Core Protein of Pestiviruses Is Processed at the C Terminus by Signal Peptide Peptidase

Abstract

The core protein of pestiviruses is released from the polyprotein by viral and cellular proteinases. Here we report on an additional intramembrane proteolytic step that generates the C terminus of the core protein. C-terminal processing of the core protein of classical swine fever virus (CSFV) was blocked by the inhibitor (Z-LL) 2 -ketone, which is specific for signal peptide peptidase (SPP). The same effect was obtained by overexpression of the dominant-negative SPP D 265 A mutant. The presence of (Z-LL) 2 -ketone reduced the viability of CSFV almost 100-fold in a concentration-dependent manner. Reduction of virus viability was also observed in infection experiments using a cell line that inducibly expressed SPP D 265 A. The position of SPP cleavage was determined by C-terminal sequencing of core protein purified from virions. The C terminus of CSFV core protein is alanine 255 and is located in the hydrophobic center of the signal peptide. The intramembrane generation of the C terminus of the CSFV core protein is almost identical to the processing scheme of the core protein of hepatitis C viruses.Pestiviruses are small, enveloped RNA viruses that account for important diseases in farm animals, e.g., classical swine fever (also known as hog cholera) and bovine viral diarrhea. Pestiviruses constitute one genus within the Flaviviridae and contain a message sense RNA of about 12.3 kilobases. The genome is translated into a single polyprotein that is processed by cellular and viral proteases into 12 mature proteins. The virion consists of four structural proteins, the core protein and the glycoproteins E rns , E1, and E2 (19). The core protein of all members of the Flaviviridae is a small protein rich in basic amino acids and locates at or near the N terminus of the polyprotein (11). In the pestivirus polyprotein, the core protein is located between the N-terminal protease N pro and the glycoprotein E rns . N pro generates the N terminus (Ser 169 ) of the core protein by autocatalytic cleavage of the polyprotein (15, 18). Core protein is followed by E rns , whose N terminus (Asp 268 ) is generated by signal peptidase (signalase, or SP) (16).Two different mechanisms of release of core protein from the polyprotein have been described for members of the Flaviviridae. Members of the genus Flavivirus (e.g., yellow fever virus [YFV]) employ the virally encoded NS2B-3 protease to generate the C terminus of core protein. The NS2B-3 protease of YFV cleaves the core protein precursor at a tribasic consensus sequence at the N terminus of the preM signal peptide (1). For hepatitis C virus (HCV), signal peptide peptidase (SPP) was recently determined to cleave within the C-terminal domain of core protein (7). Thus far, three different C termini have been proposed for HCV core protein. N-terminal sequencing of HCV core-dihydrofolate reductase fusion proteins revealed cleavage after Leu 179 or Leu 182 (4). In a recent report, Phe 177 was identified as the C terminus of HCV core protein by using matrix-assisted laser desorption ion...

Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment

“…Sequential processing results in discrete intermediate products, which sometimes have functions that the terminal products do not have, and thus provides a means for regulating functions needed at different stages of the virus infectious cycle. The cellular protease SPP has been co-opted by HCV, the closely related GB virus B and Classical swine fever virus for maturation of their core proteins [13,18,42]. Recent data in the HCV model have shown that SPP-catalysed liberation of mature core protein is required for production of infectious progeny virus at a step which remains to be defined, perhaps related to coupling of viral genome replication and virus assembly [19,33].…”

“…Signal peptides are indicated by hatched boxes. The scissors indicate the sites of cleavages catalysed by SP; the arrowheads indicate the sites proposed for the cleavage catalysed by signal peptide peptidase (SPP) to liberate p21 [14][15][16][17][18][19]. The sequence at the C-terminus of core protein is presented for the wild-type HCV strains of genotype 1a and 1b used in this study.…”

“…The SPP cleavage site has been proposed to lie between amino-acids 173 and 182 (Fig. 1); when expressed as a single protein in non hepatic cells, HCV core protein was recently shown to end with Phe 177 , but definitive determination of the C-terminus of mature core protein awaits confirmation in a more relevant context [14][15][16][17][18][19]. Based on their apparent molecular masses (aMMs) in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), the complete and mature forms of HCV core protein are usually referred to as p23 and p21 respectively [9].…”

Signal Peptide Peptidase Cleavage of GB Virus B Core Protein Is Required for Productive Infection in Vivo

“…HCV core is processed further by SPP within the h-region of the signal sequence to generate a mature product, which is considered to participate in virion morphogenesis (30). SPP cuts also in an analogous position in classical swine fever virus (CSFV), a pestivirus that is related to HCV (31). To determine whether GBV-B core is a substrate for SPP, a polyprotein encompassing GBV-B core to the N-terminal region of NS2 and including an epitope tag inserted within the core sequence (Fig.…”

An Overview of the Immune Evasion Strategies Adopted by Different Viruses with Special Reference to Classical Swine Fever Virus

“…Nevertheless, a recent study [62] indicated that recombination between strains is possible. The ORF is translated into a single polypeptide of about 3900 amino acids which is co-and post-translationally processed into mature peptide by a number of virus and host encoded proteases [63,64,65,66]. The virion is made up of 4 structural proteins viz., C, Erns, E1 and E2 which are encoded at the 5' end of the genome.…”