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. 2014 Sep;88(18):10778-91.
doi: 10.1128/JVI.01440-14. Epub 2014 Jul 9.

Influenza A and B virus intertypic reassortment through compatible viral packaging signals

Steven F Baker  1 , Aitor Nogales  1 , Courtney Finch  2 , Kevin M Tuffy  1 , William Domm  1 , Daniel R Perez  2 , David J Topham  3 , Luis Martínez-Sobrido  4
Affiliations

Influenza A and B virus intertypic reassortment through compatible viral packaging signals

Steven F Baker et al. J Virol. 2014 Sep.

Abstract

Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions.

Importance: Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or gene exchange between influenza A and B viruses is not well understood. Nucleotides comprising the coding termini of each influenza A virus gene segment are required for specific segment incorporation during budding. Whether influenza B virus shares a similar selective packaging strategy or if packaging signals prevent intertypic reassortment remains unknown. Here, we provide evidence suggesting a similar mechanism of influenza B virus genome packaging. Furthermore, by appending influenza A virus packaging signals onto influenza B virus segments, we rescued recombinant influenza A/B viruses that could reassort in vitro with another influenza A virus. These findings suggest that the divergent evolution of packaging signals aids with the speciation of influenza A and B viruses and is in part responsible for the lack of intertypic viral reassortment.

