Development of an infectious reporter dengue virus to study viral entry

JUAN A. MONDOTTE; DIEGO E. ALVAREZ; SILVANA FUCITO; ANDREA V. GAMARNIK

University of Wisconsin, Madison, USA

Congreso; American Society for Virology 25th Annual Meeting; 2006

American Society for Virology

Development of an infectious reporter dengue virus to study viral entry Mondotte, Juan A., Álvarez D., Fucito S. and Gamarnik A. Fundación Instituto Leloir, Patricias Argentinas 435, Buenos Aires 1405, Argentina.    Dengue is a mosquito-borne virus that belongs to the Flaviviridae family together with other important human pathogens such as yellow fever virus, West Nile virus, and Japanese encephalitis virus.  Dengue is an enveloped virus with a positive single stranded RNA genome of about 11 Kb. The viral RNA encodes a single open reading frame and the large viral polyprotein is processed by cellular and viral proteases into three structural (capsid (C), pre-membrane (prM), and envelope (E)) and seven nonstructural proteins. The first step in dengue virus (DV) infection requires interaction between the E glycoprotein with a cellular receptor present on the surface of the host cell. In addition, it has been reported that interaction of E with other molecules present at the surface of the host cell such as lectins and heparan sulphate, also contribute to an efficient entry process. Therefore, identification of molecules that could interfere with these host-virus interactions will prevent viral entry. Although reporter-based subgenomic replicons have been described for dengue virus, the lack of structural proteins makes them not amenable for studying viral entry. To overcome this limitation, monocistronic and bicistronic full-length dengue virus cDNAs were designed encoding luciferase. Transfection of cells with viral RNAs resulted in the production of infectious viral particles. Subsequent infections with these recombinant viruses allowed us to detect DV entry as early as 1 h post infection. An exponential increase of the reporter due to RNA amplification was detected after 24 hours of infection and viral propagation was observed after 40 hours. In addition, this reporter virus was successfully tested in BHK cells in a HT format employing previously characterized entry inhibitors. Moreover, to gain insight on the molecular interactions between E and components of the host cell, we introduced specific mutations at the two potential glycosilation sites of E (Asn 67 and Asn 153). The resulting viruses revealed a differential role of the two carbohydrate molecules in the E protein during DV replication. In summary, we developed a novel tool for both screening viral inhibitors and studying different aspects of DV life cycle.