Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb;86(3):1789-801.
doi: 10.1128/JVI.06003-11. Epub 2011 Nov 23.

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Huailiang Ma  1 , François Kien, Maxime Manière, Yang Zhang, Nadège Lagarde, Kong San Tse, Leo Lit Man Poon, Béatrice Nal
Affiliations

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Huailiang Ma et al. J Virol. 2012 Feb.

Abstract

The influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Identification of AnxA6 as a cellular interactant of M2 CT. (A) Diagram of structural motifs in the M2 protein and sequence of the CT used as bait for Y2H screening. ED, ectodomain; TM, transmembrane domain. (B) Schematic representation of full-length AnxA6 and of clones 196 and 270. (a) The eight repeats of AnxA6 are shown with gray numbered boxes. Among the 15 AnxA6-positive clones isolated, clone 196 (b) and clone 270 (c) contained the largest and smallest AnxA6 cDNA fragments, respectively. Numbers indicate amino acid positions.
Fig 2
Fig 2
AnxA6 interacts with M2 in virus-infected human cells. (A) Reciprocal coimmunoprecipitation assay of myc-AnxA6 and M2 in 293T cells. 293T cells were transfected with a plasmid encoding a myc-tagged AnxA6 protein and were infected 24 h later with A/WSN/33 virus at an MOI of 0.1. Cells were lysed at 24 h p.i., and a coimmunoprecipitation assay was performed using either anti-myc (middle panel) or anti-M2 (lower panel) MAb. Immunoprecipitated proteins were detected by Western blotting using either anti-M2 or anti-myc MAb. The input samples were 10-fold-diluted cell lysates (upper panel). (B) AnxA6 colocalizes with M2 protein close to the plasma membranes of infected A549 cells. A549 cells were transfected with AnxA6-GFP plasmid and then infected with influenza A/WSN/33 virus at an MOI of 5 as indicated. Cells were fixed at 14 h p.i., stained with mouse anti-M2 MAb followed by Alexa Fluor 555-conjugated goat anti-mouse IgG, and analyzed by confocal microscopy. Bars, 10 μm.
Fig 3
Fig 3
AnxA6 negatively modulates influenza virus infection. (A) siRNA-mediated gene knockdown efficiency analysis. Cell lysates of siRNA-treated cells were collected at 60 h posttransfection, and expression levels of the AnxA6 and GAPDH proteins were determined by Western blotting. (B, C, and D) AnxA6 depletion in A549 cells increases progeny virus titer. A549 cells treated with the indicated individual siRNAs were infected with influenza A/WSN/33 (H1N1) virus (B), A/HK/1/68 (H3N2) virus (C), or A/HK/54/98 (H1N1) virus (D) at an MOI of 0.01. Cell culture supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assay on MDCK cells. Data are shown as means ± standard deviations (SD) for triplicates from four independent experiments (B) and duplicates from three independent experiments (C and D). **, P < 0.01; ***, P < 0.0001 by unpaired Student's t test. (E) Overexpression of AnxA6 in A431 cells decreases progeny virus titer. A431 cells were used to establish a stable cell line overexpressing AnxA6 that was infected with influenza A/WSN/33 virus at an MOI of 0.01. Cell culture supernatants were collected at 24 h p.i., and virus titers were determined by plaque assay on MDCK cells. Data are shown as means ± SD for measurements from quadruplicates in a representative experiment. ***, P < 0.0001 by unpaired Student's t test. (F) Overexpression of AnxA6 in A431 cells impairs virus propagation. The same experiment as that in panel E was conducted with cell lysates collected at 24 h p.i. Expression levels of viral (M1 and M2) and cellular (AnxA6 and GAPDH) proteins were determined by Western blotting.
Fig 4
Fig 4
AnxA6 does not affect the early stages of the viral life cycle or viral gene expression. (A and B) Silencing of AnxA6 expression does not affect the early stages of the viral life cycle. (A) A549 cells treated with the indicated siRNAs were infected with influenza A/WSN/33 virus at an MOI of 5. At 3 h p.i., cells were fixed and immunostained for NP as a marker of infection. Nuclei were stained using DAPI. Bar, 20 μm. (B) Percentages of infected (NP-positive) cells (n, >100,000) plotted against the siRNA treatments used. (C and D) AnxA6 overexpression does not affect the early stages of the viral life cycle. The same experiment as that in panels A and B was performed with the A431-AnxA6 stable cell line and wild-type A431 cells (n, >10,000). (E and F) Silencing of AnxA6 expression does not affect viral RNA replication and transcription. A549 cells treated with the indicated siRNAs were infected with influenza A/WSN/33 virus at an MOI of 3, and total RNA was then extracted at 4 h p.i. M gene vRNA and mRNA levels were determined by quantitative RT-PCR. Data are shown as means ± SD for measurements from triplicates and are representative of 2 independent experiments. (G) Silencing of AnxA6 expression does not affect viral M gene expression. A549 cells treated with the indicated siRNAs were infected with influenza A/WSN/33 virus at an MOI of 3. Cell lysates were collected at 4 h p.i., and expression levels of viral (M1 and M2) and cellular (AnxA6 and GAPDH) proteins were determined by Western blotting. (H) Silencing of AnxA6 expression does not affect viral polymerase complex activity. 293T cells treated with the indicated siRNAs were transfected with plasmids encoding the polymerase complex components and NP derived from influenza A/WSN/33 virus, along with a reporter plasmid containing the noncoding sequence from the M segment as well as the luciferase gene driven by the human polymerase I promoter. Luciferase activity was determined at 48 h posttransfection, and relative activities were compared. Data are shown as means ± SD for measurements from triplicates and are representative of 2 independent experiments.
