RESUMO
Internalization of rotavirus in MA104 cells was found to induce coentry of alpha-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration.
Assuntos
Endorribonucleases/fisiologia , Proteínas Fúngicas , Rotavirus/fisiologia , Replicação Viral , Animais , Linhagem Celular , Regulação Viral da Expressão Gênica , Humanos , Biossíntese de Proteínas , Inibidores da Síntese de ProteínasRESUMO
It has previously been shown that trypsinized triple-layered particles of rotavirus induce destabilization of liposomes and membrane vesicles in the absence of Ca2+, a condition which leads to solubilization of the outer capsid proteins of the virus. In this work, we have studied the relationship between outer capsid solubilization and permeabilization of membrane vesicles, monitoring particle and vesicle size simultaneously by changes in light scattering. Permeabilization of intact cells induced by solubilized outer capsid proteins was monitored by following the rate of entry of ethidium bromide into the cells. Solubilized outer capsid proteins separated from double-layered particles induced vesicle permeabilization. Solubilization of the outer capsid preceded and was required for vesicle or cell permeabilization. Membrane damage induced by rotaviral outer proteins was not repaired upon addition of 1 mM Ca2+ to the medium. Rotavirus infection and cell permeabilization were correlated in six different cell lines tested. This phenomenon might be related to the mechanism of virus entry into the cell. We propose a new model for rotavirus internalization based on the permeabilizing ability of outer capsid proteins and the cycling of trapped calcium in the endosomal compartment.
Assuntos
Capsídeo/fisiologia , Permeabilidade da Membrana Celular , Infecções por Rotavirus/virologia , Rotavirus/fisiologia , Replicação Viral , Suscetibilidade a Doenças , Células HeLa , Humanos , Microscopia Eletrônica , Infecções por Rotavirus/patologiaRESUMO
It has been previously shown that rotavirus maturation and stability of the outer capsid are calcium-dependent processes. More recently, it has been hypothesized that penetration of the cell membrane is also affected by conformational changes of the capsid induced by Ca2+. In this study, we determined quantitatively the critical concentration of calcium ion that leads to solubilization of the outer capsid proteins VP4 and VP7. Since this critical concentration is below or close to trace levels of Ca2+, we have used buffered solutions based on ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and Ca-EGTA. This method allowed us to show a very high variability of the free [Ca2+] needed to stabilize, at room temperature, the outer capsid of several rotavirus strains. This concentration is about 600 nM for the two bovine strains tested (RF and UK), 100 nM for the porcine strain OSU, and only 10 to 20 nM for the simian strain SA11. Titration of viral infectivity after incubation in buffer of defined [Ca2+] confirmed that the loss of infectivity occurs at different [Ca2+] for these three strains. For the bovine strain, the cleavage of VP4 by trypsin has no significant effect on the [Ca2+] that solubilizes outer shell proteins. The outer layer (VP7) of virus-like particles (VLP) made of recombinant proteins VP2, VP6, and VP7 (VLP2/6/7) was also solubilized by lowering the [Ca2+]. The critical concentration of Ca2+ needed to solubilize VP7 from VLP2/6/7 made of protein from the bovine strain is close to the concentration needed for the corresponding virus. Genetic analysis of this phenotype in a set of reassortant viruses from two parental strains having the phenotypes of strains OSU (porcine) and UK (bovine) confirmed that this property of viral particles is probably associated with the gene coding for VP7. The analysis of VLP by reverse genetics might allow the identification of the region(s) essential for calcium binding.