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Title: ULTRASTRUCTURAL AND CONFOCAL MICROSCOPY ANALYSIS OF FOOT-AND-MOUTH VIRUS (FMDV) STRUCTURAL AND NO-STRUCTURAL PROTEINS ON REPLICATION COMPLEXES IN CULTURED CELLS

Author
item ODONNELL, VIVIAN - UNIV. OF CONN
item Koster, Marla
item Grubman, Marvin
item Baxt, Barry
item Burrage, Thomas

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/18/2005
Publication Date: 7/31/2005
Citation: Odonnell, V.K., Koster, M.J., Grubman, M.J., Baxt, B., Burrage, T.G. 2005. Ultrastructural and Confocal Microscopy Analysis of Foot-and-Mouth Virus (fmdv) Structural and No-structural Proteins on Replication Complexes in Cultured Cells. Microscopy and Microanalysis 2005. p 120.

Interpretive Summary:

Technical Abstract: Picornavirus infection of cells induces a rapid proliferation of cytoplasmic single and double membrane vesicles. These membranes associate into structures called replication complexes (RC). Visualizing mature FMDV particles has been difficult due to their fragility below pH 7. However, high-pressure freeze-substitution studies have produced clear images of mature FMDV particles in cytoplasm. Using a different approach, IBRS2 cells were infected with FMDV O1 Campos at a multiplicity of 5 and fixed at 4 hrs post infection (pi) with a solution containing 4% paraformaldehyde and 5% glutaraldehyde in a 0.2M cacodylate buffer (pH 8.0). Hexagonal particles, 28-32 in diameter, were attached to the external surface of single membrane RC vesicles. Examination of serial sections from infected cells revealed that numerous particles were present on individual membranes at 4 hrs pi. For confocal analysis, BKLF cells, were grown on glass cover slips, were infected at multiciplicty of infection 10 and were fixed with 4% paraformaldehyde. The distribution of FMDV's non-structural proteins 2B, 2C, 3A and 3C was determined at 2,3, and 4 hrs pi by incubation with monoclonal and monospecific polyclonal antisera followed by anti-species antiserum olabeled with Alexa 488 or Alexa 594. Confocal microscopy localized early binding of 2B, 2C, and 3A to small, uniformly distributed cytoplasmic vesicles that coalesced into a larger complex by 4 hrs. 3C, on the other hand showed a more diffuse distribution that did not coalesce. FMDV structural protein VP1, identified by Mabs co-localizes with 2B and 3A at 4 hrs pi. These results suggest that FMDV non-structural proteins are present with the RC and suggest that assembly of mature virions takes place at or near the surface of the RC vesicles.