Soumya Pathuri

Introduction. Chikungunya Virus (CHIKV) is an alphavirus transmitted by Aedes albopictus and aegypti species mosquitoes.³ CHIKV causes maculopapular rash, fever, lymphopenia, and chronic arthritis.⁵ The main enzyme involved in alphavirus capping is non-structural protein 1 (nsp1), which stabilizes nsP1-3 complex for genome replication.² Since viral capping mechanisms differ from host cell mechanisms, targeting nsP1 could yield beneficial results. Non-structural protein 3 (nsP3) regulates RNA synthesis and replication activity. Although they show tremendous potential for development of vaccines, specific functions of non-structural proteins 1 and 3 require further research. There are currently no approved antiviral drugs or vaccines for CHIKV.¹ Methods. Mass spectrometry was employed to determine the hydrolysis of ADP-ribose groups from mono-ADP(ribosyl)ated glutamate and aspartate residues via nsP3 macrodomain. nsP3^MD mutants were generated with ineffective MAR hydrolysis and ADP-ribose binding. They were then inserted into a plasmid with full-length cDNA of the RNA genome of CHIKV 181/25. Binding of ADP-ribose to nsP3^MD was studied by isothermal titration calorimetry.⁴ Results.  Through a process known as ADP ribosylation, PARPs (poly(ADP-ribose) polymerases) transfer ADP-ribose singly as mono(ADP-ribose) (MAR) or in branched polymeric chains as poly(ADP-ribose) (PAR). CHIKV nsP3 macrodomain removes MAR from aspartate and glutamate. G32 mutants with decreased MAR hydrolase activity demonstrated importance of MAR hydrolase activity. This was shown by weak CHIKV replication despite higher affinity for ADP-ribose. G32E mutants reverted to alanine or glycine residue seen with partial restoration of MAR hydrolase activity. T111A mutant has intermediate hydrolase activity between G32S and G32A mutants and lower affinity to ADP-ribose. Viral yield for T111A was lower in comparison which shows that limiting ADP-ribose binding (required for hydrolase activity) greatly reduces replication. Mutant CHIKV lacking hydrolase activity was unable to replicate in mammalian BHK-21 cells or mosquito Aedes Albopictus cells. Mutants with decreased enzymatic activity had slower replication in mammalian neuronal cells and displayed decreased virulence in 2-day-old mice. CHIKV 181/25 transfected with mutant R144A was not effective despite wild-type (WT) levels of MAR hydrolase levels and ADP-ribose binding ability.⁴ Conclusion. MAR hydrolase activity is critical for viral replication in both vertebrate host and invertebrate vector. This was demonstrated by G32 reversion occurring in both mammalian and mosquito cells. Targeting nsP3 hydrolase activity could yield powerful vaccines for CHIKV as well as other viruses with the same macrodomain.⁴ Viral load can be lowered efficiently and virulence could be diminished with such targeted treatments.

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