A novel interaction between the 5' untranslated region of the Chikungunya virus genome and Musashi RNA binding protein is essential for efficient virus genome replication

Abstract

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedesspp. mosquitoes-causing fever and debilitating joint pain, with frequent long-term health implications and high morbidity. The CHIKV replication cycle is poorly understood and specific antiviral therapeutics are lacking. In the current study, we identify host cell Musashi RNA binding protein-2 (MSI-2) as a proviral factor. MSI-2 depletion and small molecule inhibition assays demonstrated that MSI-2 is required for efficient CHIKV genome replication. Depletion of both MSI-2 and MSI-1 homologues was found to synergistically inhibit CHIKV replication, suggesting redundancy in their proviral function. Electromobility shift assay (EMSA) competition studies demonstrated that MSI-2 interacts specifically with an RNA binding motif within the 5' untranslated region (5'UTR) of CHIKV and reverse genetic analysis showed that mutation of the binding motif inhibited genome replication and blocked rescue of mutant virus. For the first time, this study identifies the proviral role of MSI RNA binding proteins in the replication of the CHIKV genome, providing important new insight into mechanisms controlling replication of this significant human pathogen and the potential of a novel therapeutic target.

Graphical Abstract

Figure 1.

(A) Schematic representation of CHIKV genome organization (B) Schematic representation of CHIKV RNA structures within the 5′UTR and adjacent ORF-1 region of the CHIKV genome (Kendall et al., 2019). RNA replication elements SL3, SL47, SL88, SL102, SL165, SL194 and SL246 are labelled in black type. The ORF-1 AUG start codon is labelled by a green arrow and the putative MSI binding site by a red oval.

Figure 2.

Native EMSAs between in vitro transcribed P³² 5′ radiolabeled CHIKV RNA nts 1–330 (*RNA^WT) and recombinantly expressed MSI-2 demonstrated an RNA/protein interaction that was outcompeted by increasing concentrations of equivalent unlabeled (RNA^WT) but less efficiently by the same RNA incorporating BSM mutation ₆₃AUUAAU₆₈ > ₆₃CAACUU₆₈ (RNA^BSM). (A) Increasing concentrations of MSI-2 intensified the observed band shift to the larger RNA/Protein complex and decreased the equivalent unbound RNA band. The interaction between a 1:4 ratio of *RNA^WT:MSI-2 was competed with increasing concentrations of unlabelled (B) RNA^WT or (C) RNA^mut (₆₃CAACUU₆₈-mut). (D) Band shifts in the unlabeled RNA competition EMSAs were quantified by densitometry and expressed as % change in the density of the RNA/protein complex bands, normalized to the equivalent total lane density, for each competition ratio and compared each time to ratio 1:0. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001). Grey block triangles indicate increasing concentrations of specific reactants.

Figure 3.

Ro 08-2750 signifyingly inhibits replication of infectious CHIKV and the CHIKV-SGR. (A) Schematic representations of CHIKV infectious clone (top) compared to the sub-genomic replicon (SGR) (bottom) in which a Renilla luciferase (RLuc) reporter gene is fused within the nsp3 coding sequence and the structural genes of ORF-2 are replaced by a firefly luciferase (Fluc) reporter gene. Replication is expressed in Relative Light Units [RLU]. (B) Ro significantly inhibits productive CHIKV productive replication relative to DMSO treated negative controls at 8 and 24 hpi. (C and D) Ro 08-2750 significantly inhibits CHIKV-SGR replication, measured by both ORF-1 and ORF-2 expression, relative to DMSO treated negative controls at 8 and 24 hpt. (E) Ro 08-2 750 had no significant (ns) effect on CHIKV-SGR (GDD-GAA) translation, measured by ORF-1 expression. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 4.

