EARLY STEPS OF PHLEBOVIRUS INFECTION – PHLEBO

The Phlebovirus genus constitutes a group of viruses mainly transmitted by ticks and phlebotomine sandflies. Many phleboviruses cause severe diseases – often fatal – and have been reported on different continents, including Western Europe. The increasing number of outbreaks and the apparent wide distribution of reservoirs of sandflies and ticks demonstrate that these viruses must be taken seriously as emerging disease agents. In this context, there is interest in using the prototype phleboviruses Toscana (TOSV) and Uukuniemi (UUKV), respectively spread by sandflies and ticks, to understand the biology of these emerging pathogens. Here, we propose to synergize the efforts of two highly complementary groups, one focusing in the cell biology of phleboviruses and the other in structural approaches. The Lozach lab (Heidelberg) previously showed that UUKV entry into cells occurs by acid-activated membrane fusion from late endosomal (LE) vacuoles, though not requiring proteins critical for the classical LE maturation. The Rey lab (Pasteur) has identified a not-yet described intermediate structure of the UUKV envelope fusion glycoprotein. Preliminary data from the Lozach lab additionally show that UUKV is not inactivated following exposure to low pH. In contrast to other class II viruses, the phlebovirus glycoproteins undergo no activating proteolytic cleavage. Together, these observations suggest that the priming process for phlebovirus induced membrane fusion may be fundamentally distinct from that of well-studied class II viruses such as flaviviruses and alphaviruses. Obtaining new insights into phlebovirus cell entry will require a synergistic combination of innovative cell biology and structure-function analyses. The combined expertise of the Lozach and Rey groups can bring forth new functional and structural data and characterize the non-classical priming process for membrane fusion. TOSV and UUKV will serve as model systems as they belong to the sandfly- and tick-borne phlebovirus groups and share features with the most clinically relevant isolates in the genus. We will rely on our expertise in the structural and functional analysis of virus-host cell interactions to investigate 1) the molecular and functional features of phleboviral particles derived from arthropod cells, 2) the 3D structure of phleboviral particles, and 3) the phlebovirus-induced membrane fusion mechanism. With this proposal, we expect to shed light on the mechanisms governing the phlebovirus activation and late penetration as well as the fusion of phlebovirus envelope with endosomes, both at the molecular and cellular levels. The information gleaned here has the potential of providing valuable clues regarding hard-to-investigate, basic cellular and molecular mechanisms that prime phleboviral particles for infectious entry. Together our results will also provide a handle to develop specific treatments against these emerging pathogens, the number of which is rapidly increasing.