Analysis of the capsid processing strategy of Thosea asigna virus using baculovirus expression of virus-like particles

Abstract

Thosea asigna virus (TaV), a putative member of the genus Betatetravirus of the family Tetraviridae, is predicted to have a novel capsid expression strategy compared with other characterized tetraviruses. The capsid precursor protein is cleaved twice to generate three proteins. Two of the proteins, L (58n3 kDa) and S (6n8 kDa), are incorporated into the TaV virion. The third, nonstructural protein, produced from the N terminus of the precursor protein, is up to 17 kDa in size and is of unknown function. The TaV capsid precursor protein sequence without the 17 kDa Nterminal region was modelled against the solved structure from Nudaurelia ω virus (NωV) using SwissModel. The TaV model was very similar to the solved structure determined for subunit A of NωV and had features that are conserved between tetraviruses and nodaviruses, including the positioning of the cleavage site between the L and S capsid proteins. The production of virus-like particles (VLPs) using the baculovirus expression system was used to analyse the capsid processing strategy employed by TaV. VLPs were formed in both the presence and absence of the 17 kDa Nterminal region of the capsid precursor. VLPs were not formed when the L and S regions were expressed from separate promoters, indicating that cleavage between the L and S capsid proteins was an essential part of TaV capsid assembly. Expression of the TaV 17 kDa protein in bacteria did not produce intracellular tubules similar to those formed by bacterial expression of the p17 protein from Helicoverpa armigera stunt virus.

Use of baculovirus expression system for generation of virus-like particles: Successes and challenges

“…Furthermore, the proper proteolytic processing does not impair its binding capacity but does membrane fusion [108,133]. Additionally, there were several other studies [134,135] independently to show that proteolytic processing contributed to generating VLPs in insect cells. Expression of immature precursor and subsequent proteolytic processing seem to be an effective strategy in formation of VLPs [136].…”

The Palm Subdomain-based Active Site is Internally Permuted in Viral RNA-dependent RNA Polymerases of an Ancient Lineage

“…Some viruses from the Picornavirus-like supergroup and TaV/EeV employ a 2A or 2A-like protein of the NPGP family for proteolytic autoprocessing (dots in Figure 6). 23,26,50 These correlations are yet to be rationalized in structure -function terms.…”

Drosophila A virus is an unusual RNA virus with a T=3 icosahedral core and permuted RNA-dependent RNA polymerase

“…However, in recent years a number of viruses have been characterised from insects that do not fit clearly into one of these groups (Habayeb et al , 2006; Hartley et al , 2005; van der Wilk et al , 1997). Furthermore, within the Tetraviridae family there are viruses with diverse genome organizations, which are classified together because of the T=4 symmetry of their capsid particles (Agrawal & Johnson, 1992; Gorbalenya et al , 2002; Gordon et al , 1999; Gordon et al , 1995; Hanzlik et al , 1995; Pringle et al , 2003; Pringle et al , 2001). As more invertebrate RNA viruses are fully characterised it is becoming clear that there is a previously unrecognised diversity of genome organization and expression strategies within this group of viruses.…”