Rescue of infectious Arumowot virus from cloned cDNA: Posttranslational degradation of Arumowot virus NSs protein in human cells

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa and the Middle East, affecting both humans and ruminants. There are no licensed vaccines or antivirals available for humans, whereas research using RVF virus (RVFV) is strictly regulated in many countries with safety concerns. Nonpathogenic Arumowot virus (AMTV), a mosquito-borne phlebovirus in Africa, is likely useful for the screening of broad-acting antiviral candidates for phleboviruses including RVFV, as well as a potential vaccine vector for RVF. In this study, we aimed to generate T7 RNA polymerase-driven reverse genetics system for AMTV. We hypothesized that recombinant AMTV (rAMTV) is viable, and AMTV NSs protein is dispensable for efficient replication of rAMTV in type-I interferon (IFN)-incompetent cells, whereas AMTV NSs proteins support robust viral replication in type-I IFN-competent cells. The study demonstrated the rescue of rAMTV and that lacking the NSs gene (rAMTVΔNSs), that expressing green fluorescent protein (GFP) (rAMTV-GFP) or that expressing Renilla luciferase (rAMTV-rLuc) from cloned cDNA. The rAMTV-rLuc and the RVFV rMP12-rLuc showed a similar susceptibility to favipiravir or ribavirin. Interestingly, neither of rAMTV nor rAMTVΔNSs replicated efficiently in human MRC-5 or A549 cells, regardless of the presence of NSs gene. Little accumulation of AMTV NSs protein occurred in those cells, which was restored via treatment with proteasomal inhibitor MG132. In murine MEF or Hepa1-6 cells, rAMTV, but not rAMTVΔNSs, replicated efficiently, with an inhibition of IFN-β gene upregulation. This study showed an establishment of the first reverse genetics for AMTV, a lack of stability of AMTV NSs proteins in human cells, and an IFN-β gene antagonist function of AMTV NSs proteins in murine cells. The AMTV can be a nonpathogenic surrogate model for studying phleboviruses including RVFV.
Author summary Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus endemic to Africa and the Middle East, causing devastating outbreaks affecting both humans and animals. The reverse genetics system for RVFV has contributed to the virology, vaccinology, and antiviral screening for RVFV. In this study, we generated the first reverse genetics system for a mosquito-borne nonpathogenic phlebovirus (Arumowot virus; AMTV) endemic to Africa, which is phylogenetically related to RVFV. The generation of recombinant AMTV supports the screening of broad-acting antivirals and vaccine development for RVF. The nonstructural NSs protein is known as a major virulence factor for RVF, yet this study revealed that AMTV NSs protein was rapidly degraded in human cells via cellular proteasomes. In contrast, AMTV NSs protein functioned as an antagonist of interferon-β gene upregulation in murine cells. The AMTV can be a nonpathogenic surrogate model for studying phleboviruses including RVFV.