Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures

Abstract

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the Pl-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VPO, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VPO, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VPO, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

Expression, purification and characterization of enterovirus-71 virus-like particles

“…Within the cells, clusters of virus-like particles resembling those observed in EV71-infected human rhabdomyosarcoma (RD) cells [26] and in enterovirusinfected skeletal muscle cells [33] were observed. The in vivo formation of particle aggregates was not previously reported by other researchers studying the poliovirus VLPs [23,25] , but was recently noted in BHK-38 cells infected by foot-and-mouth disease virus [34] , a relatively genetically distant picornavirus. To date, the biological significance of the particle aggregation remains to be explored, but the resemblance in particle morphology and the aggregation patterns between the VLP and the authentic enterovirus suggests that EV71 VLP adopts an assembly pathway in insect cells in a way similar to the authentic viruses do in their natural host cells.…”

“…In addition, recombinant poliovirus VLPs have been produced using the baculovirus/insect cell or vaccinia virus expression system. These particles were produced via the (1) expression of the complete ORF; (2) co-expression of individual VP0, VP1 and VP3 proteins; or (3) co-expression of P1 and 3CD proteins [23][24][25] . Since EV71 and poliovirus share identical genome organization and similar protein functions, we hypothesized that EV71 assembly occurs in a way similar to poliovirus and have previously demonstrated the formation of EV71 VLP by co-infecting insect cells with two recombinant baculovir uses, Bac-P1 expressing P1 and Bac-3CD expressing 3CD [26] .…”

Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis

“…Poliovirus is a nonenveloped virus, and its capsid proteins are produced by the processing of the capsid protein precursor P1 by the viral protease 3CD (15). Coexpression of P1 and 3CD in different systems has been shown to efficiently generate VLPs, which can induce a protective antibody response (16)(17)(18)(19)(20). While VLP-based vaccines eliminate biosafety concerns, since live polioviruses are not involved at any stage of production, the traditional approach based on administration of purified VLPs does not seem to be practical in the case of poliovirus.…”

Immunogenicity and safety of virus-like particle of the porcine encephalomyocarditis virus in pig

“…The baculovirus expression system has been widely used for generation of these VLPs due to the high productivity of the system and the ability to achieve rapid production scale implementation [19]. VLPs have been successfully generated from many other Picornaviridae family members, including enterovirus [20], poliovirus [21] and foot-and mouth disease virus (FMDV) [22]. In a previous study, we engineered a DNA vaccine that produced pEMCV-K3 VLPs and confirmed that the VLP antigen exhibited good antigenicity and protective immunity in mice [7].…”