Glutamic Acid at Residue 125 of the prM Helix Domain Interacts with Positively Charged Amino Acids in E Protein Domain II for Japanese Encephalitis Virus-Like-Particle Production

Abstract

Interaction between E and prM proteins in flavivirus-infected cells is a major factor for virus-like particle (VLP) production. The prM helical (prM-H) domain is topologically close to and may interact with domain II of the E protein (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. 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. Exchanges of oppositely charged residue side chains at prM-E125/E-K93 and prM-E125/E-H246 are recoverable for VLP production. 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. 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. IMPORTANCE Molecular interaction between E and prM proteins of Japanese encephalitis virus is a major driving force for virus-like particle (VLP) production. The current high-resolution structures available for prM-E complexes do not include the membrane proximal stem region of prM. The prM stem region contains an N-terminal loop and a helix domain (prM-H). 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. We found that the molecular interactions between prM-E125 residue and positively charged E-K93and E-H246 residues at EDII are critical for VLP production. 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. 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. J apanese encephalitis virus (JEV) is a small enveloped positivestrand RNA virus belonging to the Flavivirus genus of the Flaviviridae family (1, 2). 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). 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 th...

Post-translational regulation and modifications of flavivirus structural proteins

“…Interestingly, only E114 in DENV-1 was also demonstrated to be crucial for heterodimeric interactions between prM and E (Hsieh et al, 2014), indicating that the other residues may play a greater role in influencing the conformational rearrangement of structural proteins during virion maturation (Zhang et al, 2012). This hypothesis is particularly supported by recent evidence linking residue E125 in the JEV prM-H domain (corresponding to DENV E124) in electrostatic interactions with the K93 and H246 residues in E protein DII (Peng & Wu, 2014). The flexible linker between prM-H and the first transmembrane domain in DENV also appears to play a similar role in the secretion of virus particles, as mutation of H130 to a non-polar amino acid greatly reduces the recovery of either infectious virions or prM/E particles from culture supernatants (Pryor et al, 2004).…”

“…The pr peptide forms a tightly folded protein domain consisting of two small b-sheets linked by disulfide bridges, with the entire structure positioned at the distal end of E domain II (DII) within the heterodimer where it obscures the fusion loop and facilitates pr glycan presentation at the tip of trimeric spikes Yu et al, 2008;Zhang et al, 2003). The C-terminal region of the M protein ectodomain is also predicted to form a helical domain (prM-H) that may assist in prM/E heterodimer formation (Hsieh et al, 2011;Peng & Wu, 2014;Zhang et al, 2012).…”

“…The pr peptide forms a tightly folded protein domain consisting of two small b-sheets linked by disulfide bridges, with the entire structure positioned at the distal end of E domain II (DII) within the heterodimer where it obscures the fusion loop and facilitates pr glycan presentation at the tip of trimeric spikes Yu et al, 2008;Zhang et al, 2003). The C-terminal region of the M protein ectodomain is also predicted to form a helical domain (prM-H) that may assist in prM/E heterodimer formation (Hsieh et al, 2011;Peng & Wu, 2014;Zhang et al, 2012).Together with the E protein, prM forms an integral part of the flavivirus envelope. Proposed roles for prM include assisting in the chaperone-mediated folding of the E protein (Konishi & Mason, 1993;Lorenz et al, 2002), contributing to the pH-dependent conformational rearrangement of trimeric prM/E heterodimer spikes in immature virions via the influence of the helical stem domain of M (Zhang et al, 2012), and in the concealment of the fusion loop of E by the pr peptide to prevent premature fusion during virion egress (Yu et al, 2009).…”

A Single Amino Acid Substitution in the M Protein Attenuates Japanese Encephalitis Virus in Mammalian Hosts

“…1B]), have already been shown to be critical for proper flavivirus assembly (16, 30-35, 55, 56). Of note, a residue close to M-I36, residue M-E33, is part of an interaction network with domain II of the E protein, and mutagenesis of this residue affects the formation of JEV viral particles in insect and mammalian cells (33). We were able to show that the M-I36F mutation did not affect some of the processes known to be important for viral assembly in mammalian cells, such as heterodimer formation (Fig.…”

“…The perimembrane and transmembrane helices serve to anchor the M protein to the membrane. In the case of DV and JEV, the perimembrane helix region was shown to be involved in virus assembly (30)(31)(32)(33) and entry (31,32). Subsequent mutagenesis analysis of the M protein proved the importance of an interactive network between E and M in those processes (16,34,35).…”