Rotavirus infection of cells in culture induces activation of RhoA and changes in the actin and tubulin cytoskeleton.
Rotavirus infection induces an increase in [Ca(2+)](cyto), which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca(2+) chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca(2+) homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.
Instituto Venezolano de Investigaciones Científicas (IVIC), CMBC. Caracas, Venezuela. jlzr@ivic.gob.ve
Jose Luis
JL
Zambrano
Orlando
O
Sorondo
Ana
A
Alcala
Esmeralda
E
Vizzi
Yuleima
Y
Diaz
Marie Christine
MC
Ruiz
Fabian
F
Michelangeli
Ferdinando
F
Liprandi
Juan E
JE
Ludert
D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D057134:Antibodies, Neutralizing / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D002522:Chlorocebus aethiops; D004789:Enzyme Activation / Q000187:drug effects; D020868:Gene Silencing / Q000187:drug effects; D006023:Glycoproteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D012401:Rotavirus / Q000187:drug effects / Q000502:physiology; D012400:Rotavirus Infections / Q000201:enzymology*; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors; D014118:Toxins, Biological / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D014771:Virion / Q000276:immunology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000199:Actins; D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D002614:Chelating Agents; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D034741:RNA, Small Interfering; D014118:Toxins, Biological; D014404:Tubulin; D017361:Viral Nonstructural Proteins; D020742:rhoA GTP-Binding Protein; D002118:Calcium