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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

Decreased lipid raft associated NP in cells infected with the HAstop virus and non-raft HA virus. (A) Amount of viral proteins associated with the lipid raft in cells infected with the HAstop virus. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cells were sonicated, treated with 0.5% Triton X-100 at 0 °C or 37 °C, and subjected to membrane flotation assays. HA, NA, PB2, PB1, PA, NP, and M1 in the fractions were detected by western blotting; (B) Amount of viral membrane proteins associated with the lipid raft. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, and M2 in the fractions were detected by western blotting; (C) Amount of viral proteins associated with lipid raft at early and late phase of infection. The MDCK cells were infected with WT and HAstop virus, and were collected at 7 and 13 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, NP, and M1 in the fractions were detected by western blotting; (D) Relative amount of viral proteins in lipid raft fraction at 7, 10, and 13 hpi. The MDCK cells were infected with WT and HAstop virus, and the membrane flotation assays were performed using infected cell lysate prepared at 7, 10, and 13 hpi. NA, 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) of two independent experiments.
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viruses-08-00249-f003: Decreased lipid raft associated NP in cells infected with the HAstop virus and non-raft HA virus. (A) Amount of viral proteins associated with the lipid raft in cells infected with the HAstop virus. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cells were sonicated, treated with 0.5% Triton X-100 at 0 °C or 37 °C, and subjected to membrane flotation assays. HA, NA, PB2, PB1, PA, NP, and M1 in the fractions were detected by western blotting; (B) Amount of viral membrane proteins associated with the lipid raft. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, and M2 in the fractions were detected by western blotting; (C) Amount of viral proteins associated with lipid raft at early and late phase of infection. The MDCK cells were infected with WT and HAstop virus, and were collected at 7 and 13 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, NP, and M1 in the fractions were detected by western blotting; (D) Relative amount of viral proteins in lipid raft fraction at 7, 10, and 13 hpi. The MDCK cells were infected with WT and HAstop virus, and the membrane flotation assays were performed using infected cell lysate prepared at 7, 10, and 13 hpi. NA, 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) of two independent experiments.

Mentions: Since progeny virion budding occurs at membrane lipid rafts, it is possible that NA, M1, and progeny vRNPs did not sufficiently accumulate on the lipid raft in the absence of the HA protein. To test this hypothesis, we analyzed the association of viral polymerases, HA, NA, NP, and M1 with the lipid raft at 10 hpi by membrane flotation assays. Lipid rafts are insoluble in nonionic detergents such as TX-100, at 0 °C, but are soluble at 37 °C. Fractions of HA, NA, viral polymerases, NP, and M1 were observed in TX-100-insoluble membrane fractions in WT virus-infected cells at 0 °C (Figure 3A, fractions 2 and 3) but they became soluble at 37 °C (Figure 3A). These results confirmed the association of HA, NA, NP, and M1 with lipid rafts in WT virus-infected cells. Viral polymerases and NP were also recovered in the detergent-insoluble membrane fractions, indicating that vRNPs were associated with the lipid raft (Figure 3A). In contrast, for cells infected with the HAstop virus, a trace amount of vRNP components (viral polymerases and NP) was recovered in the detergent-insoluble membrane fractions. The amount of M1 in the fractions was comparable to that in cells infected with the WT virus. To analyze the association of viral membrane proteins, HA, NA, and M2, with the lipid raft in HAstop virus-infected cells, the membrane flotation assays were performed using WT and HAstop virus-infected cell lysate (Figure 3B). NA and M2 were still recovered in the detergent-insoluble membrane fractions, but the amount of NA and M2 in the fractions was clearly reduced compared to that in cells infected with the WT virus (Figure 3B). These results suggest that the accumulation of some viral components in the lipid raft was impaired in cells infected with the HAstop virus. Next, we analyzed the association of viral proteins with lipid raft at early (7 hpi) and late phase (13 hpi) of infection. The distributions of HA, NA, NP, and M1 in the fractions were analyzed by western blotting. The membrane-fraction band intensities and the total band intensity of NA, M1, and NP were measured and the ratio of membrane-fraction intensity to total intensity was calculated. At 7 and 10 hpi, the level of NA, NP, and M1 in the detergent-insoluble membrane fractions was about 60%–80% lower than that of WT fractions. In contrast, the level of NA, NP, and M1 in the fractions was comparable to that of WT fractions at 13 hpi (Figure 3C,D). These results suggest that the accumulation of viral components in the lipid raft was delayed in cells infected with the HAstop virus and the total number of viral components in lipid raft became comparable between cells infected with WT and HAstop virus at 13 hpi.


