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Results: 7

1.
FIG. 7

FIG. 7. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Morphological distribution of M1 and HA in HeLa cells coinfected with RVVM1 and RVVHA. HeLa cells (4 × 105) were grown on chamber slides and infected with RVVM1 and RVVHA. Cells were incubated with or without monensin, and at 4 hpi, they were double stained for M1 and HA as described in the legend to Fig. 6 and examined by confocal microscopy. (A to C) Cells infected with wild-type vaccinia virus; (D to F) cells without monensin treatment; (G to I) cells with monensin treatment. Image analysis was done as follows: panels A, D, and G for M1; panels B, E, and H for HA; and panels C, F, and I superimposed for both HA and M1. Cells in panels J (without monensin) and K (with monensin) were infected with RVVM1 only and stained for M1 (original magnification, ×600).

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.
2.
FIG. 5

FIG. 5. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Detergent resistance of membrane-associated M1 from WSN virus-infected MDBK cells. (A and B) TX-100 treatment of membrane-associated M1 protein synthesized early (2.5 hpi) in the virus replication cycle. WSN virus-infected (MOI of 10) MDBK cells (5 × 106) were labeled with 300 μCi at 2.5 hpi for 20 min (A) and chased for 3 h (B) in the presence of 1.0 mM cycloheximide. Cells were then harvested and fractionated, and membrane fractions were isolated from the 4K supernatant with a floatation gradient. The membrane fractions were then treated without (−) or with (+) 0.05% TX-100 and analyzed with a second floatation gradient. Fractions were immunoprecipitated and analyzed by SDS-PAGE. (C and D) Analysis of M1 protein synthesized late (6.5 hpi) in the virus replication cycle. WSN virus-infected MDBK cells were labeled with 300 μCi at 6.5 hpi for 20 min (C) and chased for 1 h (D) in the presence of cycloheximide as described above. Membrane fractions were isolated from virus-infected cells as described above, treated without (−) or with (+) 0.05% TX-100, and analyzed with a floatation gradient. Fractions were immunoprecipitated and analyzed by SDS-PAGE.

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.
3.
FIG. 1

FIG. 1. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Membrane association of M1 protein in WSN virus-infected cells. (A) Pulse-chase analysis of WSN virus-infected MDBK cells. At 7 hpi, WSN virus-infected MDBK cells (5 × 106) were pulse-labeled with 300 μCi of 35S Easy Tag for 15 min and chased for 1 h. Labeled cells were fractionated, and the 4K supernatant was analyzed with floatation gradients (31). Each fraction was immunoprecipitated with an anti-WSN polyclonal antibody and analyzed by SDS–10% PAGE. Fractions are numbered 1 (top) to 5 (bottom). Lines marked 1 and 2 (upper right-hand corner) denote nonspecific cellular proteins. Both panels are from the same gel and same autoradiograph. (B) Pulse-chase analysis of RVVM1-infected HeLa cells. HeLa cells were infected with RVVM1 at an MOI of 10. At 6 hpi, cells were pulse-labeled with 400 μCi of 35S Easy Tag and chased as described above, the 4K supernatant was analyzed with floatation gradients, and fractions were immunoprecipitated and analyzed by SDS-PAGE.

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.
4.
FIG. 3

FIG. 3. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Detergent resistance of membrane-associated M1 when coexpressed with influenza virus HA and NA. For coexpression studies, HeLa cells (5 × 106) were infected with RVV expressing M1 alone or M1 in combination with HA or NA or with both HA and NA proteins as stated in Materials and Methods. At 4 hpi, cells were labeled with 400 μCi of 35S Easy Tag for 30 min and chased for 90 min. Cells were fractionated, and pure membrane fractions of the 4K supernatant were isolated with a floatation gradient as stated in Materials and Methods. Aliquots of membrane fractions were either untreated (−) or treated (+) with TX-100 (0.05%) and analyzed with a second floatation gradient. Fractions were collected, immunoprecipitated with anti-WSN antibodies, and analyzed by SDS-PAGE. (A) Expression of influenza virus M1 alone. (B) Coexpression of influenza virus M1 with influenza virus HA protein. (C) Coexpression of influenza virus M1 with NA protein. (D) Coexpression of M1 with both influenza virus HA and NA. Left-hand panels are without TX-100 treatment, and right-hand panels are after TX-100 treatment.