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Figures

FIG 1
FIG 1
Characterization of influenza B virus HA-expressing MDCK cells. (A) HA protein detection by indirect immunofluorescence. HA-expressing MDCK cells were fixed and stained with influenza A virus HA MAbs 31C2 (pH1N1) and PY102 (PR8) or anti-influenza B virus HA PAb NR-3165 and counterstained with DAPI to visualize the nuclei. Parental MDCK cells were used as a negative control. Representative images obtained with a ×20 objective (actual magnification, ×200) are shown. Bars, 20 μm. (B) Western blotting results. Parental and HA-expressing MDCK whole-cell lysates were stained with MAbs 31C2 and PY102 and the PAb NR-3165. MAb against actin was used as a loading control. (C) Flow cytometry. Parental and HA-expressing MDCK stable cell lines infected with pH1N1/E3, PR8, B/Lee, B/Vic, and B/Yam were detected with antibodies 31C2, PY102, and NR-3165, an MAb generated against B/Yam (15B6), or an anti-B/Lee PAb (V-63). The percentage of positively stained cells is indicated (y axis).
FIG 2
FIG 2
Mouse-adapted PR8-sciIAV is complemented by HA proteins from prototypic influenza B virus lineages. (A and B) Multicycle growth analysis of PR8-sciIAV in parental MDCK and MDCK-HA cells. Confluent monolayers of parental and HA-expressing MDCK cells were infected (in triplicate) with PR8-sciIAV at a low MOI (0.001). (A) At various times postinfection, GFP was visualized by fluorescence microscopy (with a ×10 objective; magnification of actual representative images, ×100). Bars, 40 μm. (B) TCSs were collected for titration in MDCK-HA cells. Data represent the means ± SDs of the results determined for triplicate assays. Dashed black line, limit of detection (200 FFU/ml). (C) Plaque morphology of PR8-sciIAV in parental MDCK and MDCK-HA cells. Parental and HA-expressing MDCK cell lines were infected with 50 PFU of PR8-sciIAV, and at 3 days postinfection, monolayers were stained with crystal violet.
FIG 3
FIG 3
Pandemic 2009 pH1N1-sciIAV is complemented by influenza B virus HA-expressing cell lines. (A and B) Multicycle growth analysis of pH1N1-sciIAV in parental MDCK and MDCK-HA cells. Triplicate confluent monolayers of parental and HA-expressing MDCK cells were infected with pH1N1-sciIAV (MOI, 0.001). (A) At various times postinfection, GFP was visualized by fluorescence microscopy (with a ×10 objective; magnification of actual representative images, ×100). Bars, 40 μm. (B) TCSs were collected for titration in MDCK-HA cells. Data represent the means ± SDs of the results determined for triplicate assays. Dashed black line, limit of detection (200 FFU/ml). (C) Plaque morphology of pH1N1-sciIAV. Parental and HA-expressing MDCK cell lines were infected with 50 PFU of pH1N1-sciIAV, and at 3 days postinfection, monolayers were stained with crystal violet.
FIG 4
FIG 4
Promoter and polymerase compatibility of type A and B influenza viruses. (A) Schematic representation of influenza A and B virus MG constructs. GFP or FFluc MG plasmids driven by the influenza A virus (white box) or influenza B virus (black) NCRs are indicated. (B and C) MG assays. Human 293T cells were cotransfected with 500 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral replication and transcription (3P and NP), together with MG vRNA-like expression plasmids encoding GFP (B) or FFluc (C) flanked by influenza A or B virus NCRs (IAV MG and IBV MG, respectively) plus polymerase II-driven Renilla luciferase (C) plasmids as a transfection control. At 2 days posttransfection, cells were prepared for flow cytometry to determine the percentage and MFI of GFP+ cells (B) or lysed for luminescence evaluation (C). FFluc activity was normalized to that of Renilla luciferase. Data represent the means ± SDs of the results determined for triplicate assays. (D) Influenza B virus NP inhibits IAV MG activity. An IAV or IBV MG with the corresponding 3P plus NP was transfected as described above in the absence of NP (−) or in the presence of 250, 500, or 1,000 ng of competing protein expression plasmids encoding heterotypic influenza virus NP or LCMV NP. Normalized reporter expression provided (in percent) is relative to MG activity in the absence of competing NP. Statistical analysis was performed using Student's two-tailed t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 5
FIG 5
Type-specific packaging signals dictate segment incorporation into influenza A or B virions. (A) Schematic representation of IAV and IBV vRNA segment 4 constructs. Lines, NCRs; boxes with lines, packaging signals flanking the GFP ORF. (B) Rescue transfection and infection diagram. Ambisense pDZ expression plasmids containing seven different segments of influenza A or B virus were cotransfected together with the GFP vRNA pPolI expression plasmid (A) and the HA protein expression plasmid pCAGGS into 293T cells. At 48 h posttransfection, TCSs were collected and used to infect fresh monolayers of MDCK HA-expressing cells to evaluate virus rescue. (C) Fluorescence microscopy of rescue transfections. GFP expression from transfected human 293T cells was evaluated under a fluorescence microscope at 72 h posttransfection. (D) Virus rescues. TCSs from rescue transfections (C) were clarified and used to infect fresh MDCK-HA cells. At 48 h postinfection, infected cells were visualized for the presence of GFP under a fluorescence microscope. Representative images obtained with a ×10 objective are shown (actual magnification shown, ×100). Bars, 40 μm. (E) Virus rescues. Attempts to rescue intertypic sciIV were performed five times in triplicate. The percent rescue efficiency is indicated.
FIG 6
FIG 6
Manipulation of influenza A virus packaging signals to generate chimeric glycoprotein influenza A/B viruses. (A) Schematic representation of viral segments encoding HA and NA. Influenza type A virus vRNA (white boxes) with packaging signals (boxes with lines) and influenza type B virus vRNA (black boxes) are indicated. The BHA or BNA chimeric vRNA constructs include the influenza A virus NCR (lines) and packaging signals (boxes with lines), together with the complete ORFs of the influenza B virus HA and NA glycoproteins, respectively. The length of each vRNA segment (in nucleotides) is indicated on the right. (B) Antigenic characterization of recombinant PR8 viruses containing BHA, BNA, or both BHA and BNA (BHANA). MDCK cells were infected with the indicated viruses (MOI, 0.1) for 16 h, and IAV and IBV type-specific HA, NA, or NP antibodies were used to detect protein expression (green). Cell nuclei were stained with DAPI (blue). Anti-influenza A virus antibodies were PY102 for HA, NR-4540 for NA, and HT103 for NP. Anti-influenza B virus antibodies were NR-3165 for HA, NR-3114 for NA, and B017 for NP. Representative images obtained with a ×20 objective are shown (actual magnification shown, ×200). Bars, 20 μm. (C) Multicycle growth analysis of chimeric type A/B viruses. Confluent monolayers of MDCK cells (in triplicate) were infected with the indicated viruses (MOI, 0.001), and at the indicated times postinfection, TCSs were collected for titration in MDCK cells. Data represent the means ± SDs of the results determined for triplicate assays. Dashed black line, limit of detection (200 FFU/ml). (D) Plaque morphology of chimeric influenza A/B viruses in MDCK cells. Monolayers of MDCK cells were infected with the PR8 and B/Yam viruses and chimeric glycoprotein influenza A/B viruses (PR8 HA, PR8 NA, and PR8 HANA). At 3 days postinfection, monolayers were immunostained with influenza A virus (PR8, PR8 BHA, PR8 BNA, and PR8 BHANA) or influenza B virus (B/Yam) NP monoclonal antibodies HT103 and B017, respectively.
FIG 7
FIG 7
Pathogenicity and immunogenicity of influenza A/B viruses with recombinant glycoproteins. (A) Morbidity caused by influenza A/B viruses with chimeric glycoproteins. Female 6- to 8-week-old C57BL/6 mice were inoculated intranasally with 105 PFU of influenza B virus glycoprotein-containing viruses (B/Yam, PR8 BHA, PR8 BNA, or PR8 BHANA) or 10 PFU of PR8 (n = 4). For 2 weeks postinfection, weight loss was monitored daily. (B) Lethality of PR8 BNA. Tenfold decreasing doses or PR8 BNA were used to infect 6- to 8-week-old female C57BL/6 mice intranasally (n = 4). The MLD50 was determined using the method of Reed and Muench (54). (C and D) Humoral immune responses. Total anti-influenza virus IgG antibodies in serum collected at 2 weeks postinfection with the indicated viruses were determined by ELISA (n = 4). Plates were coated with lysates from PR8-infected (C) or B/Yam-infected (D) MDCK cells. Data represent the means ± SEMs of the results determined for individual mice. OD, optical density; Ag, antigen.
FIG 8
FIG 8
Chimeric influenza A/B virus can reassort with influenza A virus. (A) Experimental design. MDCK cells were coinfected with pH1N1 and PR8 BHANA (MOI, 3) for 16 h at 33°C. Viruses from TCSs were plaque purified, amplified once on MDCK cells, and used to infect cells to determine the phenotype (ph.) by IFA. Relative segment sizes (in descending order, PB2, PB1, PA, HA, NP, NA, M, and NS) and colors (black, pH1N1; blue, PR8; red, B/Yam) represent identity. (B) Phenotypes of three selected reassortant viruses. MDCK cells were infected with parental or plaque-purified reassortant viruses (MOI, 0.1; 24 h; 33°C) and phenotypically characterized by IFA. The antibodies 29E3 and NR-3165 were used to detect IAV and IBV HAs, respectively; 10C9 and NR-3114 were used to detect IAV and IBV NAs, respectively; 1A7 was used to detect PR8-specific NS1; and NR-4545 was used to detect PR8-specific NP. Representative images obtained at ×20 magnification are shown (actual magnification, ×200). Bars, 20 μm.
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