCV infection, we were unable to identify any link between TNF genetic polymorphisms and histological severity or response to antiviral therapy.