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Results: 7

1.
Figure 1

Figure 1. Sequence alignment of M2 residues 46–56. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

The amino acids that define the CRAC motif are in bold and substitutions that disrupt or complete the consensus are indicated. An asterisk indicates no change in sequence. The rWSN M2 R54F mutant still contains a potential CRAC consensus sequence due to the presence of another basic amino acid at position 56.

Shaun M Stewart, et al. Virology. ;405(2):530-538.
2.
Fig. 7

Fig. 7. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

The effects of altering the CRAC motif on replication in mouse tracheal epithelial cell (mTEC) cultures. mTEC cultures were infected with 3300 TCID50 (~0.01 MOI) of the indicated recombinant viruses in the rWSN (A) or rUdorn (B) genetic backgrounds. At the indicated hpi, infected cell supernatants were harvested and the number of infectious virus particles determined by TCID50 assay in MDCK cells. Data points represent the average and standard error of the mean. The horizontal dotted line is the limit of detection.

Shaun M Stewart, et al. Virology. ;405(2):530-538.
3.
Fig. 6

Fig. 6. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

Mortality and morbidity of mice infected with recombinant influenza A/WSN/33 viruses encoding M2 proteins with altered CRAC motifs. Mice were administered an intranasal dose of 103 (A and C) or 105 (B and D) TCID50 of the indicated viruses and monitored for 14 days post infection (dpi). (A and B) Mortality and (C and D) morbidity associated with infection, as judged by loss of starting weight. Data points indicate the average and standard deviation. Significant differences in (B) are between the CRAC altered viruses and rWSN while in (C) the differences are between rWSN M2 delCRAC and the other two viruses. * = p<0.05 and ** = P<0.01.

Shaun M Stewart, et al. Virology. ;405(2):530-538.
4.
Fig. 4

Fig. 4. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

The effects of completing the CRAC motif on formation of filamentous virus particles. MDCK7 cells were infected with 500,000 TCID50 (~0.9 MOI). At 15hpi, immunofluorescence staining was performed for HA and visualized by confocal microscopy. Confocal Z-sections (taken with a 100x objective) of rUd (A) or rUd M2 F54R (B) infected cells were flattened and show viral filaments for both viruses. The percentage of infected cells showing filaments was determined from 10–20 images taken with a 20x objective on an epifluorescence microscope (C). One representative experiment is shown. For TEM, MDCK cells were infected at an MOI of ~5. Samples were processed for transmission electron microscopy at 10 hpi. Cells were infected with rUd (D) or rUd M2 F54R (E). Arrows indicate microvilli, solid arrowheads indicate filamentous virus particles, and empty arrowheads mark either spherical virus particles or cross-sections of filamentous virus particles.

Shaun M Stewart, et al. Virology. ;405(2):530-538.
5.
Fig. 3

Fig. 3. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

The effects of altering the CRAC motif on replication in tissue culture. (A and D) MDCK cells were infected with the indicated viruses at an MOI of ~3 and the cell surface expression of M2 protein was measured by flow cytometry at 15 hours post infection (hpi). The relative expression of M2 represents the mean channel fluorescence of the indicated infected cells divided by the mean channel fluorescence of the wildtype infected cells. MDCK (B and E) or CaLu-3 (C and F) cells were infected at an MOI of ~0.001 with the indicated recombinant viruses. At the indicated hpi, infected cell supernatants were harvested and the number of infectious virus particles determined by TCID50 assay. Data points represent the average and standard error of the mean. The horizontal dotted line is the limit of detection.

Shaun M Stewart, et al. Virology. ;405(2):530-538.
6.
Fig. 5

Fig. 5. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

The effects of altering the CRAC motif on incorporation of structural proteins. MDCK cells were infected with the indicated viruses at an MOI of 5. At 15hpi, virus particles were collected and concentrated through a 20% sucrose cushion at 118,000 g for 1hr. Virus pellets were resuspended and incorporation of viral proteins was determined by Western blot with antibodies which detect the structural proteins HA, NP, M1, and M2. (A and B) Representative Western blots showing incorporation of viral proteins into virions. (C and D) Quantification of full length M2 and total M2 from Western blot analysis of replicate virion incorporation assays. A large amount of pelleted virions was loaded onto the SDS-PAGE gel in order to detect the relatively low amounts of incorporated M2 protein. Relative expression was determined by the negative log of M1 normalized data. Recombinant viruses based on the rWSN (A and C) or rUd virus strains (B and D) were used. * = p<0.05.

Shaun M Stewart, et al. Virology. ;405(2):530-538.
7.
Figure 2

Figure 2. Expression and function of M2 proteins with modified CRAC motifs. From: The cholesterol recognition/interaction amino acid consensus motif of the influenza A virus M2 protein is not required for virus replication but contributes to virulence.

(A and D) MDCK cells expressing the indicated M2 proteins were analyzed by Western blotting to detect disulfide linked oligomeric forms of the M2 protein. Monomers, dimers and trimers are indicated and some higher order oligomers can be detected. (B and E) The number of cells expressing M2 at the cell surface was quantified by flow cytometry from the indicated stably transfected MDCK cell lines. (C and F) The ability of the indicated stably transfected MDCK cells to complement infection with a recombinant influenza virus that does not encode the full length M2 protein was assessed by infecting the cells with the indicated recombinant virus and quantifying infectious virus production at various hours post infection (hpi). The average and standard error of the mean are graphed. The standard error bars are smaller than the size of the individual points. The limit of detection is marked by a dotted line. Proteins or recombinant viruses based on the rWSN (A–C) and rUd virus strains (D–F) were used.

Shaun M Stewart, et al. Virology. ;405(2):530-538.

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