Andrew Tuplin

Chikungunya virus (CHIKV) remains a significant global public health threat, yet antiviral discovery is limited by experimental systems that provide little mechanistic resolution and rely heavily on transformed cell lines of uncertain physiological relevance. Here, we developed complementary live-cell CHIKV reporter systems comprising a dual-fluorescent subgenomic replicon and fluorescent infectious viruses that enable real-time monitoring of distinct stages of the viral replication cycle. By coupling an nsP3-tagged reporter to genomic RNA translation and replication with a subgenomic reporter of structural gene expression, we achieved temporal resolution of early and late replication events in living cells. Functional validation using replication-defective and subgenomic RNA-deficient mutants confirmed that the system distinguishes between viral RNA replication and downstream structural gene expression. Application of these reporters revealed distinct antiviral responses across transformed and physiologically relevant human cell models. Ribavirin and the nsP2 inhibitor RA-0002034 potently inhibited CHIKV replication in transformed cell lines. Unexpectedly, ribavirin activity was markedly diminished in primary human skeletal muscle myoblasts and dermal fibroblasts, despite robust antiviral effects in commonly used screening cell lines. Mechanistic studies showed that ribavirin-mediated inhibition was rescued by guanosine but not inosine supplementation and was recapitulated by mycophenolic acid, consistent with guanine nucleotide depletion as the predominant antiviral mechanism under the conditions tested. Notably, inhibition through this pathway was substantially less effective in primary human cells. Together, these findings establish versatile live-cell CHIKV reporter systems for mechanistic studies of viral replication and reveal striking cell-type-dependent differences in response to guanine nucleotide depletion. Our results demonstrate that host-cell context can profoundly influence both CHIKV replication and antiviral susceptibility, highlighting the importance of physiologically relevant models for understanding virus-host interactions and antiviral mechanism. Author Summary: Chikungunya virus is a mosquito-borne pathogen that causes fever, severe joint pain, and chronic musculoskeletal disease. Although considerable progress has been made in understanding the viral replication cycle, important questions remain regarding how interactions between the virus and its host cell influence replication and susceptibility to antiviral intervention. Experimental systems that permit real-time analysis of distinct stages of infection are therefore valuable tools for advancing our understanding of chikungunya virus biology. In this study, we developed complementary fluorescent chikungunya virus reporter systems that enable live-cell monitoring of viral replication and gene expression. These tools distinguished between early replication-associated processes and downstream subgenomic gene expression, providing mechanistic resolution of the viral replication cycle in living cells. Using these systems, we investigated the activity of ribavirin and found that its antiviral effects were highly dependent on host-cell context. While ribavirin potently inhibited chikungunya virus replication in transformed cell lines, this activity was largely absent in primary human muscle and skin cells that represent physiologically relevant targets of infection. Mechanistic studies showed that inhibition was linked to depletion of intracellular guanine nucleotides and could be reversed by guanosine supplementation. Together, our findings reveal how host-cell-dependent processes influence both chikungunya virus replication and antiviral susceptibility and provide new tools for dissecting virus-host interactions.

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.

Chikungunya virus (CHIKV) is an alphavirus, transmitted by Aedes species mosquitoes. The CHIKV single-stranded positive-sense RNA genome contains two open reading frames, coding for the non-structural (nsP) and structural proteins of the virus. The non-structural polyprotein precursor is proteolytically cleaved to generate nsP1-4. Intriguingly, most isolates of CHIKV (and other alphaviruses) possess an opal stop codon close to the 3′ end of the nsP3 coding sequence and translational readthrough is necessary to produce full-length nsP3 and the nsP4 RNA polymerase. Here we investigate the role of this stop codon by replacing the arginine codon with each of the three stop codons in the context of both a subgenomic replicon and infectious CHIKV. Both opal and amber stop codons were tolerated in mammalian cells, but the ochre was not. In mosquito cells all three stop codons were tolerated. Using SHAPE analysis we interrogated the structure of a putative stem loop 3′ of the stop codon and used mutagenesis to probe the importance of a short base-paired region at the base of this structure. Our data reveal that this stem is not required for stop codon translational readthrough, and we conclude that other factors must facilitate this process to permit productive CHIKV replication.

