The roles of V protein in blocking IFN expression and signaling have been reported previously. Our results confirm these findings through the study of a recombinant virus derived from a clinical isolate (genotype G) ablating the expression the V protein in the context of in vitro infection. We have found that the lack of V protein expression also led to the induction of a higher level of IL-6, a proinflammatory cytokine, suggesting that the V protein plays a role in suppressing IL-6 expression. We speculate that the lack of V protein expression in infected cells resulted in the attenuation of this strain in an animal model, suggesting that the V protein plays an essential role in viral virulence. It is possible that the inability of rMuVIowa/US/06ΔV to counter IFN action limited the replication of the virus in vivo, and the induction of a higher level of IL-6 by rMuVIowa/US/06ΔV attracted monocytes to clear the infection quickly, resulting in the attenuation of rMuVIowa/US/06ΔV in vivo.
Genetically, the closest virus to MuV is parainfluenza virus 5 (PIV5). The V proteins of MuV and PIV5 share many identical functions, including blocking IFN expression through MDA5, blocking IFN signaling through degradation of STAT1, and inhibiting expression of IL-6 in virus-infected cells. Interestingly, a recombinant PIV5 lacking the entire V protein has never been obtained in tissue culture cells, suggesting that the V protein of PIV5 plays a more critical role in virus replication (5
) than the V protein does for MuV. The viability of rMuVIowa/US/06
ΔV suggests that the role of MuVIowa/US/06
V protein in virus replication is dispensable, at least in tissue culture cells.
The most widely used mumps vaccine, Jeryl Lynn (JL) vaccine, was introduced in the United States in 1967 and shortly thereafter globally. The effectiveness of the vaccine has been estimated at 80% for one dose and 90% for two doses (4
). Vaccine effectiveness has been found to be similar for other strains (4
), but unlike the JL strain, nearly all other strains have been associated with aseptic meningitis (31
) and thus are not widely used. Recent outbreaks of mumps in vaccinated populations have highlighted the relatively lower effectiveness of mumps vaccines as compared to other vaccines, such as measles, and have stimulated interest in development of new mumps vaccines. For instance, In the 2009-2010 mumps outbreak in the state of New York and the state of New Jersey in the United States, 88% of the patients had one dose of mumps vaccine and 75% of the patients had two doses of vaccine (21
). All live attenuated MuV vaccines are obtained through serial passages of virus in embryonic eggs and/or cells. This is a time-consuming process and a strategy with a poor record of generating safe vaccines with the exception of JL vaccine.
In this work, using a reverse genetics system for MuV based on a clinical isolate from a recent outbreak, we have generated a recombinant virus incapable of producing the V protein (rMuVIowa/US/06ΔV). We have found that this virus grew to titers similar to those for wild-type virus in Vero cells, a cell line that is used for vaccine production, as well as in other cell types. Most importantly, the virus exhibited low neurotoxicity in rats, suggesting that it may be a possible vaccine candidate. Further examination of the immunogenicity and safety of the virus in vivo will be needed to determine the suitability of this virus as a vaccine candidate.
The V/P gene of MuV encodes three proteins, V, I, and P, through a process of “RNA editing,” in which nontemplate G residues are inserted into mRNA during transcription at a specific site to generate mRNAs that can be translated into three different ORFs (33
). The V protein is translated from the “unedited” copy of mRNA, P from the mRNA with two G residue insertions, and the I protein from the mRNA with one or four G residue insertions. All of these proteins have identical N termini of 155 amino acid residues. The P protein has 391 amino acid residues and plays an essential role in viral RNA synthesis. The I protein has 170 amino acid residues, and its function is unclear. It is possible that the I mRNA is a by-product of RNA editing and it may not have any significant functions. The strategy we used to generate rMuVIowa/US/06
ΔV also eliminated expression of the I protein. Because the mRNA for I counts only for less than 2% of total V, I, and P transcripts, and its sequence is very similar to the N termini of V and P (I has about 170 amino acid residues and 155 of them are identical to the N termini of V and P) (26
), we attribute the phenotypes of rMuVIowa/US/06
ΔV to the lack of V protein. However, we cannot exclude the possibility that the I protein is also responsible.
All changes except the one in the L gene occurred in the gene junction between NP and P/P genes to generate viable infectious MuV incapable of expressing the V protein. It is interesting that a mutation in the L gene was able to allow the rescue of a virus lacking the V protein. While we cannot exclude the possibility that the mutation in the L gene occurred fortuitously and is immaterial to the function of L, we speculate that the particular mutation may play a role in modulating interactions between NP-P and L, considering that all other viruses rescued had mutations to modulate the levels of NP and P. Further analysis of the virus may lead to a better understanding of the function of L.