New insights into processing of bovine viral diarrhea virus glycoproteins Erns and E1

Abstract

Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus within the family Flaviviridae. Its single-stranded RNA encodes a polyprotein that is cleaved co-and posttranslationally by viral and cellular proteases. However, the cleavage between the envelope proteins E rns and E1 is still unexplained. In this study, an E rns -E1 protein could be identified and characterized with a new E1-specific antiserum. With bicistronic constructs bearing a deletion in the E rns -encoding region and expressing E rns or the E rns -E1 protein, it could be shown that this protein is not essential for virus replication. Furthermore, two putative cleavage sites were mutated in eukaryotic expression plasmids, as well as in full-length cDNA constructs. The mutation of position P3 of a potential signal peptide peptidase site abolished cleavage completely and no infectious virus progeny could be observed, indicating that cleavage of the E rns -E1 protein is indispensable for virus growth.Bovine viral diarrhea virus (BVDV), the causative agent of an economically important disease of cattle worldwide, belongs, like classical swine fever virus and border disease virus, to the genus Pestivirus within the family Flaviviridae (Collett et al., 1988a;Fauquet et al., 2005;Houe, 1999;Korn, 1977;Pringle, 1998). The genome is a singlestranded RNA of positive polarity with a size of about 12.3 kb. One large open reading frame (ORF) encodes a polyprotein that is cleaved co-and post-translationally by viral and cellular proteases (Collett et al., 1988b; Rümenapf et al., 1993). The ORF is flanked by 59 and 39 untranslated regions (UTRs) that are important for virus replication (Collett et al., 1988a;Yu et al., 1999Yu et al., , 2000. An internal ribosomal entry site (IRES) within the 59 UTR allows capindependent translation of the virus polyprotein (Pestova & Hellen, 1999; Poole et al., 1995;Yu et al., 2000). The virions consist of four structural proteins (C, E rns , E1 and E2). The basic protein C was believed to form a capsid that surrounds the virus genome, but recent studies by Rümenapf et al. (2008) have shown that it is dispensable for virus assembly (Gray & Nettleton, 1987;Thiel et al., 1991). The three glycosylated proteins E rns , E1 and E2 are located in the lipid membrane of cellular origin (Chu & Zee, 1984;Coria et al., 1983;Fetzer et al., 2005). The envelope protein E rns , which lacks a typical membrane anchor, seems to be important for first cell contact by binding to glycosaminoglycans (Fetzer et al., 2005;Iqbal et al., 2000). The cellular receptor for BVDV is the bovine CD46 molecule and it has recently been shown that E1-E2 heterodimers are essential for virus entry (Maurer et al., 2004;Ronecker et al., 2008). The envelope proteins E rns , E1 and E2 are processed from the polyprotein in a hierarchical way, starting with the translocation of the C-terminal signal peptide downstream of the capsid protein into the endoplasmic reticulum (ER), followed by the release of the capsid and E2 protein (Heimann et al., 2006; Rümenap...

A New Type of Signal Peptidase Cleavage Site Identified in an RNA Virus Polyprotein

“…It could be hypothesized that the E rns E1 fusion protein has a biological function in the virus life cycle, but the fact that viruses with an artificial deletion of the E rns -coding sequence can be efficiently complemented with E rns in trans argues against this hypothesis (21,57,58). On the other hand, it has recently been shown that processing of E rns E1 is crucial for pestivirus viability (59).…”

“…Because the E rns /E1 processing site lacks a hydrophobic h-region, which is regarded as a crucial part of a SPase cleavage site, it was speculated whether the delayed processing at this site was due to processing in a downstream compartment of the secretory pathway (9,59) or resulted from inefficient SPase cleavage in consequence of the unusual structure. As reported here, the E rns E1 precursor is cleaved in the ER.…”

The Molecular Biology of Pestiviruses

“…Some antisera have been generated against E1 and used to elucidate E rns -E1 processing (Bintintan & Meyers, 2010;Wegelt, Reimann, Zemke, & Beer, 2009). The C-terminus of E rns contains a classical von Heijne cleavage motif at positions À3 and À1, but lacks the hydrophobic region that is normally required for SPase recognition, due to the special membrane anchor of E rns .…”

Emergence of 2.1. subgenotype of classical swine fever virus in pig population of India in 2011

“…The genome of CSFV contains a single large open reading frame (ORF), which is flanked by 5 0 and 3 0 nontranslated regions. This ORF codes for a polyprotein of approximately 3898 amino acids, which is processed co-and post-translationally by both viral and host cell proteases to obtain four structural (C, E RNS , E1 and E2) and nine non-structural (N pro , p7, NS2, NS3, NS2-3, NS4A, NS4B, NS5A and NS5B) proteins (Meyers and Theil 1996;Wegelt et al 2009). Though CSFV forms a single serotype, it can be divided into three genotypes (1, 2 and 3), each comprising three or four subgenotypes (1.1.À1.3.…”