The adenovirus L3 23-kilodalton proteinase cleaves the amino-terminal head domain from cytokeratin 18 and disrupts the cytokeratin network of HeLa cells.
Immunofluorescence studies revealed that adenovirus induces a reorganization of the cytokeratin system in lytically infected HeLa cells. At 24 h postinfection, the cytokeratin network began to disassemble into prominent spheroid globules. By 36 h postinfection, host cell lysis occurred, accompanied by the formation of perinuclear cytokeratin clumps and additional spheroid globules. Immunoblots detected 41- and 44-kDa fragments of cytokeratin 18 and reduced levels of cytokeratin 7 at 24 and 36 h postinfection. Cytokeratin proteins isolated from HeLa cells at 36 h postinfection were deficient in filament polymerization. The 41-kDa proteolytic cytokeratin 18-specific fragment was purified, and its amino-terminal sequence was determined to be GGIQNEKETM. These residues correspond to amino acids 74 through 83 of cytokeratin 18, identifying a cleavage site at the junction of the globular head domain and the alpha-helical rod domain. Moreover, this truncation event occurs at a consensus cleavage site for the adenovirus L3 23-kDa proteinase. The temperature-sensitive mutant H2-ts1, which contains a mutation in the proteinase, neither induced cleavage of cytokeratin 18 nor precipitated the formation of spheroid globules during lytic infection at the nonpermissive temperature. The active proteinase is therefore required for cleavage of cytokeratin 18 and morphological rearrangement of the cytokeratins. We suggest that disruptions in the cytokeratin system weaken the mechanical integrity of the cell, thus promoting host cell lysis and release of progeny virions.
Chen
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PH
Department of Molecular Biology, Howard Hughes Medical Institute, Princeton University, New Jersey 08544-1014.
Ornelles
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DA
Shenk
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D000260:Adenoviruses, Human / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D007633:Keratins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D013997:Time Factors
D007633:Keratins; D010450:Endopeptidases