Viral and host determinants of susceptibility of diverse hantaviruses

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Hantaviruses cause hantavirus cardiopulmonary syndrome (HCPS) and hemorrhagic fever with renal syndrome (HFRS) with case fatality rates of up to 40% and 14.5%, respectively. FDA- approved hantavirus drugs and vaccine do not exist. Recent advances in RNA sequencing technology has led to the discovery of more than 40 genetically distinct hantaviruses carried by rodents, insectivores (moles and shrews) and bats. Consequently, chances of zoonotic spillover are likely to be higher in future as deforestation, habitat destruction and climate change will bring more of the hantavirus reservoir hosts in closer proximity to the human populations. Despite their huge genetic diversity and public health importance, our understanding of the molecular determinants of hantavirus susceptibility (capacity for virus entry) and permissiveness (capacity for virus replication) at the cellular level is derived from studies of only a few hantaviruses and remains rudimentary. This is largely due to the lack of molecular tools and hantavirus isolates and the general need to study hantaviruses in biosafety level-3 (BSL3) containment. The virion surface Gn/Gc glycoproteins are the sole viral determinants of hantavirus susceptibility. Although single-cycle pseudotyped viruses carrying Gn/Gc exist for some hantaviruses, self-replicating surrogate viruses have only been generated for one hantavirus (Andes virus) by researchers other than us. Self-replicating, fluorescent-labeled rVSVs provide robust, scalable systems that are uniquely suited for novel host factor discovery using survival and/or FACS-based genome-wide genetic screens at BSL-2. Given that the N- terminal domain of Gn (Gn-NTD) forms most of the surface-exposed part of the Gn/Gc spikes on the virion surface and is the most divergent region of Gn/Gc, we hypothesize that (i) Gn- NTD diversity is a key viral determinant of hantavirus susceptibility, and (ii) unidentified non- PCDH1 receptor(s) mediate entry of at least a subset of hantaviruses. Our primary objective is to generate well-characterized molecular tools to define viral and host determinants of hantavirus susceptibility and permissivity. In collaboration with Richard Yanagihara at the University of Hawaii, we will (i) generate rVSVs bearing hantavirus Gn/Gc glycoproteins representing mammalian hantavirus diversity, (ii) define the host factor requirements for cellular entry of novel hantaviruses, and (iii) finally, we will apply this knowledge to generate engineered cell lines over-expressing the relevant host factors for the isolation of authentic non-rodent- borne hantaviruses. These tools will help us achieve our long-term goals of generating a more comprehensive picture of the viral and host determinants of hantavirus susceptibility and permissiveness at the molecular level.