Potential and action mechanism of favipiravir as an antiviral against Junin virus.

Favipiravir is a nucleoside analogue that inhibits the replication and transcription of a broad spectrum of RNA viruses, including pathogenic arenaviruses. In this study, we isolated a favipiravir-resistant mutant of Junin virus (JUNV), which is the causative agent of Argentine hemorrhagic fever, and analyzed the antiviral mechanism of favipiravir against JUNV. Two amino acid substitutions, N462D in the RNA-dependent RNA polymerase (RdRp) and A168T in the glycoprotein precursor GPC, were identified in the mutant. GPC-A168T substitution enhanced the efficiency of JUNV internalization, which explains the robust replication kinetics of the mutant in the virus growth analysis. Although RdRp-N462D substitution did not affect polymerase activity levels in a minigenome system, comparisons of RdRp error frequencies showed that the virus with RdRp-D462 possessed a significantly higher fidelity. Our next generation sequence (NGS) analysis showed a gradual accumulation of both mutations as we passaged the virus in presence of favipiravir. We also provided experimental evidence for the first time that favipiravir inhibited JUNV through the accumulation of transition mutations, confirming its role as a purine analogue against arenaviruses. Moreover, we showed that treatment with a combination of favipiravir and either ribavirin or remdesivir inhibited JUNV replication in a synergistic manner, blocking the generation of the drug-resistant mutant. Our findings provide new insights for the clinical management and treatment of Argentine hemorrhagic fever.

Topoisomerase II as a Novel Antiviral Target against Panarenaviral Diseases.

Although many arenaviruses cause severe diseases with high fatality rates each year, treatment options are limited to off-label use of ribavirin, and a Food and Drug Administration (FDA)-approved vaccine is not available. To identify novel therapeutic candidates against arenaviral diseases, an RNA polymerase I-driven minigenome (MG) expression system for Lassa virus (LASV) was developed and optimized for high-throughput screening (HTS). Using this system, we screened 2595 FDA-approved compounds for inhibitors of LASV genome replication and identified multiple compounds including pixantrone maleate, a topoisomerase II inhibitor, as hits. Other tested topoisomerase II inhibitors also suppressed LASV MG activity. These topoisomerase II inhibitors also inhibited Junin virus (JUNV) MG activity and effectively limited infection by the JUNV Candid #1 strain, and siRNA knockdown of both topoisomerases (IIα and IIß) restricted JUNV replication. These results suggest that topoisomerases II regulate arenavirus replication and can serve as molecular targets for panarenaviral replication inhibitors.