Replication of the ΔNS1-B virus in embryonated chicken eggs. Six-, eight-, and eleven-day-old embryonated chicken eggs were inoculated with 100 infectious units of either influenza B/Lee/40, recombinant wild-type (rec. WT), or ΔNS1B virus and then incubated for 72 h at 33°C. Virus titers were determined as described in Materials and Methods. The indicated values represent the average of at least three independent experiments. Error bars indicate the standard deviations.

Lack of NS1 expression in ΔNS1-B virus-infected cells. (A) Metabolic labeling of proteins in virus-infected MDCK cells. Cells were mock treated (lane “M”) or infected with the recombinant wild-type or ΔNS1-B virus at an MOI of 10 and incubated at 33°C for various time points. At 3, 5, or 7 h postinfection, the cultures were metabolically labeled with [³⁵S]methionine for 1 h, and cell extracts were prepared at 4, 6, and 8 h postinfection, followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The positions of molecular mass markers and of the viral NP and NS1-B proteins are indicated on the left and right sides, respectively. (B) Immunoblot analysis of virus-infected cells. MDCK cells were either mock treated or infected for 8 h with influenza B/Lee/40 or ΔNS1-B virus. Lysates were prepared and analyzed by immunoblotting with NP- and NS1-specific antibodies.

Generation of recombinant influenza B/Lee wild-type virus and NS1 deletion mutant (ΔNS1-B). (A) Structure of the NS segments of the wild-type and ΔNS1-B viruses. The plasmid pHW-Lee-NS-XhoI expresses the NS wild-type segment that encodes both the NS1 and, from a spliced transcript, the NEP/NS2 proteins. This construct was used with the other seven plasmids encoding the residual viral gene segments for recovery of the recombinant B/Lee wild-type virus in transfected 293T cells. In the plasmid pHW-Lee-ΔNS1-B, the coding region for NS1 was deleted. (B) RT-PCR analysis of viral NS segments. After the extraction of viral RNAs from the natural influenza B/Lee/40, the recombinant wild-type and ΔNS1-B viruses the viral NS segments were reverse transcribed (lanes “+”) and amplified by PCR. The resulting products were separated on a 2% agarose gel and stained with ethidium bromide. DNA size marker fragments were run in parallel on the very left lane. In control reactions, the RT step or an RNA template was omitted (lanes “RT:−” and “−”). The NS segment of the recombinant wild-type (rec. WT) was distinguished from the B/Lee/40 segment by digestion at the introduced XhoI site. Deletion of the NS1 coding region reduced the length of the NS segment from 1,096 to 441 nucleotides. The positions of the 1.0- and 0.5b-kb size marker fragments are indicated to the left.

The NS1-B protein antagonizes viral activation of the IFN-β gene. (A) IFN-β gene expression is strongly upregulated in ΔNS1-B virus-infected lung epithelial cells. Human A549 cells were infected with the recombinant B/Lee wild-type virus or the ΔNS1-B mutant at an MOI of 1 or were mock infected. Total RNA was extracted from the cells 10 h postinfection, and equal amounts were analyzed by RT-PCR with DNA oligonucleotide primers specific for IFN-β mRNA (upper panel), the viral NP segment (middle panel), or human β-actin transcripts (lower panel). The amplified products were separated by agarose gel electrophoresis and stained with ethidium bromide. In control reactions, no RNA template was added (lanes H₂O). (B) Influenza A and B viruses lacking the NS1 proteins are strong activators of the IFN-β promoter. MDCK-C3 cells that contain a stable firefly luciferase reporter gene under the control of the IFN-β promoter were infected with influenza B virus strain Md/59 or Lee/40, the recombinant Lee wild-type virus (rec. WT), or ΔNS1-B virus at an MOI of 1. For a comparison, the influenza A/PR/8/34 strain and the isogenic A/delNS1 mutant virus were used in parallel infections. Luciferase activity was determined 8 h postinfection and is presented as the fold induction compared to mock-infected cells after normalization of extracts for equal amounts of protein. The graph shows average values of a typical experiment conducted in duplicate that was repeated three times. Error bars indicate the standard deviations. (C) Normal MDCK cells were transfected with the IFN-β promoter luciferase reporter plasmid p125-Luc and expression plasmids for the NS1-B or NS1-A protein or empty vector (pcDNA). In all assays, the plasmid pRL-TK-Luc encoding a Renilla luciferase under the control of a constitutive promoter was cotransfected and used as an internal control to normalize the results. One day posttransfection cells were stimulated by infection with the ΔNS1-B or the A/delNS1A virus at an MOI of 1 or were mock treated. Luciferase activities in cell extracts were determined 8 h postinfection. The value for a virus-induced increase in vector-transfected cells was arbitrarily set to 100% and compared to the induction observed in NS1-expressing cells.