Ro signifyingly inhibits CHIKV genome replication. (A) Schematic representation of CHIKV trans-complementation assay showing codon optimised pCHIKV-nsP1234 (top) from which the CHIKV nsPs were translated and pCHIK-Fluc/Gluc (bottom) in which ORF-1 was replaced by an Fluc reporter gene, fused to the first 77 nts or CHIKV ORF-1 (N77) downstream of the authentic CHIKV 5′UTR. ORF-2, flanked by the authentic intragenic (SG) and 3′ UTRs, was replaced by a Gluc reporter gene. (B) and (C) Ro significantly inhibited CHIKV genome replication of the trans-complementation assay, measured by both ORF-1 and ORF-2 expression, relative to DMSO treated negative controls at 8 and 24 hpt. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 5.

shRNA suppression of MSI-2 significantly inhibits replication of infectious CHIKV. (A) Western blot analysis of total cellular protein extracted from Rd cells and compared to negative control scrambled shRNA, demonstrated consistent shRNA knockdown of MSI-2 over 3 serial passages (P1–P3). MSI-2 suppression significantly inhibited productive CHIKV replication, relative to scrambled shRNA at 8 and 24 hpi measured by plaque assay (B) and strand specific qRT-PCR for the the virus genomic (C) and negative intermediate (D) strands. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 6.

shRNA suppression of MSI-2 significantly inhibits CHIKV-SGR replication and CHIKV genome replication. (A and B) shRNA suppression of MSI-2 significantly inhibited CHIKV-SGR replication, measured by both ORF-1 and ORF-2 expression, relative to scrambled shRNA negative controls at 8 and 24 hpt. (C and D) shRNA suppression of MSI-2 significantly inhibited CHIKV genome replication of the trans-complementation assay, measured by both ORF-1 and ORF-2 expression, relative to DMSO treated negative controls at 8 and 24 hpt. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 7.

siRNA Depletion of either MSI-1 or MSI-2 significantly inhibited CHIKV replication in Huh7 cells and co-depletion of both MSI-1 and MSI-2 had a synergistic effect on CHIKV inhibition in Huh7 cells. siRNA depletion of MSI-2 significantly inhibited CHIKV replication in RD cells and co-depletion of both MSI-1 and MSI-2 did not increase the level of CHIKV inhibition. siRNA depletion of MSI-1 and MSI-2 significantly inhibited CHIKV replication in Huh7 cells relative to scrambled siRNA at 8 and 24 hpi measured by plaque assay (A) and strand specific qRT-PCR for the the virus genomic (B) and negative intermediate (C) strands. siRNA depletion of MSI-2 in RD cells significantly inhibited CHIKV replication in relative to scrambled siRNA at 8 and 24 hpi measured by plaque assay (D) and strand specific qRT-PCR for the the virus genomic (E) and negative intermediate (F) strands. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 8.

Substitutions disrupting the predicted MSI binding site (₆₃CAACUU₆₈-mut) but maintaining local RNA structure prevented virus rescue and significantly inhibited CHIKV genome replication. Substitution _A67_G maintained the predicted MSI-2 binding motif but disrupted local RNA structure and significantly inhibited CHIKV replication. (A and B) Mutation ₆₃CAACUU₆₈ significantly inhibited CHIKV genome replication of the trans-complementation assay, measured by both ORF-1 and ORF-2 expression, relative to wild-type positive controls at 8 and 24 hpt. (C) Mutation ₆₃CAACUU₆₈-mut prevented rescue of CHIKV following transfection of capped in vitro transcribed RNA into BHK cells. Released virus was measured by plaque assay of supernatant 24 hpt and compared to positive control wild-type infectious CHIKV in vitro transcribed RNA, which was transfected and analysed in parallel. (D) Mutation ^A67^G significantly inhibited CHIKV replication at 8 hpi and 24 hpi relative to wild type CHIKV following infection of RD cells (MOI 0.1). N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).

Figure 9.

Combining substitutions within the predicted MSI binding site (₆₃CAACUU₆₈-mut) and Ro 08–2750 had a synergistic affect, significantly increasing inhibition of CHIKV genome replication. (A and B) Mutation of the MSI binding site combined with Ro 08-2750 significantly increased inhibition of the CHIKV trans-complementation assay, measured by both ORF-1 and ORF-2 expression, relative to ₆₃CAACUU₆₈-mut in the absence of Ro 08-2750 at 8 and 24 hpt. N = 3, error bars represent standard error from the mean and significance was measured by two-tailed t-test (* P < 0.05, ** P < 0.01, *** P < 0.001).