VP5 autocleavage is required for efficient infection by in vitro-recoated aquareovirus particles

Abstract

Grass carp reovirus (GCRV) is a member of the genus Aquareovirus in the family Reoviridae, and contains five core proteins (VP1-VP4 and VP6) and two outer-capsid proteins (VP5 and VP7) in its particle. Previous studies have revealed that the outer-capsid proteins of reovirus are responsible for initiating infection, but the mechanism is poorly understood. Using baculovirusexpressed VP5 and VP7 to recoat purified cores, in vitro assembly of GCRV was achieved in this study. Recoated GCRV (R-GCRV) closely resembled native GCRV (N-GCRV) in particle morphology, protein composition and infectivity. Similar to N-GCRV, the infectivity of R-GCRV could be inhibited by treating cells with the weak base NH 4 Cl. In addition, recoated particles carrying an AsnAAla substitution at residue 42 of VP5 (VP5 N42A /VP7 R-GCRV) were no longer infectious. These results provide strong evidence that autocleavage of VP5 is critical for aquareovirus to initiate efficient infection.

Comparative Proteomic Analysis of Lysine Acetylation in Fish CIK Cells Infected with Aquareovirus

“…The expression levels of cell histone acetylation or viral protein were analyzed by Western blotting. Viral yields were determined by plaque assay on CIK cells as previously described [ 8 ].…”

“…In addition, GCRV replicates well in the Ctenopharyngodon idellus kidney (CIK) cell line at 25–30 °C and produces a typical cytopathic effect consisting of large syncytia in its sensitive cells [ 3 , 4 ]. Previous studies on GCRV are mainly focused on viral biological and biochemical properties as well as viral three-dimensional structure based functions [ 5 , 6 , 7 , 8 ]. However, detailed biochemical events of biological and metabolic processes of host cells upon aquareovirus infection are poorly understood.…”

Characterization of viral entry and infection of quantum dot-labeled grass carp reovirus

“…In order to realize stable surface labeling of the GCRV outer capsid proteins depicted in Figure 1A, GCRV was propagated in Ctenopharyngodon idellus kidney (CIK) cells and purified by ultracentrifugation as previously described (Fang et al, 2008;Yan et al, 2015). The concentrated GCRV particles were incubated with sulfosuccinimidyl-6-biotinamidohexanoate (sulfo-NHS-LC-biotin; Thermo) at 28 °C for 2 h according to the manufacturer's instructions, and the unbound biotinylating agent was removed using a spin desalting column (Thermo).…”

“…The inhibitor NH 4 Cl can specifically block the reovirus entry pathway (Sturzenbecker et al, 1987;Yan et al, 2015); thus, to further elucidate whether the modified GCRV retained its native particle properties, we assessed the effects of the inhibitor on QD-GCRV infectivity using plaque assays. As shown in Figure 1I, typical plaques in infected CIK cells were observed with intact QD-GCRVs at different multiplicities of infection (MOIs), showing a pattern similar to that of unmodified GCRV.…”

N-Terminal Myristoylated VP5 is Required for Penetrating Cell Membrane and Promoting Infectivity in Aquareoviruses

“…proteins VP5 and VP7 are homologs of the mammalian reovirus (MRV) proteins l1 and r3, respectively. Furthermore, the autocleavage of outer capsid protein VP5 at its N-terminus is proposed to be required for viral entry into host cells during infection (Li and Fang 2013;Yan et al 2015).…”