Addition of exogenous polypeptides on the mammalian reovirus outer capsid using reverse genetics

Abstract

Addition of exogenous peptide sequences on viral capsids is a powerful approach to study the process of viral infection or to retarget viruses toward defined cell types. Until recently, it was not possible to manipulate the genome of mammalian reovirus and this was an obstacle to the addition of exogenous sequence tags onto the capsid of a replicating virus. This obstacle has now been overcome by the advent of the plasmid-based reverse genetics system. In the present study, reverse genetics was used to introduce different exogenous peptides, up to 40 amino acids long, at the carboxyl-terminal end of the σ1 outer capsid protein. The tagged viruses obtained were infectious, produce plaques of similar size, and could be easily propagated at hight titers. However, attempts to introduce a 750 nucleotides-long sequence failed, even when it was added after the stop codon, suggesting a possible size limitation at the nucleic acid level.

Amino acids substitutions in σ1 and μ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly

“…Cells were grown in MEM for suspension culture with 10% fetal bovine serum, proline (20μg/ml) and β-mercaptoethanol (50μM) and antibodies were recovered, as previously described (Brochu-Lafontaine and Lemay, 2012). The polyclonal antiserum directed against the carboxyl-terminal head domain of σ1 was produced originally in the laboratory of Dr. Terence Dermody (Vanderbilt University, Tennessee) and was a generous gift from Dr. Earl Brown (University of Ottawa).…”

“…Infected cells were recovered by scraping in a small volume of medium and processed for immunoblotting, as previously described (Brochu-Lafontaine and Lemay, 2012). Images were obtained using either autoradiography on Kodak BioMax Light films or on a Typhoon Trio™ imager (GE Healthcare Life Sciences).…”

“…To obtain the virus mutant harboring the amino acids substitutions of VeroAV in the defined background of the reverse genetics system, a fragment of the gene encompassing all mutations was obtained by RT-PCR amplification on the viral VeroAV genome, essentially as previously described (Brochu-Lafontaine and Lemay, 2012;Jabre et al, 2013). PCR fragments were recovered and subcloned to replace the corresponding fragment in the M2 or S1 reverse genetics plasmid.…”

“…Recovery of infectious reovirus stocks by transfection of the baby hamster kidney (BHK) cell line constitutively expressing the T7 RNA polymerase (Buchholz et al, 1999) was done essentially as previously described (Brochu-Lafontaine and Lemay, 2012).…”

Characterization of a Replicating Mammalian Orthoreovirus with Tetracysteine-Tagged μNS for Live-Cell Visualization of Viral Factories

“…Both replicating and nonreplicating recombinant viruses have also been recovered with fluorescent proteins iLOV and UnaG independently expressed downstream of the N-terminal half of viral spike protein 1 ( 1-N), as well as a fusion of 1-N with UnaG, which could infect cells but was unable to replicate in the absence of wild-type MRV (19,20). In addition, recombinant reoviruses in which small protein-coding sequences (6His, hemagglutinin [HA] tag, and 3HA tag) were added to the 1 protein have been recovered (21). However, there is currently no other published evidence of a replicating, recombinant virus in which NS or other viral proteins have been successfully tagged with fluorescent or other tags.…”