Supplementary MaterialsS1 Fig: A. ppat.1007239.s003.pptx (381K) GUID:?6F8617FD-DC4F-4C12-B784-0A53DACC2A75 S4 Fig: The P247A/V248A mutation did not revert or result in compensatory mutations elsewhere in the genome. ICRES-P247A/V248A RNA was electroporated into C2C12 cells and cytoplasmic RNA was TRIzol extracted at 48 h.p.e. cDNA was generated from the extracted cell RNA with random primers before PCR was performed with specific primers (see supplementary S1 Table). A. PCR fragments used for CHIKV whole genome sequencing. B. Sequencing alignment result between wildtype and P247A/V248A mutant using CC-5013 kinase activity assay DNA Dynamo software. Red underlined sequences show changes from P247 (CCG) and V248 (GTG) to alanine (GCGGCG)(PPTX) ppat.1007239.s004.pptx (380K) GUID:?3D863BC9-0CB4-4121-A3F2-E1B39B17AF5D S5 Fig: Sequencing analysis of virus passage P0. P0: supernatant virus stock obtained from C2C12 cells at 48 h.p.e. nsP3 coding sequence was amplified by RT-PCR and sequenced. The region spanning the indicated mutations is shown. Note that for both E225A and R243A/K245A the sequence traces shown are from the negative strand, hence the colour of the trace does not match the colour code of the sequence below. R243A/K245A had already reverted to wildtype, whereas the other mutants had not reverted.(PPTX) ppat.1007239.s005.pptx (192K) GUID:?B187F201-2076-40D1-9F01-A462A35B5A0D S1 Table: Primers used to amplify cDNA and sequence the ICRES-P247A/V248A virus for complementary mutations. (PPTX) ppat.1007239.s006.pptx (42K) GUID:?4179B60A-FE04-4270-B0B7-D678D84E8ECF Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Chikungunya virus (CHIKV) is a re-emerging causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the nonstructural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus CC-5013 kinase activity assay lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for CC-5013 kinase activity assay virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production Alox5 of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA and genus and is absent from the closely related Rubella virus (the sole member of the genus within the genus we first aligned the AUD amino acid sequences of a range of both Old World and New World alphaviruses (S1A Fig). As the AUD sequences between SINV and CHIKV are highly conserved (118 of 243 CC-5013 kinase activity assay residues are identical), the nsP2/nsP3 protein structure of SINV  was referenced to identify the putative location of each of the conserved residues. Following from the above analysis, 10 residues were chosen for further study as they were located on the surface of the protein (S1B Fig) and were either absolutely conserved throughout the alphaviruses, CC-5013 kinase activity assay or in other cases were substituted by residues with similar physical characteristics (specifically the corresponding residue for both Met219 and Val260 in CHIKV is leucine in SINV) (Fig 1B and.