Crimean-Congo hemorrhagic fever virus glycoprotein proteolytic processing by subtilase SKI-1

Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne member of the genus Nairovirus, family Bunyaviridae. The mature virus glycoproteins, Gn and Gc (previously referred to as G2 and G1), are generated by proteolytic cleavage from precursor proteins. The amino termini of Gn and Gc are immediately preceded by tetrapeptides RRLL and RKPL, respectively, leading to the hypothesis that SKI-1 or related proteases may be involved (A. J. Sanchez, M. J. Vincent, and S. T. Nichol, J. Virol. 76:7263-7275, 2002). In vitro peptide cleavage data show that an RRLL peptide representing the Gn processing site is efficiently cleaved by SKI-1 protease, whereas an RKPL peptide representing the Gc processing site is cleaved at negligible levels. The efficient cleavage of RRLL peptide is consistent with the known recognition sequences of SKI-1, including the sequence determinants involved in the cleavage of the Lassa virus (family Arenaviridae) glycoprotein precursor. These in vitro findings were confirmed by expression of wild-type or mutant CCHF virus glycoproteins in CHO cells engineered to express functional or nonfunctional SKI-1. Gn processing was found to be dependent on functional SKI-1, whereas Gc processing was not. Gn processing occurred in the endoplasmic reticulum-cis Golgi compartments and was dependent on an R at the -4 position within the RRLL recognition motif, consistent with the known cleavage properties of SKI-1. Comparison of SKI-1 cleavage efficiency between peptides representing Lassa virus GP2 and CCHF virus Gn cleavage sites suggests that amino acids flanking the RRLL may modulate the efficiency. The apparent lack of SKI-1 cleavage at the CCHF virus Gc RKPL site indicates that related proteases, other than SKI-1, are likely to be involved in the processing at this site and identical or similar sites utilized in several New World arenaviruses.     

Crimean-Congo hemorrhagic fever virus glycoprotein processing by the endoprotease SKI-1/S1P is critical for virus infectivity

Crimean-Congo hemorrhagic fever virus (CCHFV) causes severe human disease. The CCHFV medium RNA encodes a polyprotein which is proteolytically processed to yield the glycoprotein precursors PreGn and PreGc, followed by structural glycoproteins Gn and Gc. Subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P) plays a central role in Gn processing. Here we show that CCHFV-infected cells deficient in SKI-1/S1P produce no infectious virus, although PreGn and PreGc accumulated normally in the Golgi apparatus, the site of virus assembly. Only nucleoprotein-containing particles which lacked virus glycoproteins (Gn/Gc or PreGn/PreGc) were secreted. Complementation of SKI-1/S1P-deficient cells with a SKI-1/S1P expression vector restored release of infectious virus (>10⁶ PFU/ml), confirming that SKI-1/S1P processing is required for incorporation of viral glycoproteins. SKI-1/S1P may represent a promising antiviral target.     

Long-lived signal peptide of lymphocytic choriomeningitis virus glycoprotein pGP-C

Signal peptides (SPs) direct nascent secretory and membrane proteins to the membrane of the endoplasmic reticulum. They are usually cleaved from the nascent polypeptide by signal peptidase and then further proteolytically processed. The SP of the pre-glycoprotein (pGP-C) of the lymphocytic choriomeningitis virus SPGP-C (signal peptide of pGP-C) shows different properties: 1) The SPGP-C is unusually long (58 amino acid residues) and contains two hydrophobic segments interrupted by a lysine residue. 2) The SPGP-C is cleaved only from a subset of pGP-C proteins. A substantial portion of pGP-C accumulates that still contains the SPGP-C.3)The cleaved SPGP-C is rather long-lived (t(1/2) of more than 6 h). 4) The cleaved SPGP-C resides in the membrane and is resistant to digestion with proteinase K even in the presence of detergents, suggesting a very compact structure. 5) SPGP-C accumulates in virus particles. These unusual features of the cleaved SPGP-C suggest that SPGP-C not only targets the nascent pGP-C to the endoplasmic reticulum membrane but also has additional functions in lymphocytic choriomeningitis virus life cycle.     

Biosynthesis and enzymatic characterization of human SKI-1/S1P and the processing of its inhibitory prosegment

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca(2+)-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK(137)- and RRLL(186)-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P' residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100-200 nM.     

