Signal peptidase cleavage at the flavivirus C-prM junction: dependence on the viral NS2B-3 protease for efficient processing requires determinants in C, the signal peptide, and prM

Abstract

Signal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this regulation of a luminal cleavage by a cytoplasmic protease. We found that the inefficiency of signal peptidase cleavage in the absence of the viral protease is not attributable solely to features of the C protein. Inhibition of cleavage still occurred when charged residues in C were mutated to uncharged residues or when an unrelated protein sequence (that of ubiquitin) was substituted for C. Also, fusion of the C protein did not inhibit processing of an alternative adjacent signal sequence. The cleavage region of the flavivirus prM translocation signal is unusually hydrophobic, and we established that altering this characteristic by making three point mutations near the signal peptidase cleavage site in MVE prM dramatically increased the extent of cleavage without requiring removal of the C protein. In addition, we demonstrated that luminal sequences downstream from the signal peptidase cleavage site contributed to the inefficiency of cleavage.

FIG. 1

Schematic diagram showing the locations of alterations to charged residues in derivatives of the MVE C protein. Plasmid pSTR encodes the C-prM-E-NS1-NS2A-6%NS2B region of the MVE polyprotein. The C protein is represented by a solid line, and the beginning of the prM translocation signal is indicated by a dashed line. Positions of charged residues are indicated by + for Arg or Lys and − for Asp or Glu. Residues 1 to 23 and 97 to 105 of the C protein encoded by this construct are shown in single-letter amino acid code. Residues in these regions which are unchanged in the charge mutant derivatives of pSTR are denoted by dots. Sequences outside the marked regions were identical in all constructs. The site of cleavage by the viral NS2B-3 protease is marked with an arrow.

FIG. 2

Substitution of basic residues in the C protein does not influence signal peptidase cleavage at the C-prM junction. (A) COS cells were transfected with pSTR (lanes 1 and 5), pSTR.Cmut2aa (lanes 2 and 6), pSTR.Cmut4aa (lanes 3 and 7), or pSTR.Cmut11aa (lanes 4 and 8), with (+) or without (−) pNS3/T. Lane 9 shows the results of transfection with pNS3/T only. Two days after transfection, the monolayers were metabolically labelled for 30 min and cell lysates were subjected to immunoprecipitation with anti-MVE mouse ascitic fluid. Polypeptides were separated by SDS-PAGE (12% polyacrylamide). Bands corresponding to MVE E, NS1, prM, and putative C-prM glycoproteins are labelled, and the numbers indicate the sizes (in kilodaltons) of marker proteins in lane M. (B) Schematic diagrams showing the expected topology at the ER membrane of polyproteins encoded by pSTR variants in the presence and absence of NS2B-3. M denotes the ER membrane, L denotes the ER lumen, and Cy denotes the cytoplasmic side of the membrane. Sites of efficient cleavage by signal peptidase are indicated by arrowheads, the proteolytic cleavage catalyzed by the viral NS2B-3 complex is denoted by an arrow, and regions of the MVE C protein which were altered in the various pSTR.Cmut constructs are indicated by circles in parentheses.

FIG. 3

Replacement of the C protein with ubiquitin at the NH₂ terminus of the prM signal sequence does not alter the efficiency of prM cleavage by signal peptidase. (A) COS cells were transiently transfected with the ubiquitin fusion construct pSTR.UbmutV (lanes 1 and 4) or pSTR.Ub (lanes 2 and 5), in which the cleavage site for ubiquitin-specific proteases was destroyed or left intact, respectively, or with pNS3/T (lanes 3 and 6). The cells were metabolically labelled for 30 min, and polypeptides immunoprecipitated from lysates with anti-MVE mouse ascitic fluid (lanes 1 to 3) or antiserum against a ubiquitin–glutathione S-transferase fusion protein (lanes 4 to 6) were separated by SDS-PAGE (12% polyacrylamide) (lanes 1 to 3) or SDS-PAGE (15% polyacrylamide) (lanes 4 to 6). Bands corresponding to MVE E, NS1, and prM glycoproteins, as well as ubiquitin (Ub) and putative Ub-prM fusion products, are labelled, and the numbers indicate the sizes (in kilodaltons) of marker proteins in lane M. (B) Schematic diagrams showing the expected topology at the ER membrane of polyproteins encoded by pSTR.UbmutV and pSTR.Ub. Abbreviations are as listed in the legend to Fig. 2. Ubiquitin-specific protease cleavage is indicated by a dotted arrow, and a solid circle shows the approximate position of the Gly⁷⁶→Val substitution mutation.

