An A14U Substitution in the 3' Noncoding Region of the M Segment of Viral RNA Supports Replication of Influenza Virus with an NS1 Deletion by Modulating Alternative Splicing of M Segment mRNAs

Abstract

The NS1 protein of influenza virus has multiple functions and is a determinant of virulence. Influenza viruses with NS1 deletions (DelNS1 influenza viruses) are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3' noncoding region of the M segment of viral RNA (vRNA) which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus showed that the lack of NS1 has no apparent effect on expression of other viral proteins, with the exception of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3, and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus-infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts and provide strategic information important to construction of vaccine strains with NS1 deletions.

Importance: Nonstructural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus replication, but mechanistic details are not clear. The NS1 protein is a virulence determinant which inhibits both innate and adaptive immunity and live attenuated viruses with NS1 deletions show promise as effective vaccines. However, deletion of NS1 causes severe attenuation of virus replication during infection, impeding functional studies and vaccine development. We characterized a replication-competent DelNS1 virus which carries an A14U substitution in the 3' noncoding region of the vRNA M segment. We found that M-A14U mutation supports virus replication through modulation of alternative splicing of mRNAs transcribed from the M segment. Our findings give insight into the role of NS1 in influenza virus replication and provide an approach for constructing replication-competent strains with NS1 deletions for use in functional and vaccine studies.

Copyright © 2015, Zheng et al.

Figures

FIG 1

Construction and establishment of stabilized WSN-DelNS1 virus. (A) Schematic illustration of the NS segment transcripts and the NS mutant with an NS1 deletion (DelNS1). The DelNS1 plasmid was constructed by deleting the intron region ranging from nt 57 to 528 in the NS segment. NCR, noncoding region. (B) Confirmation of NS1 deletion in rescued DelNS1 viruses. Viral RNA was extracted from P1 virus, and the NS segment was amplified by RT-PCR and analyzed using agarose gel electrophoresis. (C) Plaque sizes of DelNS1 and wild-type A/WSN/33 viruses. (D) Titer (PFU/ml) of DelNS1 virus after each passage. (E) Sequence analysis of the DelNS1 virus genome revealed an A-to-U substitution at nucleotide position 14 in the M segment noncoding region. The noncoding region is marked by a black line, and the A14U mutation is indicated with an arrowhead. (F) Comparison of rescue efficiency for WSN-DelNS1 viruses containing M-WT and M-A14U, M-A14U-CM15, M-A14C, and M-A14G substitutions. NR, not rescued. (G) Rescue efficiency of PR8-DelNS1 viruses containing M-WT or M-A14U. The DelNS1 viruses were rescued with the indicated M-WT or M mutant plasmids in mixed HEK293T and MDCK cell cultures and then titrated by plaque assay. Values plotted are means (± standard deviations [SD]) (n = 3) and are representative of data from at least 5 independent experiments.

FIG 2

Growth kinetics of the M-A14U-DelNS1 virus in MDCK and Vero cells. (A and B) Column-purified reverse genetic WSN-WT, WSN-DelNS1-M-A14U, and WSN-DelNS1-M-WT viruses were used to infect MDCK cells (A) or Vero cells (B) at a multiplicity of infection (MOI) of 0.1. Supernatants were collected at the indicated time points and virus titrated by plaque assay. (C) Column-purified reverse genetic PR8-WT and PR8-DelNS1-M-A14U viruses were used to infect MDCK cells (left panel) and Vero cells (right panel) at an MOI of 0.1. Supernatants were collected at 24 h postinfection and titrated by plaque assay. The values (mean ± SD; n = 3) plotted are representative data from at least 3 independent experiments.

FIG 3

Loss of IFN-β suppression activity in WSN-DelNS1 viruses. (A) HEK293T cells were transfected with an IFN-β reporter plasmid 24 h prior to infection with either WSN-WT or WSN-DelNS1-M-A14U at an MOI of 1 or with the positive control, Sendai virus (SeV), at 50 HA units. After 24 h, cells were harvested and cell lysates prepared for estimation of luciferase activity. The luciferase assays were performed in triplicate, and values were normalized to the Renilla luciferase control. (B) IFN-β and viral NP mRNA levels were quantified by qRT-PCR after MDCK cells were infected with WSN-WT or WSN-DelNS1-M-A14U at an MOI of 0.1 and cultured for 16 h. Values were normalized against canine actin. (C) Suppression of IRF3 dimerization in HEK293T cells infected with WSN-WT, WSN-DelNS1-M-A14U, or Sendai virus was analyzed by native gel electrophoresis. (D) Activity in suppressing IFN-β expression was compared in MDCK cells infected with WSN-DelNS1-M-WT or WSN-DelNS1-M-A14U virus. Similar to the experiment described for panel B, MDCK cells were infected with the indicated viruses at an MOI of 0.1, and at 16 h postinfection, IFN-β and NP mRNA levels were quantified by qPCR. Values were normalized to canine actin. (E) Groups of six BALB/c mice, aged 6 to 8 weeks, were intranasally inoculated with 5 × 104 PFU of WSN-WT or WSN-DelNS1-M-A14U mutant virus, and body weight was monitored daily for 14 days postinfection. (F) Replication efficiency of viruses in lung tissues of infected mice. Groups of three mice were infected with 104 PFU of WSN-WT, WSN-DelNS1-M-WT, or WSN-DelNS1-M-A14U mutant viruses and then euthanized at 72 h postinfection, with lung tissues from each mouse being collected and homogenized for virus titration by plaque assay using MDCK cells. Statistical significance was analyzed by one-way analysis of variance (ANOVA) or Student's t test (**, P < 0.01). The bars plotted show means ± SD (n = 3), and the results represent at least three independent experiments.