A rapid fluorometric assay for the proteolytic activity of SKI-1/S1P based on the surface glycoprotein of the hemorrhagic fever Lassa virus

The subtilase subtilisin kexin isozyme-1 (SKI-1)/site 1 protease (S1P), has been implicated in the processing of Lassa virus glycoprotein C (GP-C) precursor into GP1 and GP2 that are responsible for viral fusion with the host cell membrane. Here, we studied in vitro the kinetics of this cleavage by hSKI-1 using an intramolecularly quenched fluorogenic (IQF) peptide, Q-GPC(251-263) [Abz-(251)Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu/Gly-Thr-Phe-Thr(263)-3-NitroTyr-Ala-CONH(2)], containing the identified site. The measured V(max (app))/K(m (app)) was compared to those for other IQF SKI-substrates. Q-GPC(251-263) is cleaved 10-fold more efficiently than the previously known best SKI-substrate, Q-hproSKI(134-142). This study confirmed the role of SKI-1 in GP-C processing and provides a novel, rapid and efficient enzymatic assay of SKI-1.     

Prosomatostatin is proteolytically processed at the amino terminal segment by subtilase SKI-1

Processing of prohormones to generate active products typically occurs at basic residues via cleavage by proprotein convertases. A less common type of cleavage is mediated at hydrophobic (L, V, F, N) or small amino acid (A, T, S) residues. Efforts to identify the proteinases responsible for processing precursors at their hydrophobic amino acids has led to the recent cloning of a new type-1 membrane-bound subtilase called SKI-1. The NH₂-terminal region of prosomatostatin, previously shown to contain a sorting signal for the regulated secretory pathways, is processed to generate PSST_[1–10]. The exact cleavage mechanism is unknown, but has been assumed to involve monobasic processing at Lys¹³ followed by carboxypeptidase trimming. We found that K13A mutation did not block PSST_[1–10] production. Since the prosomatostatin sequence R⁸–Q⁹–F¹⁰–L¹¹↓ qualifies as a potential SKI-1 substrate, using a vaccinia virus expression system along with HPLC and radioimmunoassays, we observed that overexpression of recombinant SKI-1 in COS-1 and HEK-293 cells significantly increased the production of PSST_[1–10]. Additionally, in CHO cells lacking SKI-1, there was a significant reduction in PSST_[1–10] production which could be increased upon SKI-1 stimulation. Mutagenesis studies showed that efficient processing of PSST to PSST_[1–10] required the RXRXXL motif. However, this NH₂-terminal cleavage was not a prerequisite for the formation of SST-14 and SST-28.     

Signal peptide requirements for lymphocytic choriomeningitis virus glycoprotein C maturation and virus infectivity

Insertion of the lymphocytic choriomeningitis virus (LCMV) precursor glycoprotein C (GP-C) into the membrane of the endoplasmic reticulum is mediated by an unusual signal peptide (SP(GP-C)). It is comprised of 58 amino acid residues and contains an extended hydrophilic N-terminal region, two hydrophobic regions, and a short C-terminal region. After cleavage by signal peptidase, SP(GP-C) accumulates in cells and virus particles. In the present study, we identified the LCMV SP(GP-C) as being an essential component of the GP complex and show that the different regions of SP(GP-C) are required for distinct steps in GP maturation and virus infectivity. More specifically, we show that one hydrophobic region of SP(GP-C) is sufficient for the membrane insertion of GP-C, while both hydrophobic regions are required for the processing and cell surface expression of the GPs. The N-terminal region of SP(GP-C), on the other hand, is essential for pseudoviral infection of target cells. Furthermore, we show that unmyristoylated SP(GP-C) exposes its N-terminal region to the exoplasmic side. This SP(GP-C) can promote GP-C maturation but is defective in pseudoviral infection. Myristoylation and topology of SP(GP-C) in the membrane may thus hold the key to an understanding of the role of SP(GP-C) in GP-C complex maturation and LCMV infectivity.     

