Biomedical subjects

Bruno Canard

Publications and source records attributed to Bruno Canard.

At least 37 records · Page 2Linked to original sources

PubMed2006

A novel, highly selective inhibitor of pestivirus replication that targets the viral RNA-dependent RNA polymerase.

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.

Antiviral Agents↗

PubMed2005

The modeled structure of the RNA dependent RNA polymerase of GBV-C virus suggests a role for motif E in Flaviviridae RNA polymerases.

BACKGROUND: The Flaviviridae virus family includes major human and animal pathogens. The RNA dependent RNA polymerase (RdRp) plays a central role in the replication process, and thus is a validated target for antiviral drugs. Despite the increasing structural and enzymatic characterization of viral RdRps, detailed molecular replication mechanisms remain unclear. The hepatitis C virus (HCV) is a major human pathogen difficult to study in cultured cells. The bovine viral diarrhea virus (BVDV) is often used as a surrogate model to screen antiviral drugs against HCV. The structure of BVDV RdRp has been recently published. It presents several differences relative to HCV RdRp. These differences raise questions about the relevance of BVDV as a surrogate model, and cast novel interest on the "GB" virus C (GBV-C). Indeed, GBV-C is genetically closer to HCV than BVDV, and can lead to productive infection of cultured cells. There is no structural data for the GBV-C RdRp yet. RESULTS: We show in this study that the GBV-C RdRp is closest to the HCV RdRp. We report a 3D model of the GBV-C RdRp, developed using sequence-to-structure threading and comparative modeling based on the atomic coordinates of the HCV RdRp structure. Analysis of the predicted structural features in the phylogenetic context of the RNA polymerase family allows rationalizing most of the experimental data available. Both available structures and our model are explored to examine the catalytic cleft, allosteric and substrate binding sites. CONCLUSION: Computational methods were used to infer evolutionary relationships and to predict the structure of a viral RNA polymerase. Docking a GTP molecule into the structure allows defining a GTP binding pocket in the GBV-C RdRp, such as that of BVDV. The resulting model suggests a new proposition for the mechanism of RNA synthesis, and may prove useful to design new experiments to implement our knowledge on the initiation mechanism of RNA polymerases.

Binding Sites↗

PubMed2005

Hsp72 recognizes a P binding motif in the measles virus N protein C-terminus.

The major inducible 70-kDa heat shock protein (hsp72) binds measles virus (MV) nucleocapsids and increases MV gene expression. The cytoplasmic tail of the MV N protein (N(TAIL)) contains three hydrophobic domains (Box-1-3) that are potential targets of hsp72 interaction. Low affinity binding to Box-3 is correlated to hsp72-dependent stimulation of MV minireplicon reporter gene expression whereas interactions between hsp72 and Box-1 and/or -2 have not been documented. The present work showed that virus deficient in Box-3/hsp72 interaction retains the ability to form nucleocapsid/hsp72 complexes, identifying Box-2 but not Box-1 as a mediator of high affinity hsp72 binding. Box-2 is the binding site for the viral P protein X domain (XD), where P tethers the viral polymerase to nucleocapsid in support of transcription and genome replication, and competitive inhibition of XD binding to N(TAIL) by hsp72 was shown. Recognition of a common binding site by P and hsp72 represents a potential mechanism for host cell modulation of viral gene expression.

Amino Acid Motifs↗

PubMed2005

Effects of HIV Q151M-associated multi-drug resistance mutations on the activities of (-)-beta-D-1',3'-dioxolan guanine.