FIG 4

Effect of M-A14U mutation on M1 and M2 protein expression. (A) MDCK cells were infected with WSN-WT or WSN-DelNS1-M-A14U virus at an MOI of 2. Cells were harvested at the indicated time points and cell lysates analyzed by Western blotting with specific antibodies, as described in Materials and Methods. (B) MDCK cells were infected with WSN-DelNS1-M-WT or WSN-DelNS1-M-A14U virus at an MOI of 0.1. At 16 h postinfection, cell lysates were collected for Western blotting with specific antibodies. (C) MDCK cells were infected with WSN-WT or WSN-M-A14U virus at an MOI of 5. Cell lysates were prepared at the indicated time points for Western blotting with specific antibodies. β-Tubulin was included as a loading control. All of the results are representative of three independent experiments.

FIG 5

Effect of M-A14U substitution on alternative splicing of M transcripts. (A) Schematic illustration of the M segment transcripts. The sequence of the 3′ noncoding region of the vRNA M segment is shown in red, with the mRNA3 splicing donor (SD) site highlighted in yellow. Splicing consensus sequences for the donor site are indicated in green above the noncoding region sequence (M, A or C; R, A or G). (B) Analysis of levels of different M mRNAs in virus-infected cells. MDCK cells were infected with the indicated viruses at an MOI of 0.1. Total RNA was isolated at 16 h postinfection. The mRNA levels for M1, M2, mRNA3, and M4 were determined by quantitative RT-PCR, as described in Materials and Methods. (C) HEK293T cells were transfected with pHW2000-WSN-M-WT or pHW2000-WSN-M-A14U plasmid with or without cotransfection of pCX-WSN-NS1 for coexpression of NS1. At 48 h posttransfection, total RNAs were isolated. After DNase treatment, the mRNA levels were determined by quantitative RT-PCR. (D) MDCK cells were infected with PR8-WT, PR8-M-A14U, or PR8-DelNS1-M-A14U virus at an MOI of 0.1. At 16 h postinfection, total RNAs were isolated and mRNA levels measured by qPCR. M2/M1 mRNA and mRNA3/M1 mRNA ratios are shown. All the results plotted indicate means ± SD (n = 3) and are representative of three independent experiments.

FIG 6

Effect of M-A14U substitution on M2/M1 mRNA ratio. (A) MDCK cells were infected with WSN-WT, WSN-M-A14U, WSN-M-A14G, or WSN-M-G12C-CM13 virus at an MOI of 5. Total RNAs were extracted at the indicated time points and levels of mRNA3 determined by quantitative RT-PCR. (B) Analysis of growth kinetics of WSN-WT, WSN-M-A14U, WSN-M-A14G, and WSN-M-G12C-CM13 viruses. MDCK cells were infected with these viruses at an MOI of 0.001. Supernatants were collected at the indicated time points and titrated by plaque assay. (C to E) MDCK cells were infected with WSN-WT, WSN-M-A14U, WSN-DelNS1-M-WT, or WSN-DelNS1-M-A14U virus at an MOI of 0.1. At 16 h after infection, total RNAs were isolated, and the M2/M1, mRNA3/M1, and M4/M1 mRNA ratios were determined by quantitative RT-PCR. (F) Effect of mRNA3 on virus replication. HEK293T cells were transfected with pCX-WSN-mRNA3 or a control vector 24 h prior to infection with WSN-DelNS1-M-A14U virus at an MOI of 0.1. Supernatants were collected at the indicated time points and virus titrated by plaque assay. All of the bars and points plotted indicate means ± SD (n = 3) from three independent experiments. **, P = 0.0013 by Student's t test.

FIG 7

Regulation of M mRNA splicing host machinery by viral NS1 and polymerase proteins. (A) HEK293T cells were transfected with increasing amounts of pCX-WSN-NS1. At 24 h after transfection, cells were infected with WSN-DelNS1-M-WT, WSN-DelNS1-M-A14U, or WSN-WT virus at an MOI of 0.5. Total RNAs were isolated at 8 h postinfection and M1 mRNA, M2 mRNA, and mRNA3 levels analyzed by quantitative RT-PCR. (B) HEK293T cells were transfected with WT and various mutants of the M segment cloned into pHW2000 plasmids, which express M vRNA, with or without the pCX-WSN-NS1 plasmid. Cell lysates were prepared at 48 h posttransfection for Western blotting with specific antibodies. β-Tubulin was included as a loading control. (C) Effect of A14U substitution on M vRNA replication. MDCK cells were infected with WSN-WT, WSN-M-A14U, or WSN-M-A14G virus at an MOI of 5. At 4 and 10 h postinfection, total RNAs were isolated for qRT-PCR analysis, and relative amounts of vRNA were determined as described above. (D) The A14U substitution enhances M vRNA replication. MDCK cells were infected with WSN-DelNS1-M-WT or WSN-DelNS1-M-A14U virus at an MOI of 0.1. Total RNAs were isolated at 16 h after infection. M and NP vRNA levels were determined by quantitative RT-PCR, as described above. All bars plotted show means ± SD (n = 3). The results represent three independent experiments. **, P < 0.01; ***, P = 0.0002 (by Student's t test).