Comprehensive characterization of phosphorylation sites of Newcastle disease virus proteins by a multitude of fragmentation techniques — ASN Events

Comprehensive characterization of phosphorylation sites of Newcastle disease virus proteins by a multitude of fragmentation techniques (#104)

Keyur A Dave 1 , Buddhika A Jayakody 1 , Jeffrey J Gorman 1
  1. Protein Discovery Centre, QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia

Paramyxoviruses are a family of negative stranded RNA viruses whose members comprise of serious human pathogens, such as measles virus, mumps virus and respiratory syncytial virus; and in animals, such as Newcastle disease virus (NDV) and rinderpest virus. Apart from its potential economic impact on the poultry industry worldwide, NDV is becoming a promising agent for oncolytic virotherapy of mammalian cancers.

 For productive infection viruses must hijack the host cellular machinery as they cannot self-reproduce. To avoid elimination they have to weaken host antiviral responses but at the same time utilize the host intracellular trafficking and biosynthetic systems to enter and exit infected cells while producing new viral particles. These variegated interactions expose viral proteins to numerous host signaling pathways that might post-translationally modify them leading to a change in their functions either to favour host cell preservation or proliferation of infection. In either case these post-translational modifications (PTMs) on viral proteins serve as markers that reflect viral-host protein-protein interactions and provide a useful means of identifying them, regardless of the functional implications of these modifications. In addition, cellular localization of these interacting host proteins indicates intracellular trafficking behaviour of the viral proteins during the infectious cycle.

 In this pursuit, we have extensively characterized phosphorylation sites of the avian paramyxovirus-1, NDV, proteins by using an array of fragmentation techniques on several advanced mass spectrometry (MS) platforms. This includes analysis of unfractionated and phosphopeptide enriched digests from lysates of purified virions by ion trap (IT) CID with multi-stage activation, IT-ETD, IT-ETciD, IT-EThcD, Orbitrap (OT)-HCD and IT-HCD. These multitudes of fragmentation techniques on different MS platforms have provided common and unique phosphopeptide identifications in NDV proteins. Importantly, diverse and distinctive peptide backbone cleavage favoured by different fragmentation techniques has enabled unequivocal assignment of phosphosites detected in NDV proteins.