scholarly journals An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus in Mice

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 12
Author(s):  
Jan Haviernik ◽  
Ludek Eyer ◽  
Antoine Nougairède ◽  
Marie Uhlířová ◽  
Jean-Sélim Driouich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Encephalitis Virus ◽  
Viral Fitness ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Cross Resistance ◽  
Potential Contribution ◽  
Tick Borne Encephalitis ◽  
Single Amino Acid Change ◽  
Tick Borne Encephalitis Virus

Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia for which there is currently no specific therapy. The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of TBEV and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. In our study, we performed serial in vitro passaging of TBEV in the presence of increasing concentrations of galidesivir (up to 50 μM), which resulted in the generation of two drug-resistant TBEV mutants. The first TBEV mutant was characterized by a single amino acid change, E460D. The other carried two amino acid changes, E460D and Y453H. Both mutations mapped to the active site of the viral RNA-dependent RNA polymerase (RdRp). Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2′-C-methyladenosine, 2′-C-methyladenosine, and 4′-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. By contrast, infection with wild-type virus resulted in fatal infections for all animals. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence.

scholarly journals Changing the Protease Specificity for Activation of a Flavivirus, Tick-Borne Encephalitis Virus

2008 ◽  
Vol 82 (17) ◽  
pp. 8272-8282 ◽  
Author(s):  
Wolfgang Fischl ◽  
Sigrid Elshuber ◽  
Sabrina Schrauf ◽  
Christian W. Mandl
Keyword(s):  
Cell Culture ◽  
Cleavage Site ◽  
Encephalitis Virus ◽  
Wild Type Virus ◽  
Sequence Motif ◽  
Amino Acid Residues ◽  
Tick Borne Encephalitis ◽  
Protease Specificity ◽  
Tick Borne Encephalitis Virus ◽  
Cleavage Motif

ABSTRACT The infectivity of flavivirus particles depends on a maturation process that is triggered by the proteolytic cleavage of the precursor of the M protein (prM). This activation cleavage is naturally performed by ubiquitous cellular proteases of the furin family, which typically recognize the multibasic sequence motif R-X-R/K-R. Previously, we demonstrated that a tick-borne encephalitis virus (TBEV) mutant with an altered cleavage motif, R-X-R, produced immature, noninfectious particles that could be activated by exogenous trypsin, which cleaves after single basic residues. Here, we report the adaptation of this mutant to chymotrypsin, a protease specific for large, hydrophobic amino acid residues. Using selection pressure in cell culture, two different mutations conferring a chymotrypsin-dependent phenotype were identified. Surprisingly, one of these mutations (Ser85Phe) occurred three positions upstream of the natural cleavage site. The other mutation (Arg89His) arose at the natural cleavage position but involved a His residue, which is not a typical chymotrypsin cleavage site. Efficient cleavage of protein prM and activation by the heterologous protease were confirmed using various recombinant TBEV mutants. Mutants with only the originally selected mutations exhibited unimpaired export kinetics and were genotypically stable during at least six cell culture passages. However, in contrast to the wild-type virus or trypsin-dependent mutants, chymotrypsin-dependent mutants were not neurovirulent in suckling mice. Our results demonstrate that flaviviruses with altered protease specificities can be generated and suggest that this approach can be used for the construction of viral mutants or vectors that can be activated on demand and have restricted tissue tropism and virulence.

scholarly journals Spontaneous Mutations Restore the Viability of Tick-Borne Encephalitis Virus Mutants with Large Deletions in Protein C

2003 ◽  
Vol 77 (1) ◽  
pp. 443-451 ◽  
Author(s):  
Regina M. Kofler ◽  
Agnes Leitner ◽  
Gabriel O'Riordain ◽  
Franz X. Heinz ◽  
Christian W. Mandl
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Deletion Mutant ◽  
Hydrophobic Interactions ◽  
Encephalitis Virus ◽  
Sequence Element ◽  
Hydrophobic Domain ◽  
Large Deletions ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

ABSTRACT The capsid protein, C, of tick-borne encephalitis virus has recently been found to tolerate deletions up to a length of 16 amino acid residues that partially removed the central hydrophobic domain, a sequence element conserved among flaviviruses which may be crucial for virion assembly. In this study, mutants with deletion lengths of 19, 21, 27, or 30 residues, removing more or all of this hydrophobic domain, were found to yield viable virus progeny, but this was without exception accompanied by the emergence of additional mutations within protein C. These point mutations or sequence duplications were located downstream of the engineered deletion and generally increased the hydrophobicity, suggesting that they may compensate for the loss of the central hydrophobic domain. Two of the second-site mutations, together with the corresponding deletion, were introduced into a wild-type genetic backbone, and the analysis of these “double mutants” provided direct evidence that the viability of the deletion mutant indeed depended on the presence of the second-site mutation. Our results corroborate the notion that hydrophobic interactions of protein C are essential for the assembly of infectious flavivirus particles but rule out the possibility that individual residues of the central hydrophobic domain are absolutely required for infectivity. Furthermore, the double mutants were found to be highly attenuated and capable of inducing a protective immune response in mice at even lower inoculation doses than the previously characterized 16-amino-acid-residue deletion mutant, suggesting that the combination of large deletions and second-site mutations may be a superior way to generate safe, attenuated flavivirus vaccine strains.

