Rift Valley fever virus polymerase: from antivirals discovery to resistance selection

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F25%3A00643117" target="_blank" >RIV/61388963:_____/25:00643117 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216224:14740/25:00142752

Výsledek na webu

<a href="http://www.ccsss.cz/index.php/ccsss/issue/view/52/92" target="_blank" >http://www.ccsss.cz/index.php/ccsss/issue/view/52/92</a>

DOI - Digital Object Identifier

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Jazyk výsledku

angličtina

Název v původním jazyce

Rift Valley fever virus polymerase: from antivirals discovery to resistance selection

Popis výsledku v původním jazyce

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen of the family Phenuiviridae that causes severe and often fatal disease in humans and domesticated animals. Outbreaks occur sporadically but can have devastating consequences for public health and agriculture. In livestock, RVFV infection results in high rates of abortion and mortality among young animals, with economic losses estimated in the hundreds of millions of U.S. dollars1. Despite this major impact, no approved treatment or prevention strategies are available for human use. Attenuated vaccines have been developed for veterinary application, yet their safety and efficacy remain uncertain, and the risk of reversion to apathogenic form is an ongoing concern. RVFV replication depends on the multifunctional L protein, a ~250 kDa viral polymerase that integrates three enzymatic activities: an endonuclease, an RNA-dependent RNA polymerase, and a cap-binding domain2. This organization closely mirrors the functional modules of the heterotrimeric influenza A virus polymerase (PA–PB1–PB2)3. Both viruses initiate transcription via a cap-snatching mechanism, whereby host mRNAs are cleaved by the endonuclease to prime viral RNA synthesis. The high level of conservation across bunyaviral polymerases further underscores the L protein as a promising target for therapeutic intervention. To investigate this, we established two RVFV strains under biosafety level 3 conditions: the wild-type ZH-548 strain and the attenuated MP-12, which is used in experimental veterinary vaccines. With these systems, we screened and identified several small-molecule inhibitors of the L protein. Their antiviral efficacy was validated using live-virus assays. Resistance studies were performed by serial passaging of RVFV in the presence of these inhibitors. Resistant viral variants emerged, displaying either strongly increased or complete resistance to their respective compounds. Sequencing of the resistant strains revealed multiple non-synonymous mutations in the L protein. In collaboration with the group of Gabriel Demo, we further explored the structural basis of resistance. Using cryo-electron microscopy, a 3.5 Å structure of the wild-type RVFV L protein was obtained in its apo form. Mapping of resistance-associated mutations onto this structure provided valuable mechanistic insight into how amino acid substitutions influence inhibitor binding, polymerase conformation, and overall enzymatic function. This structural framework not only clarifies the resistance mechanisms but also guides the rational design of next-generation inhibitors with improved efficacy and resilience to viral escape. Beyond antiviral development, we also investigated the stability of the attenuated MP-12 vaccine strain. Reversion of attenuating mutations is a critical safety concern for all live-attenuated vaccines, as illustrated by the well-documented case of oral polio vaccine reversion. Our analysis of MP-12 focused on identifying mutations most susceptible to reversion, thereby assessing the long-term stability of the vaccine strain. Understanding these dynamics is essential for ensuring the safety profile of MP-12 and for informing the design of future live-attenuated vaccines with reduced reversion risk. In collaboration with the group of Petr Volf, we plan to study the interactions between resistant RVFV variants and their insect vectors. Since vector competence plays a key role in shaping viral evolution and outbreak dynamics, such studies will provide important insights into how resistance mutations may affect transmission and fitness in natural hosts. In summary, our study highlights the RVFV L protein as a central target for antiviral development, demonstrates the emergence and structural basis of resistance mutations, and provides critical data on the genetic stability of the MP-12 vaccine strain.nThis work was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) – Funded by the European Union – Next Generation EU.

Název v anglickém jazyce

Rift Valley fever virus polymerase: from antivirals discovery to resistance selection

Popis výsledku anglicky

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen of the family Phenuiviridae that causes severe and often fatal disease in humans and domesticated animals. Outbreaks occur sporadically but can have devastating consequences for public health and agriculture. In livestock, RVFV infection results in high rates of abortion and mortality among young animals, with economic losses estimated in the hundreds of millions of U.S. dollars1. Despite this major impact, no approved treatment or prevention strategies are available for human use. Attenuated vaccines have been developed for veterinary application, yet their safety and efficacy remain uncertain, and the risk of reversion to apathogenic form is an ongoing concern. RVFV replication depends on the multifunctional L protein, a ~250 kDa viral polymerase that integrates three enzymatic activities: an endonuclease, an RNA-dependent RNA polymerase, and a cap-binding domain2. This organization closely mirrors the functional modules of the heterotrimeric influenza A virus polymerase (PA–PB1–PB2)3. Both viruses initiate transcription via a cap-snatching mechanism, whereby host mRNAs are cleaved by the endonuclease to prime viral RNA synthesis. The high level of conservation across bunyaviral polymerases further underscores the L protein as a promising target for therapeutic intervention. To investigate this, we established two RVFV strains under biosafety level 3 conditions: the wild-type ZH-548 strain and the attenuated MP-12, which is used in experimental veterinary vaccines. With these systems, we screened and identified several small-molecule inhibitors of the L protein. Their antiviral efficacy was validated using live-virus assays. Resistance studies were performed by serial passaging of RVFV in the presence of these inhibitors. Resistant viral variants emerged, displaying either strongly increased or complete resistance to their respective compounds. Sequencing of the resistant strains revealed multiple non-synonymous mutations in the L protein. In collaboration with the group of Gabriel Demo, we further explored the structural basis of resistance. Using cryo-electron microscopy, a 3.5 Å structure of the wild-type RVFV L protein was obtained in its apo form. Mapping of resistance-associated mutations onto this structure provided valuable mechanistic insight into how amino acid substitutions influence inhibitor binding, polymerase conformation, and overall enzymatic function. This structural framework not only clarifies the resistance mechanisms but also guides the rational design of next-generation inhibitors with improved efficacy and resilience to viral escape. Beyond antiviral development, we also investigated the stability of the attenuated MP-12 vaccine strain. Reversion of attenuating mutations is a critical safety concern for all live-attenuated vaccines, as illustrated by the well-documented case of oral polio vaccine reversion. Our analysis of MP-12 focused on identifying mutations most susceptible to reversion, thereby assessing the long-term stability of the vaccine strain. Understanding these dynamics is essential for ensuring the safety profile of MP-12 and for informing the design of future live-attenuated vaccines with reduced reversion risk. In collaboration with the group of Petr Volf, we plan to study the interactions between resistant RVFV variants and their insect vectors. Since vector competence plays a key role in shaping viral evolution and outbreak dynamics, such studies will provide important insights into how resistance mutations may affect transmission and fitness in natural hosts. In summary, our study highlights the RVFV L protein as a central target for antiviral development, demonstrates the emergence and structural basis of resistance mutations, and provides critical data on the genetic stability of the MP-12 vaccine strain.nThis work was supported by the project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103) – Funded by the European Union – Next Generation EU.

Druh

O - Ostatní výsledky

CEP obor

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OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LX22NPO5103" target="_blank" >LX22NPO5103: Národní institut virologie a bakteriologie</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2025

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů