Inhibition of Na+/H+ Exchanger Activity by Parvovirus B19 Protein NS1

Abstract

Infection with parvovirus B19 (B19) may induce apoptosis resulting in anemia, acute fulminant liver failure, placental insufficiency and myocarditis. Apoptosis has been attributed to proapoptotic activity of the non-structural viral protein NS1, which is known to trigger a signaling cascade eventually leading to activation of caspases. In several cell types apoptosis was found to be paralleled by profound cytosolic acidification, which may be secondary to inhibition of the Na⁺/H⁺ exchanger. The acidification has been considered to support the activation of pH sensitive caspases and endonucleases. However, nothing is known about the effect of NS1 on Na⁺/H⁺ exchanger activity and cytosolic pH. The present study thus explored whether NS1 expression affects cytosolic pH (pHi) and Na⁺-dependent realkalinization (ΔpHi) following acidification by an ammonium pulse. According to FACS analysis, overexpression of NS1 in RXR-10SW cells led within 72 hours to activation of caspase 3 and DNA fragmentation. NS1 overexpression resulted within 24 hours in a significant decline of pHi from 6.93 ± 0.03 (n = 6) to 6.78 ± 0.04 (n = 7), and to a significant decrease of ΔpHi from 0.159 ± 0.017 (n = 6) to 0.039 ± 0.004, (n = 7). The decrease of pHi and of ΔpHi following NS1 expression could be significantly blunted by inhibition of caspase 3 with zVAD. Western blot analysis revealed degradation of NHE1 following NS1 expression. In vitro, caspase 3, but not caspase 6, caspase 7 and caspase 8 degraded NHE1 protein of cell lysates. In conclusion, overexpression of NS1 triggers a signaling cascade eventually leading to activation of caspase 3 and subsequent degradation of NHE1. The effect contributes to cytosolic acidification which may in turn favor activation of caspases and endonucleases and thus participate in the pathophysiology of B19-infection.