Nuclear Localization of Japanese Encephalitis Virus Core Protein Enhances Viral Replication

Abstract

Japanese encephalitis virus (JEV) belongs to the genus Flavivirus within the family Flaviviridae. Members of the genus Flavivirus are predominantly arthropodborne viruses and frequently cause significant morbidity and mortality in mammals and birds (6). JEV is distributed in the south and southeast regions of Asia and kept in a zoonotic transmission cycle between pigs or birds and mosquitoes (6,50,57). JEV spreads to dead-end hosts, including humans, through the bite of JEVinfected mosquitoes and causes infection of the central nervous system, with a high mortality rate (6, 57). JEV has a single-stranded positive-strand RNA genome approximately 11 kb in length, which is capped at the 5Ј end but lacks modification of the 3Ј terminus by polyadenylation (34). The genomic RNA encodes a single large open reading frame, and a polyprotein translated from the genome is cleaved co-and posttranslationally by host and viral proteases to yield three structural proteins, the core, precursor membrane (prM), and envelope (E) proteins, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (53). Although the core protein has very little amino acid homology to other flaviviruses-for example, the core protein of JEV has only 25% homology to that of tick-borne encephalitis virus (TBEV)-the structural properties, such as the hydrophobicity profile, abundances of basic amino acid residues, and secondary structures, are very similar (11,20,36). The flavivirus core proteins commonly contain two hydrophobic sequences in the center and a carboxyl-terminal end, and the carboxyl-terminal hydrophobic region serves as a signal sequence of prM. The signal-anchor sequence is cleaved off by the viral protease NS2B-3, and this cleavage is required for the subsequent liberation of the amino terminus of prM by the host signal peptidase (35,52,63). The mature core protein, released from the endoplasmic reticulum (ER) membrane, is believed to bind to the genomic RNA via the basic amino acid clusters at the amino and carboxyl termini and forms nucleocapsids (23). The central hydrophobic region of the core protein may be associated with the ER membrane, and this interaction is believed to facilitate the assembly of nucleocapsid and two membrane proteins, prM and E, and to bud into the ER lumen as virions (39). The removal of the central hydrophobic region of the TBEV core protein increased the production of the subviral particles that consist of (pr)M and E proteins but that lack a core protein and genomic RNA (26,27).In addition to their role as structural proteins, core proteins of dengue virus (DEN) and Kunjin virus (KUN) are localized not only in the cytoplasm but also in the nucleus, especially in the nucleoli of several infected cell lines (4,38,55,59,61). Transport from the cytoplasm to the nucleus occurs through nuclear pore complexes that penetrate the double lipid layers of the nuclear envelope. Small molecules up to 9 nm in diameter (Ͻ50 kDa) can freely diffuse through the nuclear pore complexes, while most macromolecu...

Multiple regions in dengue virus capsid protein contribute to nuclear localization during virus infection

“…This feature appears to be conserved among flaviviruses, as other investigators have detected C proteins of Kunjin virus, West Nile virus and Japanese encephalitis virus (JEV) in both the cytoplasm and nuclei of infected mammalian cells (Westaway et al, 1997;Mori et al, 2005;Oh et al, 2006). Three stretches of Arg/Lys-rich sequence in the dengue virus C protein have been proposed to serve as nuclear-localization signals (NLSs): KKAR, located at aa 6-9; KKSK, located at aa 73-76; and the bipartite sequence RKEIGRMLNILNRRRR, located at aa 85-100 (Bulich & Aaskov, 1992).…”

“…Kinetics of C protein localization to nuclei and nucleoli of dengue virus-infected cells Several investigators have reported different patterns of dengue virus and other flavivirus C protein nuclear localization, or lack thereof, in many cell lines, which may reflect cellular differences and methodological variations (Tadano et al, 1989;Bulich & Aaskov, 1992;Westaway et al, 1997;Ng et al, 2001;Wang et al, 2002;Mori et al, 2005;Oh et al, 2006). To affirm the phenomenon in this study, subcellular localization of dengue virus C protein was assessed during infection of two mammalian cell lines (PS and Vero) and a mosquito cell line (C6/36) with a dengue serotype 2 virus, strain 16681, by using indirect IFA.…”

Post-translational regulation and modifications of flavivirus structural proteins

“…The C protein localizes to three distinct cellular compartments: the ER membrane and lipid droplets in the cytoplasm, as well as the nucleus (specifically the nucleolus) (Bhuvanakantham et al, 2010;Bulich & Aaskov, 1992;Colpitts et al, 2011; Martins et al, 2012;Mori et al, 2005;Samsa et al, 2009; Westaway et al, 1997). The primary role of the C protein in the flavivirus life cycle is in the assembly of the nucleocapsid and its incorporation into nascent virions, whilst other functions of C, including those performed in the nucleus, are less explored.…”

“…Phosphorylation of WNV C has conversely been shown to promote nuclear localization and thus removes the protein from sites of genomic RNA interaction (Bhuvanakantham et al, 2010). The Kunjin virus (KUNV; a naturally attenuated strain of WNV) and DENV C structures place a-1 in different orientations; thus, it is hypothesized that this helix rotates in order to change the position of the flexible N terminus for optimal RNA interaction (Dokland et al, 2004;Ma et al, 2004).The C protein localizes to three distinct cellular compartments: the ER membrane and lipid droplets in the cytoplasm, as well as the nucleus (specifically the nucleolus) (Bhuvanakantham et al, 2010;Bulich & Aaskov, 1992;Colpitts et al, 2011; Martins et al, 2012;Mori et al, 2005;Samsa et al, 2009; Westaway et al, 1997). The primary role of the C protein in the flavivirus life cycle is in the assembly of the nucleocapsid and its incorporation into nascent virions, whilst other functions of C, including those performed in the nucleus, are less explored.…”

Interactions between the West Nile virus capsid protein and the host cell-encoded phosphatase inhibitor, I2PP2A

“…Studies with Japanese encephalitis virus, a related flavivirus, indicate that nuclear localization of C is important for virus replication and neuroinvasiveness, but interestingly not for neurovirulence (Mori et al, 2005). The WNV C protein has also been implicated as a pathogenic determinant; expression of WNV C in the absence of other viral proteins has been reported to cause apoptosis in neurons both in vitro and in vivo (Yang et al, 2002).…”