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 ...
Like other enveloped viruses pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxyterminus of the signal sequence preceding Erns, and the E1/E2 and E2/P7 sites, the Erns-E1 precursor is cleaved by signal peptidase at a highly unusual structure, in which the transmembrane sequence upstream of the cleavage site is replaced by an amphipathic helix. As shown before, the integrity of the amphipathic helix is crucial for efficient processing. The data presented here demonstrate that also the E1 sequence downstream of this cleavage site is important for the cleavage. Carboxyterminal truncation of the E1 moiety as well as internal deletions in E1 reduced the cleavage efficiency to less than 30% of the wt level. Moreover, the C-terminal truncation by more than 30 amino acids resulted in strong secretion of the uncleaved fusion proteins. The reduced processing and increased secretion was even observed when 10 to 5 aminoterminal residues or E1 were left whereas extensions by 1 or 3 E1 residues resulted in reduced processing but no significantly increased secretion. In contrast to the E1 sequences, a 10 amino acid c-myc tag fused to the Erns C-terminus had only marginal effect on secretion but was also not processed efficiently. Mutation of the von Heijne sequence upstream of E2 not only blocked the cleavage between E1 and E2 but also prevented the processing between Erns and E2. Thus, processing at the ErnsE1 site is a highly regulated process. IMPORTANCE Cellular signal peptidase (SPase) cleavage represents an important step in maturation of viral envelope proteins. Fine tuning of this system allows for establishment of concerted folding and processing processes in different enveloped viruses. We report here on SPase processing of the Erns-E1-E2 glycoprotein precursor of pestiviruses. Erns-E1 cleavage is delayed and only executed efficiently when the complete E1 sequence is present. C-terminal truncation of the Erns-E1 precursor impairs processing and leads to significant secretion of the protein. The latter is not detected when internal deletions preserving the E1 carboxyterminus are introduced, but also these constructs show impaired processing. Moreover, Erns-E1 is only processed after cleavage at the E1/E2 site. Thus, processing of the pestiviral glycoprotein precursor by SPase is done in an ordered way and depends on the integrity of the proteins for efficient cleavage. The functional importance of this processing scheme is discussed in the paper.
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