Abstract
ABSTRACTMosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family 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 infection in vitro. Additionally, 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.
Highlights
Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans
chikungunya fever (CF) is usually self-limiting and nonfatal; fatalities and severe forms of CHIKV disease have been reported in recent outbreaks, with complications during infection often found in patients with comorbidities
Previous studies have shown a substantial amount of alphavirus capsid protein translocates to nucleolus-like structures in host cells [10, 11]
Summary
Mosquito-transmitted chikungunya virus (CHIKV) is an arthritogenic alphavirus of the Togaviridae family responsible for frequent outbreaks of arthritic disease in humans. 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 sitedirected mutants of the capsid protein NoLS, and examine the effect of the NoLS mutation on CHIKV pathogenesis in vivo and its potential to influence CHIKV vaccine design. Signal motifs in the N terminus have been shown to be important in the nuclear/cytoplasmic translocation of capsid protein and nucleolar targeting [10,11,12] In encephalitic alphaviruses, such as Venezuelan and eastern equine encephalitis viruses, the multifunctional capsid protein is responsible for host transcription inhibition and subsequent cytopathic effect [13, 14]. In the arthritogenic alphaviruses examined, host transcriptional shutoff and translational shutoff are thought to be dependent on nsP2 rather than capsid protein [16, 17]
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