title: Mutation of the N-Terminal Region of Chikungunya Virus Capsid Protein: Implications for Vaccine Design.
abstract: Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of theTogaviridaefamily responsible for frequent outbreaks of arthritic disease in humans. Capsid protein, a structural protein encoded by the CHIKV RNA genome, is able to translocate to the host cell nucleolus. In encephalitic alphaviruses, nuclear translocation induces host cell transcriptional shutoff; however, the role of capsid protein nucleolar localization in arthritogenic alphaviruses remains unclear. Using recombinant enhanced green fluorescent protein (EGFP)-tagged expression constructs and CHIKV infectious clones, we describe a nucleolar localization sequence (NoLS) in the N-terminal region of capsid protein, previously uncharacterized in CHIKV. Mutation of the NoLS by site-directed mutagenesis reduced efficiency of nuclear import of CHIKV capsid protein. In the virus, mutation of the capsid protein NoLS (CHIKV-NoLS) attenuated replication in mammalian and mosquito cells, producing a small-plaque phenotype. Attenuation of CHIKV-NoLS is likely due to disruption of the viral replication cycle downstream of viral RNA synthesis. In mice, CHIKV-NoLS infection caused no disease signs compared to wild-type CHIKV (CHIKV-WT)-infected mice; lack of disease signs correlated with significantly reduced viremia and decreased expression of proinflammatory factors. Mice immunized with CHIKV-NoLS, challenged with CHIKV-WT at 30 days postimmunization, develop no disease signs and no detectable viremia. Serum from CHIKV-NoLS-immunized mice is able to efficiently neutralize CHIKV infectionin vitroAdditionally, CHIKV-NoLS-immunized mice challenged with the related alphavirus Ross River virus showed reduced early and peak viremia postchallenge, indicating a cross-protective effect. The high degree of CHIKV-NoLS attenuation may improve CHIKV antiviral and rational vaccine design.IMPORTANCECHIKV is a mosquito-borne pathogen capable of causing explosive epidemics of incapacitating joint pain affecting millions of people. After a series of major outbreaks over the last 10 years, CHIKV and its mosquito vectors have been able to expand their range extensively, now making CHIKV a human pathogen of global importance. With no licensed vaccine or antiviral therapy for the treatment of CHIKV disease, there is a growing need to understand the molecular determinants of viral pathogenesis. These studies identify a previously uncharacterized nucleolar localization sequence (NoLS) in CHIKV capsid protein, begin a functional analysis of site-directed mutants of the capsid protein NoLS, and examine the effect of the NoLS mutation on CHIKV pathogenesisin vivoand its potential to influence CHIKV vaccine design. A better understanding of the pathobiology of CHIKV disease will aid the development of effective therapeutic strategies.
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia a.taylor1@griffith.edu.au s.mahalingam@griffith.edu.au.
authors.forename: Adam
authors.initials: A
authors.lastname: Taylor
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia.
authors.forename: Xiang
authors.initials: X
authors.lastname: Liu
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia.
authors.forename: Ali
authors.initials: A
authors.lastname: Zaid
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Queensland, Australia.
authors.forename: Lucas Y H
authors.initials: LY
authors.lastname: Goh
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Queensland, Australia.
authors.forename: Jody
authors.initials: J
authors.lastname: Hobson-Peters
authors.affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, Queensland, Australia.
authors.forename: Roy A
authors.initials: RA
authors.lastname: Hall
authors.affiliation: Institute of Technology, University of Tartu, Tartu, Estonia.
authors.forename: Andres
authors.initials: A
authors.lastname: Merits
authors.affiliation: Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia a.taylor1@griffith.edu.au s.mahalingam@griffith.edu.au.
authors.forename: Suresh
authors.identifier: 0000-0003-3141-8410
authors.initials: S
authors.lastname: Mahalingam
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism*; D065632:Chikungunya Fever / Q000473:pathology / Q000517:prevention & control / Q000821:virology*; D002646:Chikungunya virus / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D014644:Genetic Variation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D021382:Protein Sorting Signals; D014765:Viral Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D014774:Virulence
chemical_list: D036022:Capsid Proteins; D050505:Mutant Proteins; D021382:Protein Sorting Signals; D014765:Viral Vaccines