Recombinant Echovirus E6 Capsid Protein [His]

Human parechovirus 1 (HPeV-1) (family Picornaviridae) is a global cause of pediatric respiratory and CNS infections for which there is no treatment. Although biochemical and in vitro studies have suggested that HPeV-1 binds to alpha V beta 1, alpha V beta 3 and alpha V beta 6 integrin receptor(s), the actual cellular receptors required for infectious entry of HPeV-1 remain unknown. In this paper we analyzed the expression profiles of alpha V beta 1, alpha V beta 3, alpha V beta 6 and alpha 5 beta 1 in susceptible cell lines (A549, HeLa and SW480) to identify which integrin receptors support HPeV-1 internalization and/or replication cycle. We demonstrate by antibody blocking assay, immunofluorescence microscopy and RT-qPCR that HPeV-1 internalizes and replicates in cell lines that express alpha V beta 1 integrin but not alpha V beta 3 or alpha V beta 6 integrins. To further study the role of beta 1 integrin, we used a mouse cell line, GE11-KO, which is deficient in beta 1 expression, and its derivate GE11-beta 1 in which human integrin beta 1 subunit is overexpressed. HPeV-1 (Harris strain) and three clinical HPeV-1 isolates did not internalize into GE11-KO whereas GE11-beta 1 supported the internalization process. An integrin beta 1-activating antibody, TS2/16, enhanced HPeV-1 infectivity, but infection occurred in the absence of visible receptor clustering. HPeV-1 also co-localized with beta 1 integrin on the cell surface, and HPeV-1 and beta 1 integrin co-endocytosed into the cells. In conclusion, our results demonstrate that in some cell lines the cellular entry of HPeV-1 is primarily mediated by the active form of alpha V beta 1 integrin without visible receptor clustering.

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2A(pro) and 3C(pro)), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2A(pro) cause direct damage to the infected heart and tools to investigate 2A(pro) and 3C(pro) expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2A(pro) and 3C(pro); Two monoclonal 2A(pro) antibodies and one 3C(pro) antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3C(pro) antibody detects all of the EV species B (EV-B) viruses tested and that the 2A(pro) antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2A(pro) and 3C(pro), and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.