Further analysis of the effect of Ca2+ on morphogenesis of HVJ (Sendai virus) in LLC-MK2 cells: effects on phosphorylated M protein associated with viral morphogenesis.
Previously we have reported that Ca2+ concentration in culture medium affects the morphogenesis of HVJ from infected cells. We further investigated the effects of Ca2+ on HVJ morphogenesis in LLC-MK2 cells, focusing on M (matrix) protein which plays an important role in the assembly of viral components. On culture in standard medium (containing 1.8 mM Ca2+), most of the M protein accumulated on the apical surface in patches. On double immunofluorescent staining with anti-M protein monoclonal antibody and polyclonal antibody against HVJ glycoproteins, M protein and viral glycoproteins were observed in the same patches at the cell surface. By contrast, in low Ca2+ medium, M protein was evenly dispersed over the cell surface, especially in the regions of cell-cell contact, and was not seen in patches. Immunoblot analysis for M protein in the infected cells showed that the total amount of M protein was almost the same irrespective of Ca2+ concentration in the culture medium. But, there was a distinct difference in the forms of M protein found in the cells: most of the M protein in the cells cultured in standard medium was of the non-phosphorylated form, whereas the phosphorylated form of M protein was distinctly increased in the cells cultured in low Ca2+ medium. These results suggest that Ca(2+)-deficiency in the medium blocks dephosphorylation of phosphorylated M protein necessary for patch formation, resulting in disturbance of viral morphogenesis.
Kim
J
J
Institute of Molecular and Cellular Biology for Pharmaceutical Sciences, Kyoto Pharmaceutical University, Japan.
Kamata
M
M
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism; D000882:Haplorhini; D015151:Immunoblotting; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism / Q000821:virology*; D009024:Morphogenesis / Q000187:drug effects / Q000502:physiology; D010222:Parainfluenza Virus 1, Human / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D010766:Phosphorylation; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014763:Viral Matrix Proteins / Q000032:analysis / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects* / Q000502:physiology
D006023:Glycoproteins; D007546:Isoquinolines; D010879:Piperazines; D014763:Viral Matrix Proteins; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium