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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 packaged vRNPs in virions from cells infected with the HAstop virus. (A) Detection of each viral RNA (vRNA) segments in HAstop virions. The virions were concentrated from the supernatant of infected cells at 24 hpi. The vRNA was extracted from the virions and primer extension assay was performed; (B–D) Relative amount of vRNA in HAstop virions. The vRNA was extracted from the same samples used for western blotting in Figure 1A and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in the HAstop virus relative to that in the WT virus was calculated (B). The amounts of each vRNA segment were normalized by the amount of M1 (C) and NP (D) in the virions shown in Figure 1A. The graphs indicate average values with standard deviations of three independent experiments. ** p < 0.01 and * p < 0.05 by Student’s t-tests; (E) Detection of each vRNA segment in cells infected with HAstop virus. Total RNA was extracted from the infected cells at 10 hpi and primer extension assay was performed using [32P]-labeled primer specific to each vRNA segment or to HA and M mRNA; (F) Relative amount of vRNA in cells infected with HAstop virus. The vRNA was extracted from the same samples used for western blotting in Figure 1B and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in cells infected with HAstop virus relative to that in cells infected with WT virus was calculated. The graph indicates average values with standard deviations of three independent experiments.
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viruses-08-00249-f002: Decrease in packaged vRNPs in virions from cells infected with the HAstop virus. (A) Detection of each viral RNA (vRNA) segments in HAstop virions. The virions were concentrated from the supernatant of infected cells at 24 hpi. The vRNA was extracted from the virions and primer extension assay was performed; (B–D) Relative amount of vRNA in HAstop virions. The vRNA was extracted from the same samples used for western blotting in Figure 1A and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in the HAstop virus relative to that in the WT virus was calculated (B). The amounts of each vRNA segment were normalized by the amount of M1 (C) and NP (D) in the virions shown in Figure 1A. The graphs indicate average values with standard deviations of three independent experiments. ** p < 0.01 and * p < 0.05 by Student’s t-tests; (E) Detection of each vRNA segment in cells infected with HAstop virus. Total RNA was extracted from the infected cells at 10 hpi and primer extension assay was performed using [32P]-labeled primer specific to each vRNA segment or to HA and M mRNA; (F) Relative amount of vRNA in cells infected with HAstop virus. The vRNA was extracted from the same samples used for western blotting in Figure 1B and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in cells infected with HAstop virus relative to that in cells infected with WT virus was calculated. The graph indicates average values with standard deviations of three independent experiments.

Mentions: To analyze vRNA packaging in the HAstop virus, vRNA was purified from the virion fractions and subjected to a primer extension assay. All eight vRNA segments were detected at once using 32P-labeled specific primer mixtures. Non-specific bands were detected in total RNA from mock-infected cells, but these bands were reduced or not detected using total RNA from infected cells because binding affinity of labeled primers to vRNAs is higher than that to cellular RNAs. All eight vRNA segments were detected in HAstop virus (Figure 2A). The amount of vRNA in the supernatant from cells infected with HAstop virus was determined by qPCR. The amount of vRNA in the supernatant was approximately 80% lower than that from cells infected with WT virus (Figure 2B). To examine the packaging efficiency of vRNP into a virion, the amount of vRNA in the virions was normalized by the amount of M1 protein in the virions (the same sample used in Figure 1A). It is speculated that the amount of M1 in a WT and HAstop virion is comparable because the shape of both virions are comparable (Figure 1D). The amount of packaged vRNA was approximately 50% lower in HAstop virions than in WT virions (Figure 2C). The observed reductions were similar for the eight vRNA segments. To confirm that the vRNA quantification accurately reflects vRNA packaging efficiency, the band intensity was normalized by the amount of NP (Figure 2D). If the quantification of viral proteins and vRNA in virions is appropriate, the calculated ratio of vRNA to NP should be 1. The ratio of vRNA to NP was near 1 for the most of the segments (Figure 2D). To confirm the amount of vRNPs and viral mRNA in cells infected with HAstop virus, total RNA was purified from infected cells and subjected to primer extension and qPCR assay. The amounts of M mRNA, HA mRNA, and vRNAs were comparable in WT and HAstop virus infected cells (Figure 2E,F). These results suggest that the packaging efficiency of vRNPs is decreased in cells infected with the HAstop virus.


Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft
Decrease in packaged vRNPs in virions from cells infected with the HAstop virus. (A) Detection of each viral RNA (vRNA) segments in HAstop virions. The virions were concentrated from the supernatant of infected cells at 24 hpi. The vRNA was extracted from the virions and primer extension assay was performed; (B–D) Relative amount of vRNA in HAstop virions. The vRNA was extracted from the same samples used for western blotting in Figure 1A and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in the HAstop virus relative to that in the WT virus was calculated (B). The amounts of each vRNA segment were normalized by the amount of M1 (C) and NP (D) in the virions shown in Figure 1A. The graphs indicate average values with standard deviations of three independent experiments. ** p < 0.01 and * p < 0.05 by Student’s t-tests; (E) Detection of each vRNA segment in cells infected with HAstop virus. Total RNA was extracted from the infected cells at 10 hpi and primer extension assay was performed using [32P]-labeled primer specific to each vRNA segment or to HA and M mRNA; (F) Relative amount of vRNA in cells infected with HAstop virus. The vRNA was extracted from the same samples used for western blotting in Figure 1B and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in cells infected with HAstop virus relative to that in cells infected with WT virus was calculated. The graph indicates average values with standard deviations of three independent experiments.
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viruses-08-00249-f002: Decrease in packaged vRNPs in virions from cells infected with the HAstop virus. (A) Detection of each viral RNA (vRNA) segments in HAstop virions. The virions were concentrated from the supernatant of infected cells at 24 hpi. The vRNA was extracted from the virions and primer extension assay was performed; (B–D) Relative amount of vRNA in HAstop virions. The vRNA was extracted from the same samples used for western blotting in Figure 1A and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in the HAstop virus relative to that in the WT virus was calculated (B). The amounts of each vRNA segment were normalized by the amount of M1 (C) and NP (D) in the virions shown in Figure 1A. The graphs indicate average values with standard deviations of three independent experiments. ** p < 0.01 and * p < 0.05 by Student’s t-tests; (E) Detection of each vRNA segment in cells infected with HAstop virus. Total RNA was extracted from the infected cells at 10 hpi and primer extension assay was performed using [32P]-labeled primer specific to each vRNA segment or to HA and M mRNA; (F) Relative amount of vRNA in cells infected with HAstop virus. The vRNA was extracted from the same samples used for western blotting in Figure 1B and the amount of each vRNA segment was determined by qPCR. The amount of each vRNA segment in cells infected with HAstop virus relative to that in cells infected with WT virus was calculated. The graph indicates average values with standard deviations of three independent experiments.
Mentions: To analyze vRNA packaging in the HAstop virus, vRNA was purified from the virion fractions and subjected to a primer extension assay. All eight vRNA segments were detected at once using 32P-labeled specific primer mixtures. Non-specific bands were detected in total RNA from mock-infected cells, but these bands were reduced or not detected using total RNA from infected cells because binding affinity of labeled primers to vRNAs is higher than that to cellular RNAs. All eight vRNA segments were detected in HAstop virus (Figure 2A). The amount of vRNA in the supernatant from cells infected with HAstop virus was determined by qPCR. The amount of vRNA in the supernatant was approximately 80% lower than that from cells infected with WT virus (Figure 2B). To examine the packaging efficiency of vRNP into a virion, the amount of vRNA in the virions was normalized by the amount of M1 protein in the virions (the same sample used in Figure 1A). It is speculated that the amount of M1 in a WT and HAstop virion is comparable because the shape of both virions are comparable (Figure 1D). The amount of packaged vRNA was approximately 50% lower in HAstop virions than in WT virions (Figure 2C). The observed reductions were similar for the eight vRNA segments. To confirm that the vRNA quantification accurately reflects vRNA packaging efficiency, the band intensity was normalized by the amount of NP (Figure 2D). If the quantification of viral proteins and vRNA in virions is appropriate, the calculated ratio of vRNA to NP should be 1. The ratio of vRNA to NP was near 1 for the most of the segments (Figure 2D). To confirm the amount of vRNPs and viral mRNA in cells infected with HAstop virus, total RNA was purified from infected cells and subjected to primer extension and qPCR assay. The amounts of M mRNA, HA mRNA, and vRNAs were comparable in WT and HAstop virus infected cells (Figure 2E,F). These results suggest that the packaging efficiency of vRNPs is decreased in cells infected with the HAstop virus.

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