Fig 5
Fig 5
AnxA6 does not affect vRNP export or M2 protein expression and trafficking. (A and B) NP subcellular localization in AnxA6-depleted A549 cells. A549 cells treated with the indicated siRNAs were infected with influenza A/WSN/33 virus at an MOI of 5. Cells were fixed at 4, 6, and 8 h p.i. for immunostaining of NP as a marker for vRNP complex export, and nuclei were stained using DAPI. Bar, 20 μm. Cytoplasmic and nuclear NP localization was quantified using Metamorph software, and the plotted histogram represents the percentage of infected cells with nuclear NP only. Data are shown as means ± SD for measurements from at least 200 cells in duplicates of a representative experiment. (C) M2 subcellular localization in AnxA6-depleted A549 cells. A549 cells treated with the indicated siRNAs were infected with influenza A/WSN/33 virus at an MOI of 5. Cells were fixed at 4, 6, and 8 h p.i. for immunostaining of M2 protein. Bar, 20 μm. (D) AnxA6 overexpression does not affect M2 cell surface expression. A431 cells and the A431-AnxA6 stable cell line were infected with influenza A/WSN/33 virus at an MOI of 3. Cells were fixed at 8 and 10 h p.i., left nonpermeabilized, and labeled with mouse anti-M2 MAb followed by Alexa Fluor 488-conjugated goat anti-mouse IgG for M2 cell surface analysis by flow cytometry. The graph shows the amount of cell surface expression and fluorescence intensity of the M2 viral antigen.
Fig 6
Fig 6
AnxA6 colocalizes with M2 at the plasma membrane of virus-infected human cells. A431 cells were transfected with a plasmid encoding the AnxA6-GFP fusion protein and then infected 24 h later with influenza A/WSN/33 virus at an MOI of 5. After fixation at 14 h p.i., cells were stained with a mouse anti-M2 MAb followed by Alexa Fluor 555-conjugated goat anti-mouse IgG. Single optical sections (upper panels) and representative z-plane reconstructions (lower panels) are shown. The inset in panel d shows a magnified (×3.7) region at the plasma membrane. Arrows indicate colocalization of AnxA6 and M2 at the plasma membrane. Bar, 10 μm.
Fig 7
Fig 7
AnxA6 overexpression impairs influenza virus budding. (A) Analysis of budding virions from AnxA6-overexpressing cells by TEM. A431 cells (upper panels) and the A431-AnxA6 stable cell line (lower panels) were infected with influenza A/WSN/33 virus at an MOI of 5. At 10 h p.i., cells were fixed and processed for thin sectioning and TEM. Asterisks indicate spherical virions, and arrowheads indicate elongated virions. Noninfected controls are shown on the left. (B) Semiquantitation of total numbers of virions. Virions observed in infected cells (n = 13 cells with >100 virions) of each cell type were manually counted blinded by five different investigators, and the total numbers of virions were compared. ***, P < 0.0001 by unpaired Student's t test. (C) Semiquantitation of virions according to morphology. During counting, virions were categorized into three types according to their morphology and size, as indicated. The numbers of virions for each type were determined, and their percentages were subjected to unpaired Student's t test and plotted as means ± SD (***, P < 0.0001; **, P < 0.01; *, P < 0.05). (D) Progeny virus titration of released virions. The same experiment as that in panel A was performed with cell culture supernatants collected at 9 h p.i. Virus titers were determined by plaque assay on MDCK cells. Data are shown as means ± SD for triplicates in a representative experiment. ***, P < 0.0001 by unpaired Student's t test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Barman S, Adhikary L, Kawaoka Y, Nayak DP. 2003. Influenza A virus hemagglutinin containing basolateral localization signal does not alter the apical budding of a recombinant influenza A virus in polarized MDCK cells. Virology 305:138–152 - PubMed
    1. Barman S, Nayak DP. 2007. Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells. J. Virol. 81:12169–12178 - PMC - PubMed
    1. Bruce EA, Digard P, Stuart AD. 2010. The Rab11 pathway is required for influenza A virus budding and filament formation. J. Virol. 84:5848–5859 - PMC - PubMed
    1. Carlton JG, Martin-Serrano J. 2009. The ESCRT machinery: new functions in viral and cellular biology. Biochem. Soc. Trans. 37:195–199 - PubMed
    1. Chen BJ, Lamb RA. 2008. Mechanisms for enveloped virus budding: can some viruses do without an ESCRT? Virology 372:221–232 - PMC - PubMed

Publication types