The multi-drug resistance HIV-1 genotype A62V/V75I/F77L/F116Y/Q151M is associated with resistance to many nucleoside reverse transcriptase inhibitors including AZT, ddI, ddC, d4T, abacavir, and 3TC. In this study, we evaluated the antiviral activity of (-)-beta-D-1',3'-dioxolan guanine (DXG) towards mutant HIV-1 containing V75I/F77L/F116Y/Q151M (V75Icomplex) and A62V/V75I/F77L/F116Y/Q151M (A62Vcomplex) in MT-2 cells. We further investigated the mechanism of resistance by studying the incorporation of DXG 5'-triphosphate (DXG-TP) during DNA synthesis by mutant enzymes containing single mutations at Q151M or A62V, and the V75Icomplex and A62Vcomplex using pre-steady state kinetic analysis. Our studies showed that mutant virus containing V75Icomplex and A62Vcomplex were both more than 23-fold resistant to DXG, and this correlated with the 68- and 20-fold resistance changes observed in the enzymatic assay. Compared to the wild-type enzyme, DXG-TP was incorporated 39- and 21-fold less efficiently by the mutant enzyme containing V75Icomplex and A62Vcomplex, mainly due to decreases in the rate of incorporation. The A62V mutation significantly increased the rate of incorporation (k(pol)) for both dGTP (3-fold) and DXG-TP (7.9-fold), while the binding affinity of A62V HIV-1 RT for DXG-TP was decreased 14-fold. At the enzyme level, the addition of the A62V mutation to V75I/F77L/F116Y/Q151M moderately (3.4-fold) reversed the resistance to DXG-TP.

Amino Acid Substitution↗

PubMed2005

Suppression of multidrug-resistant HIV-1 reverse transcriptase primer unblocking activity by alpha-phosphate-modified thymidine analogues.

A dipeptide insertion between codons 69 and 70 together with the amino acid substitution T215Y in the reverse transcriptase (RT)-coding region of human immunodeficiency virus type 1 (HIV-1) strains are known to confer phenotypic resistance to zidovudine (AZT) and stavudine (d4T). Phenotypic resistance correlates with an increased ATP-dependent phosphorolytic activity. Nucleoside alpha-boranophosphate diastereoisomers derived from AZT and d4T were tested as substrates of a multidrug-resistant HIV-1 RT (designated as SS RT) bearing a Ser-Ser insertion at codons 69-70 and other drug resistance-related mutations, in DNA polymerization assays and ATP-mediated excision reactions. Using pre-steady-state kinetics, we show that SS RT can incorporate both R(p) and S(p) diastereoisomers, although R(p) is the preferred isomer. Chirality at the internucleotidic linkage formed upon incorporation of nucleoside alpha-boranophosphate did not affect ATP-mediated excision. As reported for AZT and d4T-terminated primers, substituting Thr, Asn or Ser for Tyr215 abrogates the ATP-dependent phosphorolytic activity on primers terminated with alpha-boranophosphate derivatives of thymidine analogues. However, unlike in the case of AZT, eliminating the dipeptide insertion in SS RT had no effect on the ATP-mediated excision of primers terminated with alpha-boranophosphate derivatives of d4T. Studies with ATP analogues showed that exchanging a non-bridging oxygen atom at the gamma-phosphate group for sulfur causes a significant reduction of the ATP-dependent phosphorolytic activity of SS RT. Interestingly, SS RT's excision activity is completely eliminated upon phosphorothioate substitution at the 3' end of primers terminated with AZT. These results suggest that phosphorothioate derivatives of currently approved drugs could be useful against excision-proficient HIV-1 strains.

Adenosine Triphosphate↗

PubMed2005

HIV-1-associated uracil DNA glycosylase activity controls dUTP misincorporation in viral DNA and is essential to the HIV-1 life cycle.

Uracilation of DNA represents a constant threat to the survival of many organisms including viruses. Uracil may appear in DNA either by cytosine deamination or by misincorporation of dUTP. The HIV-1-encoded Vif protein controls cytosine deamination by preventing the incorporation of host-derived APOBEC3G cytidine deaminase into viral particles. Here, we show that the host-derived uracil DNA glycosylase UNG2 enzyme, which is recruited into viral particles by the HIV-1-encoded integrase domain, is essential to the viral life cycle. We demonstrate that virion-associated UNG2 catalytic activity can be replaced by the packaging of heterologous dUTPase into virion, indicating that UNG2 acts to counteract dUTP misincorporation in the viral genome. Therefore, HIV-1 prevents incorporation of dUTP in viral cDNA by UNG2-mediated uracil excision followed by a dNTP-dependent, reverse transcriptase-mediated endonucleolytic cleavage and finally by strand-displacement polymerization. Our findings indicate that pharmacologic strategies aimed toward blocking UNG2 packaging should be explored as potential HIV/AIDS therapeutics.

