The Hemagglutinin Protein of Highly Pathogenic H5N1 Influenza Viruses Overcomes an Early Block in the Replication Cycle To Promote Productive Replication in Macrophages
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Fig 1H5N1 influenza viruses productively infect RAW cells. RAW264.7 cells were infected in triplicate with a panel of influenza viruses representing all 16 HA subtypes (A [the viruses are listed in Table 1]) or with the indicated viruses (B) at an MOI of 0.01. At the indicated times postinfection, the medium was collected, and virus titers were determined by TCID50 analysis in triplicate. (C) RAW cells were infected with the indicated influenza viruses (MOI = 3). The medium was collected at 24 and 48 hpi, and virus titers were determined by TCID50 analysis. (D) Primary murine alveolar macrophages were infected with the indicated viruses (MOI = 3), cell supernatants were collected at 24 and 48 h postinfection, and virus titers were determined as described above (limit of detection = 102 TCID50/ml). The data are representative of duplicate experiments. Error bars represent the mean TCID50 value ± the standard deviation. Titers determined at 1 hpi were below the limit of detection for the H5 viruses and ranged from below the limit of detection to 102 TCID50/ml for H1 and H3 viruses.
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Fig 2NP staining decreases in macrophages infected with CA/09. RAW264.7 macrophages (A) or MDCK cells (B) seeded onto coverslips were incubated with CA/09 or HK/483 (MOI = 5) on ice for 1 h. At time zero, warm infection medium was added, and the cells were incubated at 37°C. At the indicated time points, the cells were fixed and processed for immunofluorescent staining to detect viral NP. The slides were viewed by confocal microscopy as described in Materials and Methods. The mean percent NP+ macrophages was quantified from three independent images and is indicated in the lower right corner of each panel (A). Nuclei were visualized by DAPI staining, and representative images from two independent experiments are shown.
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Fig 3RNA synthesis is inhibited in CA/09-infected macrophages. A549 (A) or RAW (B) cells were incubated with HK/483 or CA/09 at 4°C for 1 h. At time zero, warm medium was added, and the cells were incubated at 37°C. At the indicated time points, total RNA was isolated, and the levels of vRNA, mRNA, and cRNA were determined in triplicate using primers that amplify the NP gene as described in Materials and Methods. The RNA level in HK/483-infected cells at 12 hpi was set as a value of 1.0, and all other samples are presented relative to that amount. Viral RNA levels were normalized to GAPDH and to the amount of RNA present at 30 min postinfection. The data are representative of two independent experiments. The primer sequences are presented in Table 2.
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Fig 4Viral protein synthesis is blocked during CA/09 infection of macrophages. RAW264.7 (left panels) or MDCK (right panels) cells were infected with HK/483 or CA/09, and the cells were lysed at the indicated times postinfection. Proteins were separated by SDS-PAGE under reducing conditions, transferred to nitrocellulose, and probed with anti-NS1 (top panels) or anti-actin (bottom panels) by Western blotting.
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Fig 5The HA gene mediates replication of influenza viruses in macrophages. (A) RAW264.7 cells were infected with the parental reverse genetics (rg) viruses or with rgCA/09 expressing individual genes from HK/483 (MOI = 0.1). Cell culture supernatants were collected at 24 hpi, and virus titers were determined by TCID50 analysis. (B) RAW264.7 cells were infected with the indicated viruses, and the virus titers determined as described for panel A. (C) RAW264.7 cells were infected with the indicated reverse genetics viruses as described above. The medium was collected 24 hpi, and virus titers were determined by TCID50 analysis on MDCK cells. Error bars represent the mean value ± the SD.
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