Transdominant Inhibition of Bovine Viral Diarrhea Virus Entry

Abstract

Bovine viral diarrhea virus (BVDV) is a positive-strand RNA virus and a member of the genus Pestivirus in the family Flaviviridae. To identify and characterize essential factors required for BVDV replication, a library expressing random fragments of the BVDV genome was screened for sequences that act as transdominant inhibitors of viral replication by conferring resistance to cytopathic BVDV-induced cell death. We isolated a BVDV-nonpermissive MDBK cell clone that harbored a 1.2-kb insertion spanning the carboxy terminus of the envelope glycoprotein 1 (E1), the envelope glycoprotein E2, and the amino terminus of p7. Confirming the resistance phenotype conferred by this library clone, naïve MDBK cells expressing this fragment were found to be 100-to 1,000-fold less permissive to both cytopathic and noncytopathic BVDV infection compared to parental MDBK cells, although these cells remained fully permissive to vesicular stomatitis virus. This restriction could be overcome by electroporation of BVDV RNA, indicating a block at one or more steps in viral entry prior to translation of the viral RNA. We determined that the E2 ectodomain was responsible for the inhibition to BVDV entry and that this block occurred downstream from BVDV interaction with the cellular receptor CD46 and virus binding, suggesting interference with a yet-unidentified BVDV entry factor.

The Molecular Biology of Pestiviruses

“…In further studies, the viral binding site could be mapped to two short peptides in the complement control protein module 1 of CD46 bov (Krey et al, 2006). However, CD46 is not sufficient for pestivirus entry since cells expressing E2 are resistant to BVDV infection, although they have similar levels of CD46 expression and bind BVDV to a similar extent as control cells (Tscherne, Evans, Macdonald, & Rice, 2008). CD46 is excluded from endosomes, but BVDV enters by clathrindependent endocytosis (Grummer, Grotha, & Greiser-Wilke, 2004;Krey et al, 2005;Maurer et al, 2004).…”

Crystal structure of glycoprotein E2 from bovine viral diarrhea virus

“…In HCV, as in pestiviruses, E1 is half the size of E2, which is the immunodominant protein and binds a cellular receptor that is not efficiently internalized (8,9). HCV and pestiviruses both appear to require one or more coreceptors for postattachment internalization and membrane fusion (10,11). HCV and pestiviruses are also both unusually resistant to acid outside the cell yet depend on low pH and an additional activation step for fusion (6,7,12).…”

“…Inhibition of cell entry by endocytic inhibitors suggests that pesti-and hepaciviruses enter the cell by clathrin-mediated endocytosis (6,7). However, pestiviruses and hepaciviruses recognize host cells by binding to cell surface receptors-CD46 and CD81, respectively-that are not significantly internalized (8,9), and one or more coreceptors are required for postattachment internalization (10,11). To deliver their single-stranded positive-sense RNA genome into the cytoplasm, pestiviruses and HCV must fuse their lipid envelope with a cellular membrane.…”

“…To deliver their single-stranded positive-sense RNA genome into the cytoplasm, pestiviruses and HCV must fuse their lipid envelope with a cellular membrane. The reduced pH of endocytic compartments is required but not sufficient to initiate membrane fusion of these viruses (6,7,10,12,13). Moreover, viruses from both families are acid resistant before endocytosis, and require a poorly understood activation step to become fusogenic at endosomal pH (6,7,12).…”

Selection of Classical Swine Fever Virus with Enhanced Pathogenicity Reveals Synergistic Virulence Determinants in E2 and NS4B

“…level of viral attachment and entry, as the glycoproteins E2 and E rns of pestiviruses mediate viral attachment and entry into host cells (20,41,56). Unfortunately, there is only limited information available on the receptors and pathways involved in CSFV binding and entry.…”