Base Sequence↗

PubMed2005

Borano-nucleotides: new analogues to circumvent HIV-1 RT-mediated nucleoside drug-resistance.

Alpha-boranophosphates suppress RT-mediated resistance when the catalytic rate of incorporation (kpol) of the analogue 5'-triphosphate is responsable for drug resistance, such as in the case of K65R mutant and ddNTPs, and Q151M toward AZTTP and ddNTPs. This suppression is also observed with BH3-d4T and BH3-3TC toward their clinically relevant mutants Q151M and M184V. Moreover, the presence of the borano (BH3-) group renders the incorporation of the analogue independent from amino-acid substitutions in RT. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analogue.

Amino Acids↗

PubMed2005

VaZyMolO: a tool to define and classify modularity in viral proteins.

Viral structural genomic projects aim at unveiling the function of unknown viral proteins by employing high-throughput approaches to determine their 3D structure and to identify their function through fold-homology studies. The 'viral enzyme module localization' (VaZyMolO) tool has been developed, which aims at defining viral protein modules that might be expressed in a soluble and functionally active form, thereby identifying candidates for crystallization studies. VaZyMolO includes 114 complete viral genome sequences of both negative- and positive-sense, single-stranded RNA viruses available from NCBI. In VaZyMolO, a module is defined as a structural and/or functional unit. Modules were first identified by homology search and then validated by the convergence of results from sequence composition analysis, motif search, transmembrane region search and domain definitions, as found in the literature. The public interface of VaZyMolO, which is accessible from http://www.vazymolo.org, allows comparison of a query sequence to all VaZyMolO modules of known function.

DNA, Complementary↗

PubMed2005

The intrinsically disordered C-terminal domain of the measles virus nucleoprotein interacts with the C-terminal domain of the phosphoprotein via two distinct sites and remains predominantly unfolded.

Measles virus is a negative-sense, single-stranded RNA virus within the Mononegavirales order,which includes several human pathogens, including rabies, Ebola, Nipah, and Hendra viruses. The measles virus nucleoprotein consists of a structured N-terminal domain, and of an intrinsically disordered C-terminal domain, N(TAIL) (aa 401-525), which undergoes induced folding in the presence of the C-terminal domain (XD, aa 459-507) of the viral phosphoprotein. With in N(TAIL), an alpha-helical molecular recognition element (alpha-MoRE, aa 488-499) involved in binding to P and in induced folding was identified and then observed in the crystal structure of XD. Using small-angle X-ray scattering, we have derived a low-resolution structural model of the complex between XD and N(TAIL), which shows that most of N(TAIL) remains disordered in the complex despite P-induced folding within the alpha-MoRE. The model consists of an extended shape accommodating the multiple conformations adopted by the disordered N-terminal region of N(TAIL), and of a bulky globular region, corresponding to XD and to the C terminus of N(TAIL) (aa 486-525). Using surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and heteronuclear magnetic resonance, we show that N(TAIL) has an additional site (aa 517-525) involved in binding to XD but not in the unstructured-to-structured transition. This work provides evidence that intrinsically disordered domains can establish complex interactions with their partners, and can contact them through multiple sites that do not all necessarily gain regular secondary structure.

Binding Sites↗

PubMed2005

Essential amino acids of the hantaan virus N protein in its interaction with RNA.