scholarly journals An E460D substitution in the NS5 protein of tick-borne encephalitis virus confers resistance to the inhibitor Galidesivir (BCX4430) and also attenuates the virus for mice

2019 ◽  
Author(s):  
Ludek Eyer ◽  
Antoine Nougairède ◽  
Marie Uhlířová ◽  
Jean-Sélim Driouich ◽  
Darina Zouharová ◽  
...  
Keyword(s):  
Cell Culture ◽  
Ebola Virus ◽  
Rna Virus ◽  
Encephalitis Virus ◽  
Viral Fitness ◽  
Rna Dependent Rna Polymerase ◽  
Virus Inhibitor ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Ebola Virus Infection

AbstractThe adenosine analogue Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a Phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola virus infection. Moreover, Galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution, in the active site of TBEV RNA-dependent-RNA-polymerase (RdRp), confers resistance to Galidesivir in cell culture. Stochastic molecular simulations indicate that the steric freedom caused by the E460D substitution increases close electrostatic interactions between the inhibitor and the interrogation residue of the TBEV RdRp motif F, resulting in rejection of the analogue as an incorrect/modified nucleotide. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2’-C-methyladenosine, 2’-C-methyladenosine and 4’-azido-aracytidine. Although, the E460D substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in vivo, with 100% survival rate and no clinical signs observed in infected mice. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors and the potential contribution of viral RdRp to flavivirus neurovirulence.ImportanceTick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in large areas of Europe and Asia and for which there is currently no specific therapy. We have previously found that Galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola virus infection, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to Galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of viral RNA genome. Although, this substitution led only to a subtle decrease in viral fitness in cell culture, Galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of Galidesivir antiviral activity.

scholarly journals An E460D Substitution in the NS5 Protein of Tick-Borne Encephalitis Virus Confers Resistance to the Inhibitor Galidesivir (BCX4430) and Also Attenuates the Virus for Mice

2019 ◽  
Vol 93 (16) ◽  
Author(s):  
Ludek Eyer ◽  
Antoine Nougairède ◽  
Marie Uhlířová ◽  
Jean-Sélim Driouich ◽  
Darina Zouharová ◽  
...  
Keyword(s):  
Cell Culture ◽  
Yellow Fever Virus ◽  
Rna Virus ◽  
Encephalitis Virus ◽  
Viral Fitness ◽  
Virus Infections ◽  
Rna Dependent Rna Polymerase ◽  
Virus Inhibitor ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus

ABSTRACT The adenosine analogue galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, has entered a phase 1 clinical safety and pharmacokinetics study in healthy subjects and is under clinical development for treatment of Ebola and yellow fever virus infections. Moreover, galidesivir also inhibits the reproduction of tick-borne encephalitis virus (TBEV) and numerous other medically important flaviviruses. Until now, studies of this antiviral agent have not yielded resistant viruses. Here, we demonstrate that an E460D substitution in the active site of TBEV RNA-dependent RNA polymerase (RdRp) confers resistance to galidesivir in cell culture. Galidesivir-resistant TBEV exhibited no cross-resistance to structurally different antiviral nucleoside analogues, such as 7-deaza-2′-C-methyladenosine, 2′-C-methyladenosine, and 4′-azido-aracytidine. Although the E460D substitution led to only a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in vivo, with a 100% survival rate and no clinical signs observed in infected mice. Furthermore, no virus was detected in the sera, spleen, or brain of mice inoculated with the galidesivir-resistant TBEV. Our results contribute to understanding the molecular basis of galidesivir antiviral activity, flavivirus resistance to nucleoside inhibitors, and the potential contribution of viral RdRp to flavivirus neurovirulence. IMPORTANCE Tick-borne encephalitis virus (TBEV) is a pathogen that causes severe human neuroinfections in Europe and Asia and for which there is currently no specific therapy. We have previously found that galidesivir (BCX4430), a broad-spectrum RNA virus inhibitor, which is under clinical development for treatment of Ebola and yellow fever virus infections, has a strong antiviral effect against TBEV. For any antiviral drug, it is important to generate drug-resistant mutants to understand how the drug works. Here, we produced TBEV mutants resistant to galidesivir and found that the resistance is caused by a single amino acid substitution in an active site of the viral RNA-dependent RNA polymerase, an enzyme which is crucial for replication of the viral RNA genome. Although this substitution led only to a subtle decrease in viral fitness in cell culture, galidesivir-resistant TBEV was highly attenuated in a mouse model. Our results contribute to understanding the molecular basis of galidesivir antiviral activity.

