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

Abstract

Pestiviruses, a group of enveloped positive strand RNA viruses belonging to the family Flaviviridae, express their genes via a polyprotein that is subsequently processed by proteases. The structural protein region contains typical signal peptidase cleavage sites. Only the site at the C terminus of the glycoprotein E rns is different because it does not contain a hydrophobic transmembrane region but an amphipathic helix functioning as the E rns membrane anchor. Despite the absence of a hydrophobic region, the site between the C terminus of E rns and E1, the protein located downstream in the polyprotein, is cleaved by signal peptidase, as demonstrated by mutagenesis and inhibitor studies. Thus, E rns E1 is processed at a novel type of signal peptidase cleavage site showing a different membrane topology. Prevention of glycosylation or introduction of mutations into the C-terminal region of E rns severely impairs processing, presumably by preventing proper membrane interaction or disturbing a conformation critical for the protein to be accepted as a substrate by signal peptidase. Classical swine fever virus (CSFV)2 belongs to the genus Pestivirus, which also includes the animal viruses bovine viral diarrhea virus and border disease virus of sheep. The genus Pestivirus is part of the family Flaviviridae which also comprises the genera Flavivirus and Hepacivirus (1).Pestiviruses are positive strand RNA viruses with a singlestranded genome of ϳ12.3 kb in length that contains a single open reading frame coding for a polyprotein of about 4000 amino acids (2). The polyprotein is co-and posttranslationally processed by cellular and viral proteases into at least 12 mature proteins (3-12), arranged in the polyprotein in the following order: NH 2 -N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B-COOH. C, E rns , E1, and E2 are part of the virion (13,14), with C forming the capsid, and E rns , E1, and E2 representing glycoproteins associated with the virus envelope. E rns and E2 elicit neutralizing antibody responses that can lead to protective immunity (15-18).The pestivirus E rns protein is a highly unusual protein. It forms a disulfide-linked dimer of ϳ90 kDa, about half of which is due to glycosylation (9,13,19). The protein displays homology to the RNases of the T 2 /S superfamily (20 -22). In different test systems, it was shown that E rns indeed has RNase activity, a feature that is unique among viral glycoproteins (22-25). The protein is essential for virus growth (21), but the RNase activity is dispensable. Viruses, in which the RNase activity has been knocked out, are clinically attenuated (26, 27). A role for E rns in immune evasion has been proposed (28 -31). E rns can be found in virus-free cell culture fluid of infected cells and in the blood of infected animals (9, 29, 32). It lacks a hydrophobic region that could serve as a transmembrane anchor, and there is also no consensus sequence for glycosylphosphatidylinositol anchor addition. It is nevertheless bound to the virion and associated with ...

RNA recombination in pestiviruses: Cellular RNA sequences in viral genomes highlight the role of host factors for viral persistence and lethal disease

“…12,13 The latter enzyme also releases the structural glycoproteins E rns (envelope protein ribonuclease secreted), E1 and E2 as well as the putative viroporin p7. 12,[14][15][16] The RNase activity of E rns , which is also secreted from infected cells, is a unique feature for a structural viral glycoprotein and is believed to manipulate the innate immune response of the host. 17,18 Nonstructural protein 2 (NS2) encompasses a cysteine protease domain which catalyzes the cleavage between NS2 and NS3.…”

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

“…CSFV possesses a singlestranded positive-sense RNA genome of approximately 12.3 kb with one large open reading frame flanked by 5= and 3= untranslated regions (UTRs) and coding for a polyprotein of approximately 4,000 amino acids (30). Co-and posttranslational processing of the polyprotein by cellular and viral proteases yields the 12 cleavage products N pro , C, E rns , E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2,3,4,6,16,17,26,45,53,59,60). The structural components of the CSFV virion include the capsid (C) protein and the glycoproteins E rns , E1, and E2.…”

Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

“…SPP activities also involve the intramembrane cleavage of the core proteins of hepatitis C virus, GB virus B, and classical swine fever virus (family Flaviviridae) (20,21).…”