Downstream Sequences Control the Processing of the Pestivirus E ^rns -E1 Precursor

Abstract

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.

Charged Residues in the Membrane Anchor of the Pestiviral Erns Protein Are Important for Processing and Secretion of Erns and Recovery of Infectious Viruses

“…Loss of single charged amino acids has only minor or moderate effects and even mutation of several charged residues does not block E rns -E1 processing completely. The maximal effect on E rns secretion rate in consequence of multiple exchanges was in the same range as observed for insertions impairing the amphipathic character of the E rns carboxy-terminus [ 27 ] or carboxy-terminal deletions of the E1 moiety in the E rns -E1 precursor [ 38 ]. The general level of the observed effects on processing and retention, and especially its dependence on the position of the alteration in the amphipathic helix do not support an obvious general importance of the conserved charge distribution.…”

“…Since the in-plane orientation of the amphipathic helix stands in marked contrast to the standard arrangement of SP cleavage substrates [ 31 , 32 , 33 , 34 ], the E rns /E1 site represents a highly unusual SP cleavage site. We have shown before that the integrity of the amphipathic helix as well as the presence of a full length E1 are crucial for efficient E rns -E1 processing [ 37 , 38 ]. The finding of a conserved set of complementarily charged amino acids in the amphipathic helix able to form a so-called charge zipper seemed to shed some light on the unusual processing mechanism.…”

“…The stronger influence of the mutations on secretion might reflect a kinetic effect. As we have shown before, the E rns -E1 precursor is bound to the ER membrane via the transmembrane structure at the carboxy-terminus of E1 [ 38 ]. Thus, processing can be achieved over a longer time period so that also suboptimal sites could be cleaved with rather good activity.…”

“…The plasmids SE rns and SE rns -E1 used in expression studies (originally named SSeqE rns and SSeqE rns -E1 containing the E rns and E rns E1 coding sequences of CSFV Alfort/Tübingen [ 37 , 38 ]) were used as basis for the mutant expression constructs listed in Table 1 . Single and multiple point mutations were introduced with standard PCR based methods with thermostable Pfu polymerase (Promega, Heidelberg, Germany) and synthetic primers (QuickChange mutagenesis) in one single reaction or consecutive approaches.…”

“…This unusual site is nevertheless cleaved by SP, but cleavage is delayed and very sensible to amino acid changes affecting the amphipathic helix [ 10 , 26 , 27 , 37 ]. Moreover, the presence of a full-length E1 protein cleaved at its carboxy-terminus by SP is crucial for (efficient) processing at the E rns /E1 site [ 38 ].…”