Functional Correlation between Subcellular Localizations of Japanese Encephalitis Virus Capsid Protein and Virus Production

Abstract

The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles. IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Japanese encephalitis virus capsid protein interacts with non-lipidated MAP1LC3 on replication membranes and lipid droplets

“…These residues are highly conserved among the flaviviruses. The capsid protein has been shown to interact with LDs, and this interaction is crucial for virus replication and pathogenesis [18][19][20][21][22][23].…”

“…LDs have been described as platforms for HCV assembly, with the capsid localizing to the lipid droplets and subsequent recruitment of NS5A, which engages virus replication complexes to ld-associated membranes [18,19]. All the other flavivirus capsid proteins -DENV, Zika virus (ZIKV), West nile virus (WNV) and JEV -have been reported to localize on LDs and this association is crucial for virus particle formation [20][21][22][23]. Virions are assembled in close proximity to the ER and LDs and bud into the ER-lumen for envelopment followed by transport through the secretory pathway [24].…”

The role of capsid in the flaviviral life cycle and perspectives for vaccine development

“…Although the molecular weight of flavivirus CPs is relatively low, they are actively transported into the nucleus but not by simple diffusion [44] , [46] . Interestingly, the nuclear localization signals (NLSs) of flavivirus CPs are heterogeneous [44] , [45] , [46] , [49] , [50] , [51] . It has also been demonstrated that the nuclear localization of flavivirus CPs is functionally correlated with virus production [45] , [49] , [51] .…”

“…Interestingly, the nuclear localization signals (NLSs) of flavivirus CPs are heterogeneous [44] , [45] , [46] , [49] , [50] , [51] . It has also been demonstrated that the nuclear localization of flavivirus CPs is functionally correlated with virus production [45] , [49] , [51] . It is worth stressing that the key sites of the predicted NLS are usually basic residues, which play a role in RNA binding for CPs.…”

Understanding Flavivirus Capsid Protein Functions: The Tip of the Iceberg

“…Their ability to interact with lipid droplets is essential for efficient production of virus particles [14][15][16][17]. Capsid is also able to enter the nucleus and wreak havoc on ribosome biogenesis and the host transcriptome [18][19][20][21][22][23]. The function of these interactions within the nucleus and nucleolus in the context of the flavivirus life cycle is poorly understood.…”