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, 74 (24), 11538-47

Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding

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Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding

P Gómez-Puertas et al. J Virol.

Abstract

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.

Figures

FIG. 1
FIG. 1
Identification of VLPs by biochemical assays. COS-1 cells were infected with vTF7-3 and transfected with the nine pGEM-derived plasmids encoding all the viral structural polypeptides (Stp, −RNA) or with only eight plasmids (−NP, −NA, −M1, −M2, −NEP, −HA; the gene omitted in each sample encodes the indicated protein) or with a DNA mixture that lacked the plasmids encoding the three polymerase subunits (−Pol). All transfection mixtures, except sample −RNA, contained also plasmid pCATCA18, which drives the expression of a synthetic influenza virus-like CAT RNA. Supernatants from the transfected cultures were collected at 72 h p.i., clarified by low-speed centrifugation, and centrifuged through a 33% sucrose cushion as detailed in Materials and Methods. Extracts from the transfected COS-1 cells (A) and from the material pelleted following centrifugation of the supernatants through the 33% sucrose cushion (B) were analyzed by immunoblotting using antibodies to the NP, HA, M1, and M2 proteins as indicated. Another aliquot of the pelleted material was phenol extracted and analyzed for the presence of CAT RNA by RT-PCR as detailed in Materials and Methods (B, bottom).
FIG. 2
FIG. 2
Visualization of VLPs in transfected COS-1 cell cultures. COS-1 cell cultures were infected with vTF7-3 and transfected with plasmids as described in the legend to Fig. 1. At 60 h p.i., cells were sequentially incubated with an anti-HA MAb (M234/1/F4) and decorated with a gold-labeled antiserum before fixation and analysis by EM (details are given in Materials and Methods). Sample FLU corresponds to COS-1 cell cultures infected with influenza virus strain A/Victoria/3/75. Bar, 200 nm (all pictures are shown at the same magnification).
FIG. 3
FIG. 3
Analysis by sucrose gradient centrifugation of the particles formed in cells expressing M1 and the viral glycoproteins. (A) COS-1 cells were infected with influenza virus A/Victoria/3/75, and the supernatant from this culture was collected and split into four aliquots, which were independently centrifuged through sucrose cushions of 25, 33, 41, and 49%. The material pelleted in each case was resolved by SDS-PAGE and analyzed by immunoblotting using antibodies to the HA and M1 proteins, as indicated. An aliquot of the material that sedimented through the 33% sucrose cushion was adsorbed onto carbon-coated grids, immunolabeled with an anti-HA antibody, negatively stained, and visualized by EM (right). (B to E) COS-1 cells were infected with vTF7-3 and transfected with different combinations of plasmids, and the supernatants were analyzed as described for panel A. The plasmids transfected were those encoding M1, HA, and NA (B); HA and NA (C); M1 and HA (D); or M1 alone (E). An aliquot of the panel B sample was also analyzed by EM as described for panel A. Bar, 100 nm.
FIG. 4
FIG. 4
Visualization of the particles formed on expression of M1 and the viral glycoproteins. COS-1 cells were infected with vTF7-3, transfected with different plasmids, and analyzed by EM. (A and B) Cells were previously immunostained with an anti-HA MAb as described in the legend to Fig. 2. The samples analyzed were cultures that coexpressed M1, HA, and NA (A); M1 and HA (B); or M1 alone (C). Bar, 200 nm (all pictures are shown at the same magnification).
FIG. 5
FIG. 5
Intracellular aggregates observed in cells expressing the M1 protein. (A and B) COS-1 cells were infected with vTF7-3 and transfected with plasmid pGEM-M1Δ. At 60 h p.i., thin sections of the transfected cells were processed for EM analysis as detailed in Materials and Methods. (C) The embedded thin sections were immunogold labeled with a mixture of anti-M1 MAbs. Bar, 200 nm.
FIG. 6
FIG. 6
Flotation analysis of transiently expressed influenza virus proteins. COS-1 cells were infected with vTF7-3 and transfected with different plasmids, and cell extracts were prepared and made 80% sucrose. These extracts were laid at the bottom of a centrifuge tube, which was overlaid with layers of 65 and 10% sucrose. Following centrifugation, fractions were collected from the top and analyzed by immunoblotting with either anti-HA or anti-M1 antibodies as indicated. The transfected cultures expressed M1 alone (A); M1 and HA (B); or M1, HA, and NA (C). (D) The M1 signals detected in the films shown in panels A (open circles) and B (filled circles) were quantitated by scanning and expressed as percentages of the total amount of M1 protein.

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