dsRNA formed as an intermediate during Coxsackievirus infection does not induce NO production in a b-cell line with or without addition of IFN-c Anna-Karin Berg a, * , Asma Elshebani a , Arne Andersson b , Gun Frisk a a Department of WomenÕs and ChildrenÕs Health, Uppsala University, Uppsala, Sweden b Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden Received 26 November 2004 Available online 22 December 2004 Abstract Virus infection is one environmental factor that has been implicated as a precipitating event initiating b-cell damage during the development of type 1 diabetes. One aim of this study was to investigate how permissive an insulin-producing b-cell line, RINm5F, is to enterovirus (EV) infections. A second aim was to study if the viral replicative intermediate, double-stranded RNA (dsRNA), together with IFN-c results in nitric oxide (NO) production. Monolayer cultures of RINm5F cells were not permissive to infection with seven different strains of EV. However, when the growth pattern of the b-cell line changed and the cells started to grow as free- floating RIN cell clusters (RCC), all EV strains replicated. Immunostaining for the Coxsackie-adenovirus-receptor (CAR) detected the protein on the free-floating RIN cell clusters, but not on the RINm5F cells cultured as a monolayer of b-cells. This shows that the CAR expression can change and/or the CAR protein can be redistributed on the cell surface as a consequence of altered growth pattern thus allowing viral replication in a previously non-permissive b-cell line. As expected, NO production was significantly increased (p < 0.05) by addition of synthetic dsRNA and IFN-c to the RCC. In contrast, the dsRNA formed during virus infection with a Coxsackievirus B4 strain (E2) with or without addition of IFN-c did not induce NO production in these cells. This indicates that synthetic dsRNA does not mimic a real viral infection in that respect, and suggests an NO-independent mechanism for virus- induced b-cell damage. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Type 1 diabetes; The Coxsackie-adenovirus-receptor; Nitric oxide; dsRNA; b-cell; Enterovirus Enterovirus (EV) infections are believed to be an environmental factor involved in the development of autoimmune type 1 diabetes (T1D) [1–7]. EVs of the family Picornaviridae are small non-enveloped positive single-stranded RNA viruses. These viruses are classified based on sequences in the P1 region and the human EV cluster into four distinct groups. Coxsackievirus B (CVB) 1–6, Echovirus, Coxsackievirus A9, and enterovi- rus 69 all belong to cluster B. EV infections associated with the onset of T1D are usually caused by the cluster B viruses. The mechanisms of viral involvement in this complex disease are not yet known but a direct infection of the b-cell might be what initiates or precipitates the destruction of the b-cells that eventually leads to T1D [8]. It has been shown that many different EVs can rep- licate in isolated human pancreatic islets, including in the b-cell, in vitro. The outcome of these infections is usually cell lysis and islet degeneration within 10 days, but some virus strains can also establish a persistent infection in human islet cells and such infections in vivo might have long-term effects [9–13]. The major determinant for tropism is the receptor usage of the virus, and a successful EV infection requires 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.12.068 * Corresponding author. Fax: +46 18 611 265. E-mail address: Anna-Karin.Berg@kbh.uu.se (A.-K. Berg). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 327 (2005) 780–788 BBRC