Trypsin is associated with the rotavirus capsid and is activated by solubilization of outer capsid proteins.
The rotavirus capsid is made up of three concentric protein layers. The outer layer, consisting of VP7 and VP4, is lost during virus entry into the host cell. Rotavirus field isolates can be adapted to high-titre growth in tissue culture by treatment with trypsin and by supplementing the culture medium with trypsin, which cleaves VP4 into two fragments, VP8* and VP5*. It is known that protease inhibitors reduce the replication of rotavirus in vitro and in vivo and also diminish disease symptoms in a mouse model. To clarify the molecular basis of these observations, a series of assays were conducted on purified rotavirus particles grown in the presence of trypsin. Results of HPLC and mass spectrometry followed by N-terminal sequencing showed that viral particles contain molecules of trypsin. When associated with triple-layer particles (TLPs), trypsin is inactive and not accessible to protease inhibitors, such as aprotinin. When the outer layer is solubilized by calcium-chelating agents, VP5*, VP8* and VP7 are released and the associated trypsin is activated, allowing cleavage of the viral capsid proteins, as well as other exogenous proteins. It is shown that addition of trypsin inhibitors significantly reduces synthesis of viral mRNA and viral proteins in cells and has a major inhibitory effect if present when virus enters the cell. These data indicate that incorporation of trypsin into rotavirus particles may enhance its infectivity.
Benureau
Yann
Y
Virologie Moléculaire et Structurale, CNRS-INRA, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Huet
Jean Claude
JC
Biochimie et Structure des Protéines, INRA, 78352 Jouy-en-Josas Cedex, France.
Charpilienne
Annie
A
Virologie Moléculaire et Structurale, CNRS-INRA, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Poncet
Didier
D
Virologie Moléculaire et Structurale, CNRS-INRA, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
Cohen
Jean
J
Virologie Moléculaire et Structurale, CNRS-INRA, 1 avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France.
D000956:Antigens, Viral / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008954:Models, Biological; D012401:Rotavirus / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism*; D014771:Virion
D000956:Antigens, Viral; D036022:Capsid Proteins; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus; D014357:Trypsin