Structural characterization by transmission electron microscopy and immunoreactivity of recombinant Hendra virus nucleocapsid protein expressed and purified from Escherichia coli

Abstract

Hendra virus (family Paramyxoviridae) is a negative sense single-stranded RNA virus (NSRV) which has been found to cause disease in humans, horses, and experimentally in other animals, e.g. pigs and cats. Pteropid bats commonly known as flying foxes have been identified as the natural host reservoir. The Hendra virus nucleocapsid protein (HeV N) represents the most abundant viral protein produced by the host cell, and is highly immunogenic with naturally infected humans and horses producing specific antibodies towards this protein. The purpose of this study was to express and purify soluble, functionally active recombinant HeV N, suitable for use as an immunodiagnostic reagent to detect antibodies against HeV. We expressed both full-length HeV N, (HeV NFL), and a C-terminal truncated form, (HeV NCORE), using a bacterial heterologous expression system. Both HeV N constructs were engineered with an N-terminal Hisx6 tag, and purified using a combination of immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC). Purified recombinant HeV N proteins self-assembled into soluble higher order oligomers as determined by SEC and negative-stain transmission electron microscopy. Both HeV N proteins were highly immuno-reactive with sera from animals and humans infected with either HeV or the closely related Nipah virus (NiV), but displayed no immuno-reactivity towards sera from animals infected with a non-pathogenic paramyxovirus (CedPV), or animals receiving Equivac® (HeV G glycoprotein subunit vaccine), using a Luminex-based multiplexed microsphere assay.

Indirect ELISA based on Hendra and Nipah virus proteins for the detection of henipavirus specific antibodies in pigs

“…coli starting culture, which was considerably higher than what has been described in earlier studies using recombinant NiV N protein in E . coli obtaining yields of 1 mg per liter starting culture [ 25 ] and 0.8 mg per liter starting culture [ 51 ]. In another study, baculovirus-infected Sf9 cells were used for the expression of the recombinant NiV N protein at high yields of 2–3 mg per 1 x 10 6 infected Sf9 cells [ 23 ].…”

Structure and organization of paramyxovirus particles

“…While electron microscopy has established a basic framework for the paramyxovirus virion organization [3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23] and a number of crystal structures of paramyxovirus proteins and protein fragments have been solved (for instance [6,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42], the reconstruction of the 3D ultrastructures of paramyxovirus particles is impaired by particle size and the pleomorphic nature of the virions, which prevents single particle reconstruction approaches. To overcome the problem, recent studies have applied cryo-electron tomography (cryo-ET) to the analysis of paramyxovirus particles.…”

Characterization of parainfluenza virus 5 from diarrheic piglet highlights its zoonotic potential

“…The N protein possesses highly conserved amino acid regions among paramyxoviruses (Morgan, 1991; Lamb & Parks, 2013). Furthermore, the N protein is the most abundant and a highly immunogenic viral structural protein (Alayyoubi et al., 2015; Dortmans et al., 2011; Pearce et al., 2015). Furthermore, the N protein of isolated PIV5 contains highly homologous sequences with reference PIV5 strains from other species, sharing 98.4−99.8% amino acid identity (Table 2).…”