Creation of dual EMCV-IRES-integrated dengue virus; translation suppression of non-structural polyprotein and induction of cellular Mdm2 oncoprotein suppress the viral replication in mosquito cells and mammalian cells, respectively.

Flaviviruses in the Flaviviridae family are arthropod viruses transmitting between vertebrates and hematophagous insects. Flaviviruses possess 5prime cap genome RNAs, while the other genera in Flaviviridae are transmitted within a limited set of species and utilize an alternative translation mechanism; internal ribosome entry site (IRES) sequences. In this study, encephalomyocarditis (EMCV)-IRES sequence was inserted in dengue virus serotype 2 (DENV2) for translating non-structural (NS) polyprotein, separately from 5primecap-translated structural polyprotein. It was revealed that DENV replication by NS polyprotein required co-translating the preceding hydrophobic amino acids at C-terminal envelop protein (E). The 73 amino acids (E73) covering transmembrane domain 1 (TM1) and 2 (TM2) or the 25 amino acids (E25) corresponding to TM2 supported the virus replication. These IRES-integrated DENV2 replicates efficiently in BHK21 mammalian cells but not in C6/36 mosquito cells. Moreover, E73 or E25 can be replaced by the DENV4 corresponding. These results suggest that the IRES translation restricts DENV replication in limited hosts and that the C-terminal E plays a functional role in DENV replication. We further created dual IRES-integrated DENV2, in which a non-viral gene can be expressed by the flanking IRESs. The eGFP insertion could fluorescently visualize the virus spread, while renilla luciferase (Rluc) integration enabled the quantification of the viral replication. It was also revealed that a cellular gene, Mdm2, which antagonizes Tumor suppressor protein p53 (TP53), could terminate the viral replication in BHK21 cells. Thus, the IRES-integrations into DENV genome with/without a foreign gene could suppress the virus replication in both mammalian and mosquito cells.