Apoptosis induced by Oropouche virus infection in HeLa cells is dependent on virus protein expression.
Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis.
Acrani
Gustavo Olszanski
GO
Department of Cell and Molecular Biology, University of Sao Paulo School of Medicine, 3900, Av Bandeirantes, Ribeirão Preto, SP 14049-900, Brazil. gacrani@gmail.com, goacrani@usp.br
Gomes
Rogério
R
Proença-Módena
José Luiz
JL
da Silva
Andrei Furlan
AF
Carminati
Patricia Oliveira
PO
Silva
Maria Lucia
ML
Santos
Rodrigo Ivo Marques
RI
Arruda
Eurico
E
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D045304:Cytochromes c / Q000032:analysis; D003593:Cytoplasm / Q000737:chemistry; D053938:DNA Fragmentation; D006367:HeLa Cells; D006801:Humans; D016851:Orthobunyavirus / Q000472:pathogenicity*; D014764:Viral Proteins / Q000096:biosynthesis*; D037521:Virulence Factors / Q000096:biosynthesis*
D014764:Viral Proteins; D037521:Virulence Factors; D045304:Cytochromes c; D053148:Caspase 3; D053453:Caspase 9