Recombinant respiratory syncytial virus F protein expression is hindered by inefficient nuclear export and mRNA processing

Abstract

Studies of the fusion activity of respiratory syncytial virus (RSV) F protein are significantly hindered by low recombinant expression levels. While infection produces F protein levels detectable by western blot, recombinant expression produces undetectable to low levels of F protein. Identifying the obstacles that hinder recombinant F protein expression may lead to improved expression and facilitate the study of F protein function. We hypothesized that nuclear localization and/or inefficient RNA polymerase II-mediated transcription contribute to poor recombinant F protein expression. This study shows a combination of stalled nuclear export, premature polyadenylation, and low mRNA abundance all contribute to low recombinant F protein expression levels. In addition, this study provides an expression optimization strategy that results in greater F protein expression levels than observed by codon-optimization of the F protein gene, which will be useful for future studies of F protein function.

A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis

“…Sequence optimization of RSV cDNA for human codon bias and other optimal features enhances cDNA expression in mammalian cells (Huang et al, 2010). Plasmids encoding the wild-type (non-optimized) N, P, M2-1, and L genes have been used previously to drive minigenome expression (Grosfeld, Hill, and Collins, 1995).…”

Novel Vaccine Regimen Elicits Strong Airway Immune Responses and Control of Respiratory Syncytial Virus in Nonhuman Primates

“…The numbers of cells recovered from BAL were generally within 1 ϫ 10 6 to 6 ϫ 10 6 per ml. A total of 10 6 cells were stimulated with a mixture of two stimulation peptide pools from the hRSV-F peptide library (pools 1 to 4 and pools 5 to 7, respectively) with a final concentration of 2 g/ml/ peptide or with SEB, respectively, or with a peptide pool from HCV (16,17,34) or with anti-CD28 as negative controls. Intracellular staining was performed as described previously (21)(22)(23)29) and analyzed by flow cytometry (BD LSRII flow cytometer).…”

“…Initial interest in DNA and other gene-based vaccines against hRSV using the fusion protein of hRSV (hRSV-F) as vaccine antigen was tempered by poor immunogenicity and efficacy in NHPs (13)(14)(15). It subsequently turned out that expression of hRSV-F cDNA is impaired by premature polyadenylation and inefficient nuclear export (16,17). However, high levels of the hRSV-F protein, as well as its secreted form, could be expressed from a codon-optimized hRSV-F sequence.…”

Targeting RSV with Vaccines and Small Molecule Drugs

“…A replication-deficient adenovirus expressing aa 155–524 of the RSV F protein, preceded by a signal sequence to enable secretion, administered to the nasal mucosa of mice, protected them against RSV challenge without inducing enhanced pathology [204]. This RSV F protein sequence was codon optimized to enable expression from the nucleus, a requirement for the F protein [205]. The success of this vaccine in mice is striking because the F protein fragment used excludes F2, pep27, the fusion peptide, and the transmembrane and cytoplasmic domains [204] and therefore much of this F protein fragment would not be expected to be folded properly.…”