The NB protein of influenza B virus is not necessary for virus replication in vitro

Abstract

The NB protein of influenza B virus is thought to function as an ion channel and therefore would be expected to have an essential function in viral replication. Because direct evidence for its absolute requirement in the viral life cycle is lacking, we generated NB knockout viruses by reverse genetics and tested their growth properties both in vitro and in vivo. Mutants not expressing NB replicated as efficiently as the wild-type virus in cell culture, whereas in mice they showed restricted growth compared with findings for the wild-type virus. Thus, the NB protein is not essential for influenza B virus replication in cell culture but promotes efficient growth in mice.

FIG. 1.

Schematic diagram of mutations introduced into the NA segment. Mutations are shown in bold (-, deletion; ∗, insertion). The numbers shown are nucleotide positions.

FIG. 2.

Analysis of the expression of NB protein. (A) Detection of NB protein in infected MDCK cells by immunofluorescence assay. B/LeeRG, B/LeeRG-infected; WSN, A/WSN/33-infected; Control, uninfected; #1, #2, and #3, BLeeNBstop#1-, BLeeNBstop#2-, and BLeeNBstop#3-infected cells, respectively. (B) Detection of NB protein in virus-infected MDCK cells by immunoprecipitation assays. Radiolabeled NB proteins were immunoprecipitated with a rabbit anti-NB peptide serum and analyzed on 4 to 20%-gradient polyacrylamide gels as described in Materials and Methods. Lane #1, BLeeNBstop#1-infected; lane #2, BLeeNBstop#2-infected; lane #3, BLeeNBstop#3-infected; lane C, uninfected cell lysate. Molecular mass markers (kDa) are indicated.

FIG. 2.

Analysis of the expression of NB protein. (A) Detection of NB protein in infected MDCK cells by immunofluorescence assay. B/LeeRG, B/LeeRG-infected; WSN, A/WSN/33-infected; Control, uninfected; #1, #2, and #3, BLeeNBstop#1-, BLeeNBstop#2-, and BLeeNBstop#3-infected cells, respectively. (B) Detection of NB protein in virus-infected MDCK cells by immunoprecipitation assays. Radiolabeled NB proteins were immunoprecipitated with a rabbit anti-NB peptide serum and analyzed on 4 to 20%-gradient polyacrylamide gels as described in Materials and Methods. Lane #1, BLeeNBstop#1-infected; lane #2, BLeeNBstop#2-infected; lane #3, BLeeNBstop#3-infected; lane C, uninfected cell lysate. Molecular mass markers (kDa) are indicated.

FIG. 3.

Growth curves for B/LeeRG and mutant viruses. MDCK cells were infected with virus (0.001 PFU) and incubated at 37°C. At the indicated times after infection, titers of virus were determined in the supernatant. The values are means (± standard deviation) of three determinations.