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.
ChemelloM. E.,
AristimunoO. C.,
MichelangeliF.,
RuizM. C.2002; Requirement for vacuolar H+-ATPase activity and Ca2+ gradient during entry of rotavirus into MA104 cells. J Virol 76:13083–13087[CrossRef]
CiarletM.,
HidalgoM.,
GorzigliaM.,
LiprandiF.1994; Characterization of neutralization epitopes on the VP7 surface protein of serotype G11 porcine rotaviruses. J Gen Virol 75:1867–1873[CrossRef]
CrawfordS. E.,
MukherjeeS. K.,
EstesM. K.,
LawtonJ. A.,
ShawA. L.,
RamigR. F.,
PrasadB. V.2001; Trypsin cleavage stabilizes the rotavirus VP4 spike. J Virol 75:6052–6061[CrossRef]
DormitzerP. R.,
NasonE. B.,
PrasadB. V.,
HarrisonS. C.2004; Structural rearrangements in the membrane penetration protein of a non-enveloped virus. Nature 430:1053–1058[CrossRef]
KaljotK. T.,
ShawR. D.,
RubinD. H.,
GreenbergH. B.1988; Infectious rotavirus enters cells by direct cell membrane penetration, not by endocytosis. J Virol 62:1136–1144
MendezI. I.,
HermannL. L.,
HazeltonP. R.,
CoombsK. M.2000; A comparative analysis of freon substitutes in the purification of reovirus and calicivirus. J Virol Methods 90:59–67[CrossRef]
OverberghL.,
ValckxD.,
WaerM.,
MathieuC.1999; Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11:305–312[CrossRef]
ParasharU. D.,
HummelmanE. G.,
BreseeJ. S.,
MillerM. A.,
GlassR. I.2003; Global illness and deaths caused by rotavirus disease in children. Emerg Infect Dis 9:565–572[CrossRef]
RuizM. C.,
AbadM. J.,
CharpilienneA.,
CohenJ.,
MichelangeliF.1997; Cell lines susceptible to infection are permeabilized by cleaved and solubilized outer layer proteins of rotavirus. J Gen Virol 78:2883–2893
RuizM. C.,
CohenJ.,
MichelangeliF.2000; Role of Ca2+ in the replication and pathogenesis of rotavirus and other viral infections. Cell Calcium 28:137–149
Schwartz-CornilI.,
BenureauY.,
GreenbergH.,
HendricksonB. A.,
CohenJ.2002; Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins. J Virol 76:8110–8117[CrossRef]
VonderfechtS. L.,
MiskuffR. L.,
WeeS. B.,
SatoS.,
TidwellR. R.,
GeratzJ. D.,
YolkenR. H.1988; Protease inhibitors suppress the in vitro and in vivo replication of rotavirus. J Clin Invest 82:2011–2016[CrossRef]