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Poliovirus infection results in structural alteration of a microtubule-associated protein
1Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66160.
Abstract:
Poliovirus infection results in profound changes in cellular metabolism and architecture. To identify alterations in cellular proteins following poliovirus infection which might account for these changes, monoclonal antibodies were prepared by screening for differences in antigen pattern in infected and uninfected cell lysates. Further characterization of the antigen of one such antibody (25 C C1) is described in this report. The 25 C C1 antigen is a cytoskeleton-associated protein which decreases in size 4 to 5 h postinfection. It copurifies with some of the protein synthesis initiation factors but not with eucaryotic initiation factor (eIF)-4F, the p220 subunit of which is cleaved following infection (D. Etchison, S. C. Milburn, I. Edery, N. Sonenberg, and J. W. B. Hershey, J. Biol. Chem. 257:14806-14810, 1982). Unlike alteration of p220, alteration of the 25 C C1 antigen is not due to a protease which can be detected by cell lysate mixing experiments. Alteration of the antigen occurs during purification, suggesting progressive proteolysis, but the alteration is more extensive in preparations from infected cells than in those from uninfected cells. A recombinant phage expressing the antigenic determinant was isolated from a human fibroblast cDNA library, and the sequence of the cDNA insert was found to be entirely contained within the established sequence of microtubule-associated protein (MAP) 4 (R. R. West, K. M. Tenbarge, and J. B. Olmsted, J. Biol. Chem. 266:21886-21896, 1991). The antigen distribution, as detected by indirect immunofluorescence, was similar to, but more diffuse than, the distribution of tubulin. The antibody recognized the largest abundant HeLa cell MAP, which copurified with tubulin after three cycles of polymerization-depolymerization, thus confirming the identity of the antigen as MAP 4. These results indicate that poliovirus infection of HeLa cells affects the structural integrity of a cytoskeletal protein, MAP 4.
Insights
Poliovirus infection alters the cellular cytoskeleton. Researchers identified microtubule-associated protein (MAP) 4 as a key protein affected, showing structural changes post-infection that impact cell architecture.
Area of Science:
- Cell Biology
- Virology
- Molecular Biology
Background:
- Poliovirus infection profoundly alters host cell metabolism and architecture.
- Understanding cellular protein changes is crucial for explaining these alterations.
- Previous studies noted cleavage of eukaryotic initiation factor (eIF)-4F subunit p220 post-infection.
Purpose of the Study:
- To identify and characterize cellular proteins altered by poliovirus infection.
- To investigate the nature of these alterations and their potential role in cellular changes.
Main Methods:
- Preparation of monoclonal antibodies to screen for antigen differences between infected and uninfected cells.
- Characterization of a specific antigen (25 C C1) using immunoprecipitation and Western blotting.
- Isolation of a recombinant phage expressing the antigenic determinant from a human fibroblast cDNA library.
- Sequence analysis of the cDNA insert and confirmation of antigen identity via immunofluorescence and protein purification.
Main Results:
- A cytoskeleton-associated protein, initially identified by antibody 25 C C1, decreases in size 4-5 hours post-poliovirus infection.
- This antigen copurifies with some protein synthesis initiation factors but not with eIF-4F.
- Sequence analysis identified the antigen as microtubule-associated protein (MAP) 4.
- Immunofluorescence showed MAP 4 distribution was similar but more diffuse than tubulin in infected cells.
- The antibody recognized the largest abundant HeLa cell MAP, confirmed by copurification with tubulin.
Conclusions:
- Poliovirus infection affects the structural integrity of microtubule-associated protein (MAP) 4 in HeLa cells.
- Alteration of MAP 4 appears to be a progressive process, more pronounced in infected cells.
- These findings suggest a mechanism by which poliovirus disrupts cellular architecture via cytoskeletal modification.