Chikungunya virus (CHIKV) is an alphavirus, transmitted by Aedes species mosquitoes. The CHIKV positive-sense RNA genome contains two open reading frames, coding for the non-structural (nsP) and structural proteins of the virus. The non-structural polyprotein precursor is proteolytically cleaved to generate nsP1-4. Intriguingly most isolates of CHIKV (and other alphaviruses) possess an opal stop codon close to the 3’ end of the nsP3 coding sequence and translational readthrough is necessary to produce full-length nsP3 and the nsP4 RNA polymerase. Here we investigate the role of this stop codon by replacing the arginine codon with all three stop codons in the context of both a subgenomic replicon and infectious CHIKV. Both opal and amber stop codons were tolerated in mammalian cells, but the ochre was not. In mosquito cells all three stop codons were tolerated. Using SHAPE analysis we interrogated the structure of a putative stem loop 3’ of the stop codon and used mutagenesis to probe the importance of a short base-paired region at the base of this structure. Our data reveal that this stem is not required for stop codon translational readthrough, and we conclude that other factors must facilitate this process to permit productive CHIKV replication.

Chikungunya virus (CHIKV) is a re-emerging, pathogenic alphavirus that is transmitted to humans by Aedes spp . 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.

Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesized that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed, this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV), and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses.

Genome replication of positive strand RNA viruses requires the production of a complementary negative strand RNA that serves as a template for synthesis of more positive strand progeny. Structural RNA elements are important for genome replication, but while they are readily observed in the positive strand, evidence of their existence in the negative strand is more limited. We hypothesised that this was due to viruses differing in their capacity to allow this latter RNA to adopt structural folds. To investigate this, ribozymes were introduced into the negative strand of different viral constructs; the expectation being that if RNA folding occurred, negative strand cleavage and suppression of replication would be seen. Indeed this was what happened with hepatitis C virus (HCV) and feline calicivirus (FCV) constructs. However, little or no impact was observed for chikungunya virus (CHIKV), human rhinovirus (HRV), hepatitis E virus (HEV) and yellow fever virus (YFV) constructs. Reduced cleavage in the negative strand proved to be due to duplex formation with the positive strand. Interestingly, ribozyme-containing RNAs also remained intact when produced in vitro by the HCV polymerase, again due to duplex formation. Overall, our results show that there are important differences in the conformational constraints imposed on the folding of the negative strand between different positive strand RNA viruses.

Most alphaviruses (family Togaviridae) including Sindbis virus (SINV) and other human pathogens, are transmitted by arthropods. The first open reading frame in their positive strand RNA genome encodes for the non-structural polyprotein, a precursor to four separate subunits of the replicase. The replicase interacts with cis-acting elements located near the intergenic region and at the ends of the viral RNA genome. A trans-replication assay was developed and used to analyse the template requirements for nine alphavirus replicases. Replicases of alphaviruses of the Semliki Forest virus complex were able to cross-utilize each other’s templates as well as those of outgroup alphaviruses. Templates of outgroup alphaviruses, including SINV and the mosquito-specific Eilat virus, were promiscuous; in contrast, their replicases displayed a limited capacity to use heterologous templates, especially in mosquito cells. The determinants important for efficient replication of template RNA were mapped to the 5' region of the genome. For SINV these include the extreme 5'- end of the genome and sequences corresponding to the first stem-loop structure in the 5' untranslated region. Mutations introduced in these elements drastically reduced infectivity of recombinant SINV genomes. The trans-replicase tools and approaches developed here can be instrumental in studying alphavirus recombination and evolution, but can also be applied to study other viruses such as picornaviruses, flaviviruses and coronaviruses. Alphaviruses are positive-strand RNA viruses, most of which use mosquitoes to spread between vertebrate hosts; many are human pathogens with potentially severe medical consequences. Some alphavirus species are believed to have resulted from the recombination between different members of the genus and there is evidence of movement of alphaviruses between continents. Here, a novel assay uncoupling viral replicase and template RNA production was developed and used to analyse cross-utilization of alphavirus template RNAs. We observed that replicases of closely related alphaviruses belonging to the Semliki Forest virus complex can generally use each other’s template RNAs as well as those of distantly related outgroup viruses. In contrast, replicases of outgroup viruses clearly preferred homologous template RNAs. These trends were observed in both mammalian and mosquito cells, with template preferences generally more pronounced in mosquito cells. Interestingly, the template RNA of the mosquito-specific Eilat virus was efficiently used by other alphavirus replicases while Eilat replicase could not use heterologous templates. Determinants for template selectivity were mapped to the beginning of the RNA genome and template recognition was more likely based on the recognition of RNA sequences than recognition of structural elements formed by the RNAs.