The nucleocapsid (N) protein of hantavirus encapsidates viral genomic and antigenomic RNAs. Previously, deletion mapping identified a central, conserved region (amino acids 175 to 217) within the Hantaan virus (HTNV) N protein that interacts with a high affinity with these viral RNAs (vRNAs). To further define the boundaries of the RNA binding domain (RBD), several peptides were synthesized and examined for the ability to bind full-length S-segment vRNA. Peptide 195-217 retained 94% of the vRNA bound by the HTNV N protein, while peptides 175-186 and 205-217 bound only 1% of the vRNA. To further explore which residues were essential for binding vRNA, we performed a comprehensive mutational analysis of the amino acids in the RBD. Single and double Ala substitutions were constructed for 18 amino acids from amino acids 175 to 217 in the full-length N protein. In addition, Ala substitutions were made for the three R residues in peptide 185-217. An analysis of protein-RNA interactions by electrophoretic mobility shift assays implicated E192, Y206, and S217 as important for binding. Chemical modification experiments showed that lysine residues, but not arginine or cysteine residues, contribute to RNA binding, which agreed with bioinformatic predictions. Overall, these data implicate lysine residues dispersed from amino acids 175 to 429 of the protein and three amino acids located in the RBD as essential for RNA binding.

Amino Acid Sequence↗

PubMed2004

A relaxed discrimination of 2'-O-methyl-GTP relative to GTP between de novo and Elongative RNA synthesis by the hepatitis C RNA-dependent RNA polymerase NS5B.

Several nucleotide analogues have been described as inhibitors of NS5B, the essential viral RNA-dependent RNA polymerase of hepatitis C virus. However, their precise mode of action remains poorly defined at the molecular level, much like the different steps of de novo initiation of viral RNA synthesis. Here, we show that before elongation, de novo RNA synthesis is made of at least two distinct kinetic phases, the creation of the first phosphodiester bond being the most efficient nucleotide incorporation event. We have studied 2'-O-methyl-GTP as an inhibitor of NS5B-directed RNA synthesis. As a nucleotide competitor of GTP in RNA synthesis, 2'-O-methyl-GTP is able to act as a chain terminator and inhibit RNA synthesis. Relative to GTP, we find that this analogue is strongly discriminated against at the initiation step ( approximately 150-fold) compared with approximately 2-fold at the elongation step. Interestingly, discrimination of the 2'-O-methyl-GTP at initiation is suppressed in a variant NS5B deleted in a subdomain critical for initiation (the "flap," encompassing amino acids 443-454), but not in P495L NS5B, which shows a selective alteration of transition from initiation to elongation. Our results demonstrate that the conformational change occurring between initiation and elongation is dependent on the allosteric GTP-binding site and relaxes nucleotide selectivity. RNA elongation may represent the most probable target of 2'-modified nucleotide analogues, because it is more permissive to inhibition than initiation.

Allosteric Site↗

PubMed2004

Mechanistic insights into the suppression of drug resistance by human immunodeficiency virus type 1 reverse transcriptase using alpha-boranophosphate nucleoside analogs.

A class of amino acid substitutions in drug-resistant HIV-1 reverse transcriptase (RT) is responsible for the selectively impaired incorporation of the nucleotide analog inhibitor into DNA. We have shown previously that alpha-boranophosphate nucleoside analogs suppress RT-mediated resistance when the catalytic rate is responsible for drug resistance such as in the case of K65R and dideoxy (dd)NTPs, and Q151M toward AZTTP and ddNTPs. Here, we extend this property to BH3-d4TTP and BH3-3TCTP toward their clinically relevant mutants Q151M and M184V, respectively. Pre-steady-state kinetics on mutants of the Q151M RT family reveal a 3-5-fold resistance to d4TTP. This resistance is suppressed using BH3-d4TTP. Likewise, resistance to 3TCTP by M184V RT (30-fold) and K65R/M184V RT (180-fold) is suppressed using BH3-3TCTP because of a 160-fold acceleration of the catalytic constant kpol. Mechanistic insights into the rate enhancement were obtained using various alpha-boranophosphate nucleotides. The presence of the BH3 group renders kpol independent of amino acid substitutions present in RT. Indeed, the approximately 100-fold decrease in polymerase activity caused by the R72A substitution is restored to wild-type levels using BH3-dTTP. Metal ion titration studies show that alpha-boranophosphate nucleoside analogs enhance 3-8-fold the binding of Mg2+ ions to the active site of the RT.DNA.dNTP complex and alleviate the requirement of critical amino acids involved in phosphodiester bond formation. To our knowledge, this is the first example of rescue of polymerase activity by means of a nucleotide analog.