Human subtilase SKI-1/S1P is a master regulator of the HCV Lifecycle and a potential host cell target for developing indirect-acting antiviral agents

HCV infection is a major risk factor for liver cancer and liver transplantation worldwide. Overstimulation of host lipid metabolism in the liver by HCV-encoded proteins during viral infection creates a favorable environment for virus propagation and pathogenesis. In this study, we hypothesize that targeting cellular enzymes acting as master regulators of lipid homeostasis could represent a powerful approach to developing a novel class of broad-spectrum antivirals against infection associated with human Flaviviridae viruses such as hepatitis C virus (HCV), whose assembly and pathogenesis depend on interaction with lipid droplets (LDs). One such master regulator of cholesterol metabolic pathways is the host subtilisin/kexin-isozyme-1 (SKI-1)--or site-1 protease (S1P). SKI-1/S1P plays a critical role in the proteolytic activation of sterol regulatory element binding proteins (SREBPs), which control expression of the key enzymes of cholesterol and fatty-acid biosynthesis. Here we report the development of a SKI-1/S1P-specific protein-based inhibitor and its application to blocking the SREBP signaling cascade. We demonstrate that SKI-1/S1P inhibition effectively blocks HCV from establishing infection in hepatoma cells. The inhibitory mechanism is associated with a dramatic reduction in the abundance of neutral lipids, LDs, and the LD marker: adipose differentiation-related protein (ADRP)/perilipin 2. Reduction of LD formation inhibits virus assembly from infected cells. Importantly, we confirm that SKI-1/S1P is a key host factor for HCV infection by using a specific active, site-directed, small-molecule inhibitor of SKI-1/S1P: PF-429242. Our studies identify SKI-1/S1P as both a novel regulator of the HCV lifecycle and as a potential host-directed therapeutic target against HCV infection and liver steatosis. With identification of an increasing number of human viruses that use host LDs for infection, our results suggest that SKI-1/S1P inhibitors may allow development of novel broad-spectrum biopharmaceuticals that could lead to novel indirect-acting antiviral options with the current standard of care.     

Cellular entry of lymphocytic choriomeningitis virus

In contrast to most enveloped viruses that enter the host cell via clathrin-dependent endocytosis, the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) enters cells via noncoated vesicles that deliver the virus to endosomes, where pH-dependent membrane fusion occurs. Here, we investigated the initial steps of LCMV infection. We found that the attachment of LCMV to its cellular receptor α-dystroglycan occurs rapidly and is not dependent on membrane cholesterol. However, subsequent virus internalization is sensitive to cholesterol depletion, indicating the involvement of a cholesterol-dependent pathway. We provide evidence that LCMV entry involves an endocytotic pathway that is independent of clathrin and caveolin and that does not require the GTPase dynamin. In addition, neither the structural integrity nor the dynamics of the actin cytoskeleton are required for infection. These findings indicate that the prototypic Old World arenavirus LCMV uses a mechanism of entry that is different from clathrin-mediated endocytosis, which is used by the New World arenavirus Junin virus, and pathways used by other enveloped viruses.     

Molecular definition of a major cytotoxic T-lymphocyte epitope in the glycoprotein of lymphocytic choriomeningitis virus.

Analyses with segmental reassortants of lymphocytic choriomeningitis virus (LCMV) RNA have shown that cytotoxic T lymphocytes (CTL) are induced by and recognize proteins encoded by the viral short segment, which specifies two virus structural proteins, glycoprotein (GP) and nucleoprotein (NP). Expression of cDNA copies of these genes in vaccinia virus vectors demonstrates that C57BL/6 (H2bb) mice mount significant CTL responses to both GP and NP. We have used LCMV-specific H2bb-restricted CTL clones and a family of serial C-terminal truncations of the LCMV GP expressed in vaccinia virus to map the precise specificities of the anti-GP clones. Of the 18 CTL clones studied, 1 recognizes NP and the other 17 recognize GP. The reactivities of 14 of the 17 anti-GP CTL clones against the deleted GP molecules have been fully characterized, and two clear patterns of anti-GP activity have emerged, defining at least two CTL epitopes. The first epitope, recognized by only two of the clones, lies within GP residues 1 to 218. The second is recognized by all 12 of the remaining clones and was mapped, by using the GP deletions, to a 22-amino-acid region comprising GP residues 272 to 293. A synthetic peptide representing this area sensitized uninfected syngeneic target cells to lysis both by bulk CTL obtained from the spleen after a primary immunization and by appropriate CTL clones. Two sets of criteria are available which are said to identify potential T-cell epitopes, one based on primary amino acid sequence and the second based on protein secondary structure. Neither of these predictive schemes would have identified region 272 to 293 as a CTL recognition motif, indicating that such programs are of limited usefulness as presently conceived. Analysis of the CTL clones shows clearly that all three families (anti-NP and anti-GP 1 to 218 and 272 to 293) direct efficient cross-reactive killing against a variety of serologically distinct strains of LCMV.     

