Downstream Sequences Control the Processing of the Pestivirus Erns-E1 Precursor.

Like other enveloped viruses, pestiviruses employ cellular proteases for processing of their structural proteins. While typical signal peptidase cleavage motifs are present at the carboxy terminus 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 the E1 sequence downstream of this cleavage site is also important for the cleavage. Carboxy-terminal truncation of the E1 moiety as well as internal deletions in E1 reduced the cleavage efficiency to less than 30% of the wild-type (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 were even observed when 10 to 5 amino-terminal residues of 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 Erns-E1 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 carboxy terminus 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.

A new type of signal peptidase cleavage site identified in an RNA virus polyprotein.

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.