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Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft

View Article: PubMed Central - PubMed

ABSTRACT

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

No MeSH data available.


Related in: MedlinePlus

Decrease in virion production from cells infected with HAstop virus. (A) Detection of viral proteins in virions from cells infected with the HAstop virus. Wild-type (WT) and HAstop viruses were infected to MDCK cells at a multiplicity of infection (MOI) of 1. The virions were concentrated from the supernatant of infected cells at 24 hpi, and nucleoprotein (NP), matrix protein 1 (M1), and hemagglutinin (HA) were detected by western blotting. The ratio of the loaded sample volumes was 1:2:4:8:16. The band intensities of NP and M1 in the WT virus and HAstop virus were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments. ** p < 0.01 by Student’s t-tests; (B) The detection of viral proteins in cells infected with the HAstop virus. The infected cell lysate was prepared at 10 hpi and NP,M1, and HA were detected by western blotting. The ratio of loaded sample volumes was 1:2:4:8:16. The band intensities of viral proteins were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments; (C) The localization of viral ribonucleoprotein complex (vRNP) in cells infected with the HAstop virus. MDCK-F11-WT cells were infected with WT or HAstop virus at an MOI of 1. At 12 hpi, NP and FLAG-Rab11 were visualized. The Pearson correlation coefficient (PCC) between pixel intensity of NP staining and FLAG-Rab11 staining was calculated. The distribution of PCC from 10 independent cells in the same well infected with each WT and HAstop virus is indicated in the box plot; (D) Defective progeny virion production from cells infected with the HAstop virus. The MDCK cells infected with the WT virus or HAstop virus were fixed at 12 hpi and the cell surfaces were observed by thin-section electron microscopy. Scale bars: 500 nm.
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viruses-08-00249-f001: Decrease in virion production from cells infected with HAstop virus. (A) Detection of viral proteins in virions from cells infected with the HAstop virus. Wild-type (WT) and HAstop viruses were infected to MDCK cells at a multiplicity of infection (MOI) of 1. The virions were concentrated from the supernatant of infected cells at 24 hpi, and nucleoprotein (NP), matrix protein 1 (M1), and hemagglutinin (HA) were detected by western blotting. The ratio of the loaded sample volumes was 1:2:4:8:16. The band intensities of NP and M1 in the WT virus and HAstop virus were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments. ** p < 0.01 by Student’s t-tests; (B) The detection of viral proteins in cells infected with the HAstop virus. The infected cell lysate was prepared at 10 hpi and NP,M1, and HA were detected by western blotting. The ratio of loaded sample volumes was 1:2:4:8:16. The band intensities of viral proteins were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments; (C) The localization of viral ribonucleoprotein complex (vRNP) in cells infected with the HAstop virus. MDCK-F11-WT cells were infected with WT or HAstop virus at an MOI of 1. At 12 hpi, NP and FLAG-Rab11 were visualized. The Pearson correlation coefficient (PCC) between pixel intensity of NP staining and FLAG-Rab11 staining was calculated. The distribution of PCC from 10 independent cells in the same well infected with each WT and HAstop virus is indicated in the box plot; (D) Defective progeny virion production from cells infected with the HAstop virus. The MDCK cells infected with the WT virus or HAstop virus were fixed at 12 hpi and the cell surfaces were observed by thin-section electron microscopy. Scale bars: 500 nm.

Mentions: To dissect the requirements for HA for progeny virion production, we generated recombinant viruses that did not express HA by introducing premature nonsense codons (HAstop virus). We made recombinant wild type and HAstop viruses that have a mutation in NA to prevent trypsin-independent cleavage of HA. This wild type virus is not capable of multi-cycle infection without trypsin (Supplemental Figure S1A). HAstop virus was infected to MDCK-HA cells and the titer of the supernatant at 48 hr post-infection (hpi) was 2.1 × 106 PFU/mL. To analyze the production of progeny HAstop virions, the WT and HAstop viruses were used to infect MDCK cells at a multiplicity of infection (MOI) of 1 and the cell culture supernatants were collected at 24 hpi. About 70% of cells were infected (1 MOI) and the ratio of cells infected with WT and HAstop virus was comparable (Supplemental Figure S1B). The virions in the supernatants were concentrated by ultracentrifugation and analyzed by western blotting. The amounts of NP and M1 were 74.4% ± 8.73% and 58.0% ± 3.63% less in the HAstop virions than in the WT virions, respectively (Figure 1A). The expression levels of the NP and M1 proteins in cells infected with the HAstop virus were comparable to those in WT-infected cells (Figure 1B). These results suggest that HA is required for efficient progeny virion production.


Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft
Decrease in virion production from cells infected with HAstop virus. (A) Detection of viral proteins in virions from cells infected with the HAstop virus. Wild-type (WT) and HAstop viruses were infected to MDCK cells at a multiplicity of infection (MOI) of 1. The virions were concentrated from the supernatant of infected cells at 24 hpi, and nucleoprotein (NP), matrix protein 1 (M1), and hemagglutinin (HA) were detected by western blotting. The ratio of the loaded sample volumes was 1:2:4:8:16. The band intensities of NP and M1 in the WT virus and HAstop virus were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments. ** p < 0.01 by Student’s t-tests; (B) The detection of viral proteins in cells infected with the HAstop virus. The infected cell lysate was prepared at 10 hpi and NP,M1, and HA were detected by western blotting. The ratio of loaded sample volumes was 1:2:4:8:16. The band intensities of viral proteins were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments; (C) The localization of viral ribonucleoprotein complex (vRNP) in cells infected with the HAstop virus. MDCK-F11-WT cells were infected with WT or HAstop virus at an MOI of 1. At 12 hpi, NP and FLAG-Rab11 were visualized. The Pearson correlation coefficient (PCC) between pixel intensity of NP staining and FLAG-Rab11 staining was calculated. The distribution of PCC from 10 independent cells in the same well infected with each WT and HAstop virus is indicated in the box plot; (D) Defective progeny virion production from cells infected with the HAstop virus. The MDCK cells infected with the WT virus or HAstop virus were fixed at 12 hpi and the cell surfaces were observed by thin-section electron microscopy. Scale bars: 500 nm.
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viruses-08-00249-f001: Decrease in virion production from cells infected with HAstop virus. (A) Detection of viral proteins in virions from cells infected with the HAstop virus. Wild-type (WT) and HAstop viruses were infected to MDCK cells at a multiplicity of infection (MOI) of 1. The virions were concentrated from the supernatant of infected cells at 24 hpi, and nucleoprotein (NP), matrix protein 1 (M1), and hemagglutinin (HA) were detected by western blotting. The ratio of the loaded sample volumes was 1:2:4:8:16. The band intensities of NP and M1 in the WT virus and HAstop virus were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments. ** p < 0.01 by Student’s t-tests; (B) The detection of viral proteins in cells infected with the HAstop virus. The infected cell lysate was prepared at 10 hpi and NP,M1, and HA were detected by western blotting. The ratio of loaded sample volumes was 1:2:4:8:16. The band intensities of viral proteins were measured and the relative amount of each viral protein was semi-quantified from the standard curves. The graph indicates average values and standard deviations of three independent experiments; (C) The localization of viral ribonucleoprotein complex (vRNP) in cells infected with the HAstop virus. MDCK-F11-WT cells were infected with WT or HAstop virus at an MOI of 1. At 12 hpi, NP and FLAG-Rab11 were visualized. The Pearson correlation coefficient (PCC) between pixel intensity of NP staining and FLAG-Rab11 staining was calculated. The distribution of PCC from 10 independent cells in the same well infected with each WT and HAstop virus is indicated in the box plot; (D) Defective progeny virion production from cells infected with the HAstop virus. The MDCK cells infected with the WT virus or HAstop virus were fixed at 12 hpi and the cell surfaces were observed by thin-section electron microscopy. Scale bars: 500 nm.
Mentions: To dissect the requirements for HA for progeny virion production, we generated recombinant viruses that did not express HA by introducing premature nonsense codons (HAstop virus). We made recombinant wild type and HAstop viruses that have a mutation in NA to prevent trypsin-independent cleavage of HA. This wild type virus is not capable of multi-cycle infection without trypsin (Supplemental Figure S1A). HAstop virus was infected to MDCK-HA cells and the titer of the supernatant at 48 hr post-infection (hpi) was 2.1 × 106 PFU/mL. To analyze the production of progeny HAstop virions, the WT and HAstop viruses were used to infect MDCK cells at a multiplicity of infection (MOI) of 1 and the cell culture supernatants were collected at 24 hpi. About 70% of cells were infected (1 MOI) and the ratio of cells infected with WT and HAstop virus was comparable (Supplemental Figure S1B). The virions in the supernatants were concentrated by ultracentrifugation and analyzed by western blotting. The amounts of NP and M1 were 74.4% ± 8.73% and 58.0% ± 3.63% less in the HAstop virions than in the WT virions, respectively (Figure 1A). The expression levels of the NP and M1 proteins in cells infected with the HAstop virus were comparable to those in WT-infected cells (Figure 1B). These results suggest that HA is required for efficient progeny virion production.

View Article: PubMed Central - PubMed

ABSTRACT

The influenza glycoproteins, hemagglutinin (HA) and neuraminidase (NA), which are associated with the lipid raft, have the potential to initiate virion budding. However, the role of these viral proteins in infectious virion assembly is still unclear. In addition, it is not known how the viral ribonucleoprotein complex (vRNP) is tethered to the budding site. Here, we show that HA is necessary for the efficient progeny virion production and vRNP packaging in the virion. We also found that the level of HA does not affect the bundling of the eight vRNP segments, despite reduced virion production. Detergent solubilization and a subsequent membrane flotation analysis indicated that the accumulation of nucleoprotein, viral polymerases, NA, and matrix protein 1 (M1) in the lipid raft fraction was delayed without HA. Based on our results, we inferred that HA plays a role in the accumulation of viral components, including bundled vRNPs, at the lipid raft.

No MeSH data available.


Related in: MedlinePlus