Apoptosis in coxsackievirus B3-caused diseases: interaction between the capsid protein VP2 and the proapoptotic protein siva

Abstract

Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis.

FIG. 1

Yeast two-hybrid filter lift assay demonstrating the interaction between VP2 of CVB3 and huSiva. Plasmids carrying Gal4BD-VP2 and Gal4AD-huSiva or the Gal4BD and Gal4AD alone were transformed into yeast reporter strain Y190. The resulting cells were analyzed for β-galactosidase activity (A). Only the interaction of Gal4BD-VP2 and Gal4AD-huSiva reconstituted an active Gal4 transcription factor, demonstrated by blue-stained yeast colonies detected after 2 h of incubation with X-Gal (lane 3). GST pull-down experiments confirmed the VP2-huSiva interaction in vitro (B). Thirty-microgram of crude extract proteins of CVB3-infected HeLa cells was incubated with 2 μg of GST (lane 3) or 2 μg of GST-huSiva (lane 4). Protein complexes were precipitated with glutathione-Sepharose and analyzed for VP2 content as described in Materials and Methods. Lane 2, 30 μg of CVB3-infected HeLa cell crude extract proteins; lane 5, 30 μg of noninfected HeLa cell crude extract proteins. A prestained protein marker was used as a size standard (lane M).

FIG. 2

CVB3-caused induction of muSiva. Sequence comparison between the human (first row) and the murine (second row) proapoptotic protein Siva reveals a 71% identity (A). The interaction between VP2 of CVB3 and muSiva was confirmed using the yeast two-hybrid system (B), demonstrated by the blue-colored yeast colonies coexpressing Gal4BD-VP2 and Gal4AD-muSiva (lane 1). Male BALB/c mice were infected with CVB3 i.p. RNA was isolated from pancreas and heart tissue 1 or 7 days p.i. Transcription of muSiva, CVB3-VP2, and β-actin in tissue of individual noninfected or CVB3-infected mice was analyzed by RT-PCR, demonstrating the induction of high levels of muSiva mRNA only in tissue of CVB3-infected mice (C).

FIG. 3

Coxsackievirus-induced pathology in pancreas and heart tissue of BALB/c mice. BALB/c mice were infected with CVB3 i.p. (A) Pancreas tissue and heart tissue was isolated from infected animals 1 day and 7 days p.i., respectively, and from noninfected animals. After hematoxylin-eosin staining, virus-caused tissue damage in the pancreas was obvious, demonstrated by massive destruction of the exocrine pancreas up to 7 days p.i. (original magnification, ×500). Only the islets of Langerhans remained unaffected (arrows). In the heart tissue, CVB3 infection caused massive inflammation accompanied by infiltration of mononuclear cells 7 days p.i. (original magnification, ×500). (B) At the indicated time points, eight mice were sacrificed and virus titers were measured by plaque formation assays. Average titers and standard deviations are shown as log₁₀ values of PFU/0.1 g of tissue.

FIG. 4

Detection of muSiva, apoptotic cells, and CD27- and CD70-positive cells in CVB3-infected tissue. BALB/c mice were infected with CVB3 i.p. Pancreas tissue and heart tissue were isolated at 1 day and 7 days after infection, respectively, and used to perform in situ hybridization studies (A and B), TUNEL assays (C), and immunohistochemistry stainings (D to F). In the area of CVB3-infected tissue (B), transcriptional activity of muSiva (A, arrows) as well as TUNEL assay (C)- and active caspase-3 (D)-positive cells were detectable. CVB3-caused inflammation also induced the accumulation of CD27- and CD70-positive cells in both tissue (E and F). Original magnification, ×1575.

FIG. 5

Model for the putative induction of apoptosis in CD27/Siva-mediated pathways (A) and possible role of VP2 in CD27/Siva-mediated apoptotic events after CVB3 infection (B).