scholarly journals Functional Analysis of Potential Carboxy-Terminal Cleavage Sites of Tick-Borne Encephalitis Virus Capsid Protein

2007 ◽  
Vol 82 (5) ◽  
pp. 2218-2229 ◽  
Author(s):  
Sabrina Schrauf ◽  
Petra Schlick ◽  
Tim Skern ◽  
Christian W. Mandl
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Protein C ◽  
Terminal Region ◽  
Encephalitis Virus ◽  
Viral Protease ◽  
Tick Borne Encephalitis ◽  
Carboxy Terminal Region ◽  
Tick Borne Encephalitis Virus ◽  
Carboxy Terminal

ABSTRACT The mature capsid protein C of flaviviruses is generated through the proteolytic cleavage of the precursor polyprotein by the viral NS2B/3 protease. This cleavage is a prerequisite for the subsequent processing of the viral surface protein prM, and the concerted progression of these events plays a key role in the process of the assembly of infectious virions. Protein C of tick-borne encephalitis virus (TBEV) contains two amino acid sequence motifs within the carboxy-terminal region that match the canonical NS2B/3 recognition site. Site-specific mutagenesis in the context of the full-length TBEV genome was used to investigate the in vivo cleavage specificity of the viral protease in this functionally important domain. The results indicate that the downstream site is necessary and sufficient for efficient cleavage and virion assembly; in contrast, the upstream site is dispensable and placed in a structural context that renders it largely inaccessible to the viral protease. Mutants with impaired C-prM cleavage generally exhibited a significantly increased cytotoxicity. In spite of the clear preference of the protease for only one of the two naturally occurring motifs, the enzyme was unexpectedly tolerant to both the presence of a noncanonical threonine residue at position P2 and the position of cleavage relative to the adjacent internal prM signal sequence. The insertion of three amino acid residues downstream of the cleavage site did not change the viral phenotype. Thus, this study further illuminates the specificity of the TBEV protease and reveals that the carboxy-terminal region of protein C has a remarkable functional flexibility in its role in the assembly of infectious virions.

scholarly journals Molecular Genetic Analysis of the Complete Genome of Tick-Borne Encephalitis Virus (Siberia Subtype): Modern Kolarovo-2008 Isolate

2011 ◽  
pp. 44-48 ◽  
Author(s):  
E. V. Chausov ◽  
V. A. Ternovoy ◽  
E. V. Protopopova ◽  
S. N. Konovalova ◽  
Yu. V. Kononova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Complete Genome ◽  
Molecular Genetic ◽  
Molecular Genetic Analysis ◽  
Encephalitis Virus ◽  
Nucleotide Sequence Analysis ◽  
Tick Borne Encephalitis ◽  
Tick Borne Encephalitis Virus ◽  
Siberian Subtype

Determined is the complete genome sequence of Kolarovo-2008 strain (Siberia subtype) of Tick-borne encephalitis virus (TBEV), isolated from a tick in the suburbs of the Tomsk city. Nucleotide sequence analysis testifies of the fact that the level of genetic differences within the Siberian subtype of TBEV amounts to 10 % of the nucleotide sequence and to 7 % of amino-acid sequence for certain virus genes. 3'-HTO of the genome of Siberian subtype has the highest rate of variability and the homology level ranging from 65 to 97 %. Kolarovo-2008 and Vasilchenko (isolated in Novosibirsk in 1969) strains have the highest level of genome homology. The level of dissimilarity between the two Tomsk strains is substantially higher: the total number of amino-acid substitutions in Tomsk Zausaev and Kolarovo-2008 strains equals to 124, and 3'HTO level of homology is 79 %. Identified genetic variability of the Siberian subtype of TBEV is of a great importance for further development and enhancement of tick-borne encephalitis virus diagnostics.

Generation of duck hepatitis B virus polymerase mutants through site-directed mutagenesis which demonstrate resistance to lamivudine [(--)-beta-L-2', 3'-dideoxy-3'-thiacytidine] in vitro.

1996 ◽  
Vol 40 (8) ◽  
pp. 1957-1960 ◽  
Author(s):  
K P Fischer ◽  
D L Tyrrell
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
Single Amino Acid Change ◽  
B Virus

Hepatitis B virus replication is very sensitive to lamivudine. A single amino acid change in human immunodeficiency virus reverse transcriptase is responsible for high-level resistance to this compound. Duck hepatitis B virus mutants were created bearing the analogous amino acid change in the duck hepatitis B virus polymerase. Viral DNA production was reduced 92% for the wild-type virus at 2 micrograms of lamivudine per ml, while the mutants required 40 micrograms of lamivudine per ml to inhibit replication by greater than 80%.

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