Entry of bovine parainfluenza virus type 3 into MDBK cells occurs via clathrin-mediated endocytosis and macropinocytosis in a acid-dependent manner.

Bovine parainfluenza virus 3 (BPIV3) is an important respiratory pathogen of both young and adult cattle. No specific therapies are available for BPIV3. Understanding the viral internalization pathway of BPIV3 will provide new strategies for the development of antiviral treatments. Here, the entry mechanism of BPIV3 into MDBK cells was analyzed using chemical inhibitors and RNA silencing. Our data demonstrated that treatment with an inhibitor targeting the clathrin-mediated pathway or clathrin heavy chain (CHC) knockdown suppressed the entry of BPIV3 into MDBK cells. In contrast, sequestration of cellular cholesterol by nystatin or silencing of caveolin-1 had no effect on viral entry. Moreover, inhibition of critical modulators of macropinocytosis significantly reduced BPIV3 uptake. In addition, fluid-phase uptake was significantly increased in cells infected with BPIV3, which is indicative of virus-induced facilitation of macropinocytosis. These results suggest that BPIV3 enters MDBK cells via macropinocytosis and clathrin- but not caveolar-dependent endocytosis. Furthermore, inhibition of endosomal acidification and activation of cathepsin blocked BPIV3 entry, demonstrating that BPIV3 entered MDBK cells in a acid-dependent manner and required cathepsin L. Finally, we demonstrated that macropinocytosis but not clathrin-mediated endocytosis is dependent on actin dynamics during BPIV3 infection.