Insight into Poliovirus Genome Replication and Encapsidation Obtained from Studies of 3B-3C Cleavage Site Mutants

Abstract

A poliovirus (PV) mutant (termed GG), which is incapable of producing 3AB, VPg, and 3CD proteins due to a defective cleavage site between the 3B and 3C proteins, replicated, producing 3BC-linked RNA rather than the VPg-linked RNA produced by the wild type (WT). GG PV RNA is quasi-infectious. The yield of infectious GG PV relative to replicated RNA is reduced by almost 5 logs relative to that of WT PV. Proteolytic activity required for polyprotein processing is normal for the GG mutant. 3BC-linked RNA can be encapsidated as efficiently as VPg-linked RNA. However, a step after genome replication but preceding virus assembly that is dependent on 3CD and/or 3AB proteins limits production of infectious GG PV. This step may involve release of replicated genomes from replication complexes. A pseudorevertant (termed EG) partially restored cleavage at the 3B-3C cleavage site. The reduced rate of formation of 3AB and 3CD caused corresponding reductions in the observed rate of genome replication and infectious virus production by EG PV without impacting the final yield of replicated RNA or infectious virus relative to that of WT PV. Using EG PV, we showed that genome replication and encapsidation were distinct steps in the multiplication cycle. Ectopic expression of 3CD protein reversed the genome replication phenotype without alleviating the infectious-virus production phenotype. This is the first report of a trans-complementable function for 3CD for any picornavirus. This observation supports an interaction between 3CD protein and viral and/or host factors that is critical for genome replication, perhaps formation of replication complexes.

A Polymerase Mechanism-based Strategy for Viral Attenuation and Vaccine Development

“…Virus Isolation, Titer, and One-step Growth Curves-Virus was harvested and titered by plaque assay as described previously (21). For TCID 50 , virus was serially diluted in medium, and each dilution was added to 10 wells of a 96-well plate previously seeded with 5 ϫ 10 3 HeLa cells.…”

“…20), and then the fragment between BglII and ApaI was cloned into either the subgenomic replicon, pRLucRA, or viral cDNA, pMovRA. Plasmids were linearized and used in RNA transcription reactions as described previously (21).…”

“…Subgenomic Replicon Assays-Subgenomic replicon assays were performed as described previously using a luciferase-expressing, subgenomic replicon (21).…”

Poliovirus Infection Transiently Increases COPII Vesicle Budding

“…Briefly, PVR cDNA (a gift from Mattias Gromeier) was cloned into the retroviral vector pLP-RevTRE (pLP-RevTRE-PVR). pLP-RevTRE-PVR-and pRevTet-off-containing retrovirus stocks were made as described previously (25). NRK cells coinfected with pLP-RevTRE-PVR-and pRevTet-off-containing retroviruses were cultured in growth medium with 300 g/ml of hygromycin B and G418.…”

“…The resultant PVR-NRK cells were routinely cultured in growth medium supplemented with 100 g/ml of hygromycin B and G418. PV type I Mahoney and eGFP-PV (a gift from Raul Andino [8]) stocks were produced in HeLa cells as described previously (25). Briefly, HeLa cells were electroporated with RNA transcribed from linearized plasmid DNA and incubated at 37°C for 2 to 3 days until cytopathic effect became apparent.…”