Influenza A Virus Hemagglutinin is Required for the Assembly of Viral Components Including Bundled vRNPs at the Lipid Raft
Decreased lipid raft associated NP in cells infected with the HAstop virus and non-raft HA virus. (A) Amount of viral proteins associated with the lipid raft in cells infected with the HAstop virus. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cells were sonicated, treated with 0.5% Triton X-100 at 0 °C or 37 °C, and subjected to membrane flotation assays. HA, NA, PB2, PB1, PA, NP, and M1 in the fractions were detected by western blotting; (B) Amount of viral membrane proteins associated with the lipid raft. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, and M2 in the fractions were detected by western blotting; (C) Amount of viral proteins associated with lipid raft at early and late phase of infection. The MDCK cells were infected with WT and HAstop virus, and were collected at 7 and 13 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, NP, and M1 in the fractions were detected by western blotting; (D) Relative amount of viral proteins in lipid raft fraction at 7, 10, and 13 hpi. The MDCK cells were infected with WT and HAstop virus, and the membrane flotation assays were performed using infected cell lysate prepared at 7, 10, and 13 hpi. NA, 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) of two independent experiments.
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viruses-08-00249-f003: Decreased lipid raft associated NP in cells infected with the HAstop virus and non-raft HA virus. (A) Amount of viral proteins associated with the lipid raft in cells infected with the HAstop virus. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cells were sonicated, treated with 0.5% Triton X-100 at 0 °C or 37 °C, and subjected to membrane flotation assays. HA, NA, PB2, PB1, PA, NP, and M1 in the fractions were detected by western blotting; (B) Amount of viral membrane proteins associated with the lipid raft. The MDCK cells were infected with WT and HAstop virus, and were collected at 10 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, and M2 in the fractions were detected by western blotting; (C) Amount of viral proteins associated with lipid raft at early and late phase of infection. The MDCK cells were infected with WT and HAstop virus, and were collected at 7 and 13 hpi. The cell lysate was prepared, and subjected to membrane flotation assays. HA, NA, NP, and M1 in the fractions were detected by western blotting; (D) Relative amount of viral proteins in lipid raft fraction at 7, 10, and 13 hpi. The MDCK cells were infected with WT and HAstop virus, and the membrane flotation assays were performed using infected cell lysate prepared at 7, 10, and 13 hpi. NA, 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) of two independent experiments.
Mentions: Since progeny virion budding occurs at membrane lipid rafts, it is possible that NA, M1, and progeny vRNPs did not sufficiently accumulate on the lipid raft in the absence of the HA protein. To test this hypothesis, we analyzed the association of viral polymerases, HA, NA, NP, and M1 with the lipid raft at 10 hpi by membrane flotation assays. Lipid rafts are insoluble in nonionic detergents such as TX-100, at 0 °C, but are soluble at 37 °C. Fractions of HA, NA, viral polymerases, NP, and M1 were observed in TX-100-insoluble membrane fractions in WT virus-infected cells at 0 °C (Figure 3A, fractions 2 and 3) but they became soluble at 37 °C (Figure 3A). These results confirmed the association of HA, NA, NP, and M1 with lipid rafts in WT virus-infected cells. Viral polymerases and NP were also recovered in the detergent-insoluble membrane fractions, indicating that vRNPs were associated with the lipid raft (Figure 3A). In contrast, for cells infected with the HAstop virus, a trace amount of vRNP components (viral polymerases and NP) was recovered in the detergent-insoluble membrane fractions. The amount of M1 in the fractions was comparable to that in cells infected with the WT virus. To analyze the association of viral membrane proteins, HA, NA, and M2, with the lipid raft in HAstop virus-infected cells, the membrane flotation assays were performed using WT and HAstop virus-infected cell lysate (Figure 3B). NA and M2 were still recovered in the detergent-insoluble membrane fractions, but the amount of NA and M2 in the fractions was clearly reduced compared to that in cells infected with the WT virus (Figure 3B). These results suggest that the accumulation of some viral components in the lipid raft was impaired in cells infected with the HAstop virus. Next, we analyzed the association of viral proteins with lipid raft at early (7 hpi) and late phase (13 hpi) of infection. The distributions of HA, NA, NP, and M1 in the fractions were analyzed by western blotting. The membrane-fraction band intensities and the total band intensity of NA, M1, and NP were measured and the ratio of membrane-fraction intensity to total intensity was calculated. At 7 and 10 hpi, the level of NA, NP, and M1 in the detergent-insoluble membrane fractions was about 60%–80% lower than that of WT fractions. In contrast, the level of NA, NP, and M1 in the fractions was comparable to that of WT fractions at 13 hpi (Figure 3C,D). These results suggest that the accumulation of viral components in the lipid raft was delayed in cells infected with the HAstop virus and the total number of viral components in lipid raft became comparable between cells infected with WT and HAstop virus at 13 hpi.

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