FIG. 4

NH₂-terminal linkage of the MVE C protein to an idealized signal sequence does not inhibit cleavage by signal peptidase. (A) COS cells transfected with pSFVΔE3.MVE-C (lane 1), pSFVΔE3 (lane 3), or pNS3/T (lane 4) or cotransfected with pSFVΔE3.MVE-C and pNS3/T (lane 2) were metabolically labelled for 3 h. The products immunoprecipitated by an anti-SFV mouse serum were separated by SDS-PAGE (10% polyacrylamide) under nonreducing conditions to separate E2 and E1. Bands corresponding to the SFV E2 and E1 glycoproteins are shown, and the numbers indicate the positions and sizes (in kilodaltons) of marker proteins. (B) Schematic diagrams showing the expected topology at the ER membrane of polyproteins encoded by pSFVΔE3.MVE-C and pSFVΔE3. Abbreviations are as listed in the legend to Fig. 2. Autocatalytic cleavage of the unmutated SFV C protein is indicated by a curved arrow.

FIG. 5

Alignment of flavivirus amino acid sequences around the C-prM junction. The MVE prM translocation signal with flanking sequences encoded by pSTR is shown at the top, with the sequences we have considered to be the h- and c-regions indicated. Corresponding sequences are also shown for yellow fever virus (YF) (32), Japanese encephalitis virus (JE) (38), Kunjin virus (KUN) (7), tick-borne encephalitis virus (TBE) (26), dengue virus type 2 (DEN2) (13) and West Nile virus (WN) (6). Large arrows indicate sites of cleavage by the viral NS2B-3 protease complex and signal peptidase. Small arrows show sites at which cleavage potential scores based on the algorithm of von Heijne (39) are greater than an arbitrary value of 4. Positively (+) and negatively (−) charged amino acid residues are labelled.

FIG. 6

Mutations in the signal sequence of prM induce efficient signal peptidase cleavage independent of the presence of the NS2B-3 protease. (A) COS cells transiently transfected with pSTR (lane 1), pSTR.mutPQAQA (lane 2), or pSTR.ΔC (lane 3) were metabolically labelled for 3 h. Polypeptides immunoprecipitated from cell lysates with anti-MVE mouse ascitic fluid were resolved by SDS-PAGE (12% polyacrylamide). Bands corresponding to MVE E, NS1, and prM glycoproteins are labelled, and the numbers indicate the positions and sizes (in kilodaltons) of marker proteins. (B) Schematic diagrams showing the expected topology at the ER membrane of polyproteins encoded by pSTR.mutPQAQA and pSTRΔC. Abbreviations are as listed in the legend to Fig. 2. The location of the altered amino acid residues encoded by pSTR.mutPQAQA is represented by a solid circle.

FIG. 7

The production of MVE E from a C_anch-E fusion protein is dependent on the viral protease. (A) COS cells transiently transfected with pC_anch-E (lanes 1 and 2) or pSTR (lanes 3 and 4), in the absence (−) or presence (+) of pNS3/T, were metabolically labelled for 30 min. Products immunoprecipitated from the lysates with anti-MVE E monoclonal antibody M2-8E7 (14) were examined by SDS-PAGE (12% polyacrylamide). Positions of E, prM (coprecipitated as part of a prM-E heterodimer), and putative C_anch-E fusion glycoproteins are labelled, and the numbers indicate the positions and sizes (in kilodaltons) of marker proteins. (B) Schematic diagram showing the expected topology at the ER membrane of the polyprotein encoded by pC_anch-E. Abbreviations are as listed in the legend to Fig. 2.

FIG. 8

The production of H-2K^d from a C_anch-K^d fusion protein is not dependent on the viral protease. (A) COS cells were transiently transfected with pK^d (lane 1), pC_anch-K^d (lanes 2 and 3), or pC_anch-K^dmutK (lanes 4 and 5), with (+) or without (−) pNS3/T. Lane 6 shows the results of transfection with pNS3/T only. Cell monolayers were pulse-labelled for 30 min, and the products immunoprecipitated from cell lysates with an anti-K^d monoclonal antibody (Hb159) were subjected to SDS-PAGE (12% polyacrylamide). Bands corresponding to the K^d and possible C_anch-K^d fusion proteins are labelled, and the numbers indicate the positions and sizes (in kilodaltons) of marker proteins. (B) Schematic diagrams showing the expected topology at the ER membrane of polyproteins encoded by pK^d, pC_anch-K^d and pC_anch-K^dmutK. Abbreviations are as listed in the legend to Fig. 2, and the Pro→Lys substitution encoded in pC_anch-K^dmutK is represented by a solid circle.