The Chikungunya virus nsP3 macro domain inhibits activation of the NF-κB pathway

Abstract

14The role of the chikungunya virus (CHIKV) non-structural protein 3 (nsP3) in the virus lifecycle 15 is poorly understood. The protein comprises 3 domains. The N-terminus is a macro domain, 16 biochemically characterised to bind both RNA and ADP-ribose, and to possess ADP-ribosyl 17 hydrolase activity -an enzymatic activity that removes ADP-ribose from mono-ADP-18 ribosylated proteins. As ADP-ribosylation is important in the signalling pathway leading to 19 activation of the transcription factor NF-кB, we sought to determine if the macro domain might 20 perturb NF-кB signalling. We first show that CHIKV infection did not induce NF-кB activation, 21 and could not block exogenous activation of the pathway via TNFα, although TNFα treatment 22 did reduce virus titres. Ectopic expression of nsP3 was able to block TNFα-mediated NF-кB 23 activation and this was dependent on the macro domain, as mutations previously shown to 24 disrupt either ADP-ribose binding or hydrolase activity lost the ability to inhibit NF-кB 25 activation. Lastly, we determined the phenotype of the macro domain mutants in the context 26 of virus infection in a range of cell types. Our data are consistent with cell-and species-27 dependent roles of the macro domain, however, these phenotypes do not correlate with the 28 ability to inhibit NF-кB activation suggesting that the macro domain plays multiple independent 29 roles in the virus lifecycle.Chikungunya virus (CHIKV) is an alphavirus of the Togaviridae family, and is increasingly 32 becoming a threat to global health. First discovered in Tanzania in 1952, CHIKV is now 33 classified into four distinct genotypes and has spread from Africa to Asia, Europe, and the 34 Americas 1 . CHIKV is transmitted by mosquitos and was previously geographically limited to 35 the distribution of the Aedes aegypti mosquito which favours tropical climates. However, 36 mutations in the E1 and E2 glycoproteins, detected in 2004, have enabled spread by the 37Aedes albopictus mosquito, which is more widely disseminated around the globe 2 . 38Infection with CHIKV causes Chikungunya fever, characterised by high fever, rash and joint 39 pain which can be debilitating and, in some cases, persistent 3 . CHIKV infection induces a 40 highly inflammatory state, inducing tissue damage 4 . In addition, individuals who have been 41 infected with CHIKV are significantly predisposed to arthritis in later life 5 . In healthy adults, 42 CHIKV is rarely fatal though the risk of death is high in infants, the elderly and the 43 immunocompromised 6 . 44The CHIKV genome is a positive-sense, single stranded (ss) RNA. It possesses a 5' cap and 45 a poly-A tail and contains two open reading frames (ORFs) 7 . The first ORF encodes for the 46 four non-structural proteins, nsP1-4, which are directly translated from the genomic RNA 8 . The 47 NF-кB pathway, and is unable to suppress exogenous activation of the pathway via TNFα 84 treatment. However, in contrast we further demonstrate that ectopic expression of nsP3 is 85 able to inhibit exogen...

Enhancement of Chikungunya virus genome replication in mammalian cells at sub-physiological temperatures

“…Reassuringly, all three AUD mutants showed replication defects compared to WT in all these cell lines (Fig 2D), although the magnitude of the defective phenotypes varied between different cell lines. In particular the defective phenotypes were less apparent in BHK-21 cells, this is consistent with our observations of other mutants in nsP3 [40]. We conclude that the three residues W220, D249 and Y324 within the CHIKV nsP3 AUD play a role in genome replication in mammalian cells but not in mosquito cells.…”

“…The phenotypes of the 3 mutants were observed in all mammalian cells tested although the magnitude of the defects varied somewhat. Intriguingly, in BHK-21 cells the mutants appeared to be better tolerated, as seen previously for both PV [15] and mutations in the nsP3 macrodomain [34]. Our study took a surprising turn when we observed that the 3 mutants exhibited no defective phenotype in mosquito cells and also when assayed in mammalian cells at sub-physiological temperatures, defining these as temperature-sensitive (ts) mutants.…”