Amino Acid Substitution↗

PubMed2004

The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core.

The nonstructural protein 3 (NS3) of Dengue virus (DV) is a multifunctional enzyme carrying activities involved in viral RNA replication and capping: helicase, nucleoside 5'-triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase). Here, a 54-kDa C-terminal domain of NS3 (DeltaNS3) bearing all three activities was expressed as a recombinant protein. Structure-based sequence analysis in comparison with Hepatitis C virus (HCV) helicase indicates the presence of a HCV-helicase-like catalytic core domain in the N-terminal part of DeltaNS3, whereas the C-terminal part seems to be different. In this report, we show that the RTPase activity of DeltaNS3 is Mg2+-dependent as are both helicase and NTPase activities. Mutational analysis shows that the RTPase activity requires an intact NTPase/helicase Walker B motif in the helicase core, consistent with the fact that such motifs are involved in the coordination of Mg2+. The R513A substitution in the C-terminal domain of DeltaNS3 abrogates helicase activity and strongly diminishes RTPase activity, indicating that both activities are functionally coupled. DV RTPase seems to belong to a new class of Mg2+-dependent RTPases, which use the active center of the helicase/NTPase catalytic core in conjunction with elements in the C-terminal domain.

Acid Anhydride Hydrolases↗

PubMed2004

A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate.

Ribavirin is one of the few nucleoside analogues currently used in the clinic to treat RNA virus infections, but its mechanism of action remains poorly understood at the molecular level. Here, we show that ribavirin 5'-triphosphate inhibits the activity of the dengue virus 2'-O-methyltransferase NS5 domain (NS5MTase(DV)). Along with several other guanosine 5'-triphosphate analogues such as acyclovir, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-carboxamide (EICAR), and a series of ribose-modified ribavirin analogues, ribavirin 5'-triphosphate competes with GTP to bind to NS5MTase(DV). A structural view of the binding of ribavirin 5'-triphosphate to this enzyme was obtained by determining the crystal structure of a ternary complex consisting of NS5MTase(DV), ribavirin 5'-triphosphate, and S-adenosyl-l-homocysteine at a resolution of 2.6 A. These detailed atomic interactions provide the first structural insights into the inhibition of a viral enzyme by ribavirin 5'-triphosphate, as well as the basis for rational drug design of antiviral agents with improved specificity against the emerging flaviviruses.

Binding Sites↗

PubMed2004

A loss of viral replicative capacity correlates with altered DNA polymerization kinetics by the human immunodeficiency virus reverse transcriptase bearing the K65R and L74V dideoxynucleoside resistance substitutions.

Mechanisms governing viral replicative capacity are poorly understood at the biochemical level. Human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) K65R or L74V substitutions confer viral resistance to 2',3'-dideoxyinosine (ddI) in vivo. The two substitutions never occur together, and L74V is frequently found in patients receiving ddI, while K65R is not. Here we show that recombinant viruses carrying K65R and K65R/L74V display the same resistance level to ddI (about 9.5-fold) relative to wild type. Consistent with this result, purified HIV-1 RT carrying K65R RT or K65R/L74V substitutions exhibits an 8-fold resistance to ddATP as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. Resistance is due to a selective decrease of the catalytic rate constant k(pol): 22-fold (from 7.2 to 0.33 s(-1)) for K65R RT and 84-fold (from 7.2 to 0.086 s(-1)) for K65R/L74V RT. However, the K65R/L74V virus replication capacity is severely impaired relative to that of wild-type virus. This loss of viral fitness is correlated to a poor ability of K65R/L74V RT to use natural nucleotides relative to wild-type RT: 15% that of wild-type RT for dATP, 36% for dGTP, 50% for dTTP, and 25% for dCTP. The order of incorporation efficiency is wild-type RT > L74V RT > K65R RT > K65R/L74V RT. Processivity of DNA synthesis remains unaffected. These results explain why the two mutations do not combine in the clinic and might give a mechanism for a decreased viral fitness at the molecular level.

