Virus Evolution and Replication
Influenza viruses undergo continuous genetic alterations to evolve and adapt to newer host species. Viral antigens harbors mutational “hotspots” which regulate species-specific fitness and “adaptation” to a new host, thus recognized as adaptation hotspots. We aim to characterize such adaptation hotspots in viral replication machinery, the RNPs, and characterize the molecular mechanism underlying such adaptation process
Bat influenza viruses are genetically divergent from the classical influenza A viruses and their replication potential in humans is largely uncharacterised. We have recently shown that bat influenza virus polymerase, the key replication machinery of the virus, is restricted in human cells, but gains fitness advantage through acquiring specific mutations at one of its adaptation hotspots
1. Identification of “adaptation hotspots” in influenza virus RNPs and elucidating and elucidating their role in host specific adaptation process
Influenza viruses constitutes its replication machinery in the form of RNPs where viral genomic RNA is enwrapped with oligomeric nucleoproteins (NP) and remains associated with a heterotrimeric RNA-dependent RNA polymerase (RdRp) composed of PB1, PB2 and PA subunits. RNPs are the self-sufficient machinery that perform both viral gene transcription at the early phases of infection and genome replication at the late phase thus serves as the key player driving virus propagation within the host. We utilize the recently discovered Bat influenza A/ H17N10, and the human adapted influenza A/ H1N1 viruses as genetically divergent systems to identify adaptation hot-spots in viral RNPs, specifically within RdRp, that may determine the replication fitness of these viruses in their respective host species.
An evolutionary conserved motif fine-tunes the activity of viral RNA-dependant RNA polymerase: A motif in the midlink region of PB2-subunit of the trimeric polymerase is “S-E-S” for classical influenza, but “S-S-T” for the naturally attenuated, bat-restricted influenza. This results in reduced fitness of the bat-virus polymerase in human cells, with a marked defect in viral genome replication. Negative-charge substitution in the bat-virus “S-S-T” motif results in fitness gain, and the reverse substitution has the opposite effect.
2. Characterizing the novel interaction between RNP and viral non-structural protein 1 (NS1)
Influenza virus non-structural protein 1 (NS1) plays a critical role in antagonizing host antiviral defenses. The NS1 protein also associates with viral RNPs through specific interaction with viral nucleoprotein (NP), but the physiological significance of the NP-NS1 interaction in context of virus life cycle remains elusive. Employing biochemical, biophysical, and molecular biology techniques, we aim to extensively characterize the NP-NS1 interaction at the molecular level and elucidate its role in the influenza virus replication cycle.
Influenza Nucleoprotein (NP) interaction with Non-structural protein-1 (NS1) observed through surface plasmon resonance (SPR) and split nanobit assay. Both wild-type and monomeric NP mutant E339A shows significant interaction with NS1 although the extent of interaction between NPWT and NS1 is greater