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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

Amount of viral proteins associated with the lipid raft in cells infected with the non-raft HA virus. The MDCK cells were infected with the WT, HAstop, and HA 529–531A virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NP, and M1 in each fraction were detected by western blotting and the band intensities of viral proteins in each fraction were measured. The graph indicates average values of the ratio of the membrane-fraction band intensities (fractions 2 and 3) to the total band intensity (fractions from 1 to 10) with the standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 by Student’s t-tests.
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viruses-08-00249-f004: Amount of viral proteins associated with the lipid raft in cells infected with the non-raft HA virus. The MDCK cells were infected with the WT, HAstop, and HA 529–531A virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NP, and M1 in each fraction were detected by western blotting and the band intensities of viral proteins in each fraction were measured. The graph indicates average values of the ratio of the membrane-fraction band intensities (fractions 2 and 3) to the total band intensity (fractions from 1 to 10) with the standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 by Student’s t-tests.

Mentions: To analyze whether the lipid raft association of HA is required for the association of vRNP with the lipid raft, we generated a recombinant virus that expresses non-raft-associated HA (HA 529–531A virus, residues 529–531 in H1 subtype numbering, corresponding to residues 530–532 in H3 subtype numbering) [16]. We performed membrane flotation assays using HA 529–531A virus-infected cell extracts. The distributions of HA, NP (as a vRNP representative), and M1 in the fractions were analyzed by western blotting. The level of HA 529–531A in the detergent-insoluble membrane fractions was much lower than that of WT fractions (Figure 4). Similarly, the level of NP in the lipid raft fractions in cells infected with HA 529–531A virus was very low, similar to that of the HAstop virus. In contrast, the amount of M1 in the fractions was comparable for cells infected with WT, HAstop, and HA 529–531A viruses. These results suggest that the lipid raft association of HA is necessary for vRNP recruitment to the lipid raft.


Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft
Amount of viral proteins associated with the lipid raft in cells infected with the non-raft HA virus. The MDCK cells were infected with the WT, HAstop, and HA 529–531A virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NP, and M1 in each fraction were detected by western blotting and the band intensities of viral proteins in each fraction were measured. The graph indicates average values of the ratio of the membrane-fraction band intensities (fractions 2 and 3) to the total band intensity (fractions from 1 to 10) with the standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 by Student’s t-tests.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035963&req=5

viruses-08-00249-f004: Amount of viral proteins associated with the lipid raft in cells infected with the non-raft HA virus. The MDCK cells were infected with the WT, HAstop, and HA 529–531A virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NP, and M1 in each fraction were detected by western blotting and the band intensities of viral proteins in each fraction were measured. The graph indicates average values of the ratio of the membrane-fraction band intensities (fractions 2 and 3) to the total band intensity (fractions from 1 to 10) with the standard deviation of three independent experiments. * p < 0.05, ** p < 0.01 by Student’s t-tests.
Mentions: To analyze whether the lipid raft association of HA is required for the association of vRNP with the lipid raft, we generated a recombinant virus that expresses non-raft-associated HA (HA 529–531A virus, residues 529–531 in H1 subtype numbering, corresponding to residues 530–532 in H3 subtype numbering) [16]. We performed membrane flotation assays using HA 529–531A virus-infected cell extracts. The distributions of HA, NP (as a vRNP representative), and M1 in the fractions were analyzed by western blotting. The level of HA 529–531A in the detergent-insoluble membrane fractions was much lower than that of WT fractions (Figure 4). Similarly, the level of NP in the lipid raft fractions in cells infected with HA 529–531A virus was very low, similar to that of the HAstop virus. In contrast, the amount of M1 in the fractions was comparable for cells infected with WT, HAstop, and HA 529–531A viruses. These results suggest that the lipid raft association of HA is necessary for vRNP recruitment to 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