Cells, Cultured↗

PubMed2004

The severe acute respiratory syndrome-coronavirus replicative protein nsp9 is a single-stranded RNA-binding subunit unique in the RNA virus world.

The recently identified etiological agent of the severe acute respiratory syndrome (SARS) belongs to Coronaviridae (CoV), a family of viruses replicating by a poorly understood mechanism. Here, we report the crystal structure at 2.7-A resolution of nsp9, a hitherto uncharacterized subunit of the SARS-CoV replicative polyproteins. We show that SARS-CoV nsp9 is a single-stranded RNA-binding protein displaying a previously unreported, oligosaccharide/oligonucleotide fold-like fold. The presence of this type of protein has not been detected in the replicative complexes of RNA viruses, and its presence may reflect the unique and complex CoV viral replication/transcription machinery.

Amino Acid Sequence↗

PubMed2004

The C-terminal domain of measles virus nucleoprotein belongs to the class of intrinsically disordered proteins that fold upon binding to their physiological partner.

The nucleoprotein of measles virus consists of an N-terminal domain, N(CORE) (aa 1-400), resistant to proteolysis, and a C-terminal domain, N(TAIL) (aa 401-525), hypersensitive to proteolysis and not visible by electron microscopy. Using two complementary computational approaches, we predict that N(TAIL) belongs to the class of natively unfolded proteins. Using different biochemical and biophysical approaches, we show that N(TAIL) is indeed unstructured in solution. In particular, the spectroscopic and hydrodynamic properties of N(TAIL) indicate that this protein domain belongs to the premolten globule subfamily within the class of intrinsically disordered proteins. The isolated N(TAIL) domain was shown to be able to bind to its physiological partner, the phosphoprotein (P), and to undergo an induced folding upon binding to the C-terminal moiety of P [J. Biol. Chem. 278 (2003) 18638]. Using a computational analysis, we have identified within N(TAIL) a putative alpha-helical molecular recognition element (alpha-MoRE, aa 488-499), which could be involved in binding to P via induced folding. We report the bacterial expression and purification of a truncated form of N(TAIL) (N(TAIL2), aa 401-488) devoid of the alpha-MoRE. We show that N(TAIL2) has lost the ability to bind to P, thus supporting the hypothesis that the alpha-MoRE may play a role in binding to P. We have further analyzed the alpha-helical propensities of N(TAIL2) and N(TAIL) using circular dichroism in the presence of 2,2,2-trifluoroethanol. We show that N(TAIL2) has a lower alpha-helical potential compared to N(TAIL), thus suggesting that the alpha-MoRE may be indeed involved in the induced folding of N(TAIL).

Circular Dichroism↗

PubMed2003

Mechanistic basis for reduced viral and enzymatic fitness of HIV-1 reverse transcriptase containing both K65R and M184V mutations.

HIV-1 drug resistance mutations are often inversely correlated with viral fitness, which remains poorly described at the molecular level. Some resistance mutations can also suppress resistance caused by other resistance mutations. We report the molecular mechanisms by which a virus resistant to lamivudine with the M184V reverse transcriptase mutation shows increased susceptibility to tenofovir and can suppress the effects of the tenofovir resistance mutation K65R. Additionally, we report how the decreased viral replication capacity of resistant viruses is directly linked to their decreased ability to use natural nucleotide substrates and that combination of the K65R and M184V resistance mutations leads to greater decreases in viral replication capacity. All together, these results define at the molecular level how nucleoside-resistant viruses can be driven to reduced viral fitness.

Adenine↗