trans-encapsidation of a poliovirus replicon by different picornavirus capsid proteins

Abstract

A trans-encapsidation assay was established to study the specificity of picornavirus RNA encapsidation. A poliovirus replicon with the luciferase gene replacing the capsid protein-coding region was coexpressed in transfected HeLa cells with capsid proteins from homologous or heterologous virus. Successful trans-encapsidation resulted in assembly and production of virions whose replication, upon subsequent infection of HeLa cells, was accompanied by expression of luciferase activity. The amount of luciferase activity was proportional to the amount of trans-encapsidated virus produced from the cotransfection. When poliovirus capsid proteins were supplied in trans, >2 x 10(6) infectious particles/ml were produced. When coxsackievirus B3, human rhinovirus 14, mengovirus, or hepatitis A virus (HAV) capsid proteins were supplied in trans, all but HAV showed some encapsidation of the replicon. The overall encapsidation efficiency of the replicon RNA by heterologous capsid proteins was significantly lower than when poliovirus capsid was used. trans-encapsidated particles could be completely neutralized with specific antisera against each of the donor virus capsids. The results indicate that encapsidation is regulated by specific viral nucleic acid and protein sequences.

FIG. 1

Schematic representation of poliovirus cDNA constructs used in this study. pT7-PV1 encodes the complete poliovirus genome, pRLuc31 encodes a poliovirus replicon in which the luciferase gene is substituted for the viral capsid coding sequence, pMonoLuc5d contains the complete poliovirus cDNA with the luciferase gene inserted upstream of the capsid coding sequences, and pMonoLuc5d contains a large out-of-frame deletion from nt 6054 to 6516 in the P3 coding region.

FIG. 2

trans-encapsidation assay. HeLa cell monolayers were cotransfected with pRLuc31 and capsid donor plasmid in the presence of recombinant vaccinia virus v-TF7-3, expressing T7 RNA polymerase. Progeny viruses recovered from the transfection are labelled passage 1 (Pass 1) virus; progeny viruses recovered from subsequent infection of HeLa cells with passage 1 virus are labelled passage 2 (Pass 2) virus.

FIG. 3

trans-encapsidation of poliovirus replicon pRLuc31 RNA with different sources of poliovirus capsid proteins. (A) Luciferase activity from trans-encapsidated poliovirus replicon with different capsid donors. Plasmid pRLuc31 was cotransfected with the three indicated poliovirus capsid protein donors or control plasmid pUC19. Progeny viruses (passage 1 [Pass 1]) were used to infect HeLa cells, and luciferase production was measured at 8 h postinfection. (B) Luciferase activity from second passage (Pass 2) virus. Virus recovered from the experiment shown in panel A was used to infect fresh HeLa cell cultures, and luciferase activity was monitored after 8 h.

FIG. 4

trans-encapsidation of the poliovirus replicon by the capsid protein from a nonreplicating construct, pMonoLuc5d. trans-encapsidation was carried out as described in the text, and luciferase activity (RLU) was monitored after 8 h of infection with passage 1 virus.

FIG. 5

Kinetics of postinfection luciferase expression after infection with trans-encapsidated virus. trans-encapsidation of the poliovirus replicon pRLuc31 was carried out as described in the text, and passage 1 virus trans-encapsidated by capsid protein expressed from pMonoLuc5 was used to infect HeLa cells. Samples were harvested at the indicated times for luciferase assay, with results expressed as RLU.

FIG. 6

Estimated infectivity of trans-encapsidated viruses. Supernatants from transfected cells containing passage 1 viruses were used to make 10-fold serial dilutions in DMEM, and 0.1 ml was used to infect HeLa cell monolayers in 24-well plates. Luciferase activity was measured 8 h postinfection.

FIG. 7

trans-encapsidation of a poliovirus replicon by different donor picornavirus coat proteins. Plasmid cDNA constructs encoding different picornavirus capsid proteins were used to cotransfect HeLa cells, and trans-encapsidation was monitored by luciferase activity after infection of HeLa cells with progeny virus.

FIG. 8

HAV VP1 immunoblot analysis following transfection with HAV cDNA. Lane 1, transfection of HeLa cells by pT7-HAV1 in the presence of recombinant vaccinia virus expressing T7 RNA polymerase; lane 2, FRhK4 cells infected for 3 weeks with HAV; lane 3, HeLa cells transfected with pUC19 plasmid as a negative control. Eighty micrograms of total protein was loaded in each gel lane. Numbers on the left are molecular masses (in kilodaltons) of marker proteins.

FIG. 9

Serum neutralization of trans-encapsidated viruses. Viruses resulting from transfection and trans-encapsidation with different picornavirus capsid donors were incubated with corresponding specific antisera or control sera diluted 1:200 in DMEM at 37°C for 20 min before inoculation of HeLa cell monolayers. Each trans-encapsidated virus was also tested against poliovirus-specific antiserum. Since different antisera were used for each trans-encapsidated virus, values are comparable only within a set of an individual virus, not between different virus sets. PV, poliovirus; CBV3, coxsackievirus B3; G. pig, guinea pig; MV, mengovirus; NC, negative control with uninfected HeLa cell lysate.