Abstract
Bovine parainfluenza virus 3 (BPIV3) is a crucial causative agent of respiratory disease in young and adult cattle. No specific therapies are available for BPIV3 infection. Understanding the internalization pathway of the virus will provide a new strategy for the development of antiviral therapy. Here, the mechanism of BPIV3 entry into HeLa cells was analyzed using RNA silencing and pharmacological inhibitors. Treatment of HeLa cells with hypertonic medium prevented BPIV3 internalization. These results indicated that BPIV3 entered HeLa cells via receptor-mediated endocytosis. Moreover, removing cell membrane cholesterol through MβCD treatment hampered viral penetration but not viral replication. In addition, BPIV3 infection was inhibited by pretreatment with dynasore or chlorpromazine (CPZ) or knockdown of dynamin II or clathrin heavy chain. However, virus entry was unaffected by nystatin, EIPA, wortmannin, or cytochalasin D treatment or caveolin-1 knockdown. These data demonstrated that the entry of BPIV3 into HeLa cells was dependent on clathrin-mediated endocytosis but not on caveolae-mediated endocytosis or the macropinocytosis pathway. Many viruses are transported to endosomes, which provide an acidic environment and release their genome upon separation from primary endocytic vesicles. However, we found that BPIV3 infection required endosomal cathepsins, but not a low pH. In summary, we show, for the first time, that BPIV3 enters HeLa cells through the clathrin-mediated endocytosis pathway, presenting novel insights into the invasion mechanism of Paramyxoviridae.
Highlights
Bovine parainfluenza virus type 3 (BPIV3), a member of the Paramyxoviridae family, is one of the most critical pathogens correlated to bovine respiratory disease complex (BRDC) in cattle [1]
These results demonstrated that BPIV3 entered HeLa cells in a dynamin-dependent manner
These results demonstrated that BPIV3 penetration into penetration into HeLa cells was clathrin-dependent
Summary
Bovine parainfluenza virus type 3 (BPIV3), a member of the Paramyxoviridae family, is one of the most critical pathogens correlated to bovine respiratory disease complex (BRDC) in cattle [1]. Viruses can utilize multiple endocytic pathways to penetrate cells These main routes of endocytosis include clathrin-mediated endocytosis (CME), caveolae-mediated endocytosis (CavME), macropinocytosis, and clathrin- and caveolin-independent endocytosis [3]. Facilitated by dynamin GTPase, CCPs separate from the plasma membrane and form mature vesicles coated with clathrin [9]. Another intensively studied pathway is caveolae-mediated endocytosis, which is characterized by the presence of caveolin [10]. Due to the smallness of the vesicles that are endocytosed in association with clathrin or caveolin-1, some large viral particles undergo cellular internalization through other mechanisms, such as macropinocytosis
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