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.
5.
FIG. 4

FIG. 4. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Detergent resistance of membrane-associated M1 when coexpressed with chimeric constructs of influenza virus HA and Sendai virus F proteins. (A) Schematic presentation of chimeric constructs between Sendai virus F and influenza virus HA. aa, amino acids; Cyt., cytoplasmic. (B to F) Detergent-resistant membrane fractions of M1 in the presence of heterologous or chimeric proteins. HeLa cells (5 × 106) were infected with RVV expressing M1 alone (B) or M1 with Sendai virus F (C), M1 with FHH (D), M1 with HHF (E), or M1 with FFH (F) as described in Materials and Methods. At 4 hpi, cells were pulse-labeled with 400 μCi of 35S Easy Tag for 30 min and chased for 90 min. Pure membrane fractions were isolated from 4K supernatants with floatation gradients, treated without (−) or with (+) TX-100 (0.05%), and analyzed again with floatation gradients. Fractions were collected, immunoprecipitated, and analyzed by SDS-PAGE. (G) Expression of F and chimeric proteins. HeLa cells (5 × 106) were infected with RVV expressing F, FHH, HHF, and FFH. At 4 hpi, cells were pulse-labeled for 30 min and chased for 90 min. The whole-cell extract was immunoprecipitated and analyzed by SDS-PAGE. Note that similar levels of F and chimeras were expressed. Lanes 1, marker; lanes 2, FHH; lanes 3, FFH; lanes 4, HHF; lanes 5, F.

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.
6.
FIG. 6

FIG. 6. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Morphological distribution of M1 and HA in influenza virus-infected MDBK cells by confocal microscopy. MDBK cells (4 × 105) were grown on chamber slides and synchronously infected with WSN virus at an MOI of 10 at 4°C. Cells were then washed and incubated at 37°C. Monensin at a 10 μM final concentration was added to some cells at 2 hpi (G to L), and the cells were incubated for another 5 h at 37°C. At 7 hpi, all virus-infected cells were fixed with ice-cold acetone, incubated with a mixture of anti-M1 rabbit polyclonal and anti-HA mouse monoclonal antibodies, and stained with anti-rabbit IgG (green) and anti-mouse IgG (red). The stained cells were examined by confocal microscopy as described in Materials and Methods. (A to C) Mock-infected cells without monensin; (D to F) virus-infected cells without monensin treatment; (G to I) mock-infected cells with monensin; (J to L) virus-infected cells with monensin. Image analysis was done as follows: panels A, D, G, and J for M1 (green); panels B, E, H, and K for HA (red); and panels C, F, I, and L superimposed for both HA and M1 (original magnification, ×1,000).

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.
7.
FIG. 2

FIG. 2. From: Influenza Virus Assembly: Effect of Influenza Virus Glycoproteins on the Membrane Association of M1 Protein.

Analysis of membrane-associated M1 expressed alone by RVV and in influenza virus-infected cells after TX-100 detergent treatment. (A) HeLa cells (5 × 106) were infected at an MOI of 10 with RVVM1. At 6.0 hpi, infected cells were labeled for 30 min with 400 μCi of 35S Easy Tag and chased for 90 min. Infected cells were harvested and fractionated for 4K supernatant. The membrane fractions were isolated from the 4K supernatant with a floatation gradient as described in Materials and Methods and were either mock treated or treated with varying concentrations (0.01, 0.02, 0.03, 0.04, 0.05, and 0.06%) of TX-100 detergent for 15 min on ice. Each sample was then analyzed again by floatation in sucrose gradients. Gradient fractions were immunoprecipitated using rabbit anti-WSN polyclonal antibodies and analyzed by SDS–10% PAGE. (B and C) Influenza virus-infected cells were labeled at 6.5 hpi for 30 min and chased for 90 min. The total 4K supernatants were prepared and analyzed with floatation gradients before (−) and after (+) TX-100 treatment at different concentrations. Note that both HA and M1 decreased in the membrane fractions (fractions 1 and 2) after treatment with higher TX-100 concentrations (0.06 and 0.08%). Results in panels B and C are from two separate experiments.

Ayub Ali, et al. J Virol. 2000 September;74(18):8709-8719.

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