Chikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitoes and causing fever, rash and arthralgia. Currently there are no vaccines or antiviral agents against CHIKV, therefore it is important to understand the molecular details of CHIKV replication. In this regard, the function of the Alphavirus non-structural protein 3 (nsP3) remains enigmatic. Building on previous studies (Gao et al, 2019), we generated a panel of mutants in a conserved and surface exposed cluster in the nsP3 alphavirus unique domain (AUD) and tested their replication phenotype using a sub-genomic replicon (SGR) in mammalian and mosquito cells. We identified three mutants that replicated poorly in mammalian cells but showed no defect in mosquito cells. We further showed that these mutants were temperature-sensitive, rather than species-specific, as they exhibited no replication defect in mammalian cells at sub-physiological temperature (28°C). Similar effects were observed in the context of infectious CHIKV as well as a closely related virus: O’Nyong Nyong virus (ONNV). Intriguingly, this analysis also revealed that the wildtype SGR replicated much more efficiently at sub-physiological temperature as compared to 37°C. This was not due to impaired interferon responses as this enhancement was also observed in Vero cells. Neither was this due to a defect in the phosphorylation of eIF2α as treatment with ISRIB, an inhibitor of global translation attenuation, did not compensate for replication defects at 37°C. However, we noticed significant differences between the sizes and numbers of virus-induced stress granules (SG) at physiological and sub-physiological temperatures. As cells in the periphery will be at sub-physiological temperatures, and these will be the first cells infected in the mammalian host following a mosquito bite, we propose that alphaviruses have evolved mechanisms to limit antiviral responses in these cells to promote viral genome replication.
Chikungunya virus (CHIKV) is a re-emerging Alphavirus transmitted by Aedes mosquitos and causing fever, rash and chronic arthralgia. There are no vaccines or antiviral agents available for CHIKV therefore it is important to understand the molecular details of virus replication. To address this, we previously conducted a mutagenic analysis of the central alphavirus unique domain (AUD) of the CHIKV non-structural protein 3 (nsP3), testing replication of a subgenomic replicon in a variety of mammalian and mosquito (Aedes albopictus) cell lines. One mutant (M219A) exhibited a different phenotype in two Aedes albopictus cell lines (U4.4 and C6/36): replicating as wildtype in C6/36 but was blocked in U4.4. As U4.4 cells have an intact RNAi response whereas C6/36 have a frameshift mutation in the Dicer-2 (Dcr2) gene and express an inactive Dcr2 protein, we proposed that the replication of M219A was suppressed by the RNAi antiviral response in U4.4 cells, while wild type nsP3 was able to counteract this response. To further investigate this hypothesis we have extended the mutagenic analysis to screen other residues in proximity with M219 within the AUD. All of these mutants retain wildtype levels of replication in mammalian and C6/36 cells, except for W220A which replicates poorly. Evaluation of the replication of these mutants in U4.4 is ongoing. We are also pursuing a CRISPR/Cas9 approach to ablate expression of Dcr2 in U4.4 cells, and generating stable C6/36 cells expressing Dcr2 to confirm our hypothesis. Our studies will shed light on how CHIKV nsP3 can antagonise mosquito innate immunity.
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