Virus-like particles (VLPs) that do not contain genomic RNA have been found in flavivirus-infected cells ( 3). The flavivirus particle assembly process entails (i) the interaction of prM and E proteins in the form of heterodimer formation in the endoplasmic reticulum (ER), (ii) genomic RNA encapsulation by the C protein and enclosure by cell membrane-derived lipid bilayers containing prM and E proteins to form immature virions, and (iii) the cleavage of prM proteins to M proteins via furin or a furin-like protease in the trans-Golgi network, thus triggering the release of viral particles. In all flaviviruses, the RNA genome encodes three structural (core C, membrane precursor prM, and envelope E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) genes, with flanking untranslated region (UTR) genes at the 5′ and 3′ ends ( 1, 2). Japanese encephalitis virus (JEV) is a small enveloped positive-strand RNA virus belonging to the Flavivirus genus of the Flaviviridae family ( 1, 2). Our findings help refine the structure and molecular interactions on the flavivirus surface and reveal a potential strategy for blocking flavivirus infections by inhibiting these electrostatic interactions. More importantly, the prM-E125 and E-H246 residues are conserved and the positive charge of the E-K93 residue is preserved in different flavivirus groups. We found that the molecular interactions between prM-E125 residue and positively charged E-K93 and E-H246 residues at EDII are critical for VLP production. Since the prM-H domain is topologically close to domain II of the E protein (EDII), this study was to determine molecular interactions between the prM-H domain and EDII. The prM stem region contains an N-terminal loop and a helix domain (prM-H). The current high-resolution structures available for prM-E complexes do not include the membrane proximal stem region of prM. IMPORTANCE Molecular interaction between E and prM proteins of Japanese encephalitis virus is a major driving force for virus-like particle (VLP) production. These findings suggest that the electrostatic attractions of prM-E125, E-K93, and E-H246 residues are important to flavivirus VLP production and that inhibiting these interactions is a potential strategy for blocking flavivirus infections.

The prM-E125 and E-H246 residues are conserved and that the positive charge of the E-K93 residue is preserved in different flavivirus groups. Exchanges of oppositely charged residue side chains at prM-E125/E-K93 and prM-E125/E-H246 are recoverable for VLP production. Our results indicate that negatively charged prM-E125 residue at the prM-H domain affected VLP production via one or more interactions with positively charged E-K93 and E-H246 residues at EDII. In this study, we investigated prM-H domain amino acid residues facing Japanese encephalitis virus EDII using site-directed mutagenesis to determine their roles in prM-E interaction and VLP production. The prM helical (prM-H) domain is topologically close to and may interact with domain II of the E protein (EDII). Interaction between E and prM proteins in flavivirus-infected cells is a major factor for virus-like particle (VLP) production.

Positively charged amino acids