The S RNA segment of lymphocytic choriomeningitis virus codes for the nucleoprotein and glycoproteins 1 and 2.

The lymphocytic choriomeningitis virus (LCMV) genome consists of a large RNA segment and a small RNA segment. The three major structural proteins of this virus are an internal nucleoprotein and two surface glycoproteins. Intertypic reassortants between the Armstrong and WE strains of LCMV were made to map proteins encoded by the LCMV genome segments. Using monoclonal antibodies specific for the nucleoprotein and the glycoproteins of WE and Armstrong, we showed that the small RNA segment of LCMV codes for the three major structural polypeptides.     

A-to-G hypermutation in the genome of lymphocytic choriomeningitis virus

The interferon-inducible adenosine deaminase that acts on double-stranded RNA (ADAR1-L) has been proposed to be one of the antiviral effector proteins within the complex innate immune response. Here, the potential role of ADAR1-L in the innate immune response to lymphocytic choriomeningitis virus (LCMV), a widely used virus model, was studied. Infection with LCMV clearly upregulated ADAR1-L expression and activity. The editing activity of ADAR1-L on an RNA substrate was not inhibited by LCMV replication. Accordingly, an adenosine-to-guanosine (A-to-G) and uracil-to-cytidine (U-to-C) hypermutation pattern was found in the LCMV genomic RNA in infected cell lines and in mice. In addition, two hypermutated clones with a high level of A-to-G or U-to-C mutations within a short stretch of the viral genome were isolated. Analysis of the functionality of viral glycoprotein revealed that A-to-G- and U-to-C-mutated LCMV genomes coded for nonfunctional glycoprotein at a surprisingly high frequency. Approximately half the GP clones with an amino acid mutation lacked functionality. These results suggest that ADAR1-L-induced mutations in the viral RNA lead to a loss of viral protein function and reduced viral infectivity. This study therefore provides strong support for the contribution of ADAR1-L to the innate antiviral immune response.     

Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells

Appropriate activation of naive CD8(+) T cells depends on the coordinated interaction of these cells with professional APC that present antigenic peptides in the context of MHC class I molecules. It is accepted that dendritic cells (DC) are efficient in activating naive T cells and are unique in their capacity to prime CD8(+) T cell responses against exogenous cell-associated Ags. Nevertheless, it is unclear whether epitopes, derived from endogenously synthesized proteins and presented by MHC class I molecules on the surface of other APC including B cells and macrophages, can activate naive CD8(+) T cells in vivo. By infecting transgenic CD11c-DTR/GFP mice that allow conditional depletion of DC with lymphocytic choriomeningitis virus (LCMV), which infects all types of APC and elicits a vigorous CTL response, we unambiguously show that priming of LCMV-specific CD8(+) T cells is crucially dependent on DC, despite ample presence of LCMV-infected macrophages and B cells in secondary lymphoid organs.     

Polymorphism of cytotoxic T-lymphocyte clones that recognize a defined nine-amino-acid immunodominant domain of lymphocytic choriomeningitis virus glycoprotein

To assess the heterogeneity of cytotoxic T lymphocytes (CTLs) directed against viral epitopes, we studied six class I major histocompatibility complex-restricted (H-2Db) CTL clones that recognize the same 9-amino-acid immunodominant epitope, amino acids 278 to 286 from envelope glycoprotein 2 (GP2) of lymphocytic choriomeningitis virus (LCMV). Using Southern blot analysis of beta-chain rearrangements, we found that each clone has a unique restriction pattern, providing evidence of the independent derivation of the clones and suggesting that the clones express different beta-chain sequences for their T-cell receptor. All these clones killed syngeneic target cells infected with strain Armstrong or WE of LCMV; however, two of the six clones failed to recognize target cells infected with the Pasteur strain of LCMV. Sequence analysis of LCMV Armstrong, WE, and Pasteur GP in the region of amino acids 272 to 293 demonstrated a single-amino-acid substitution at amino acid 278 in the region of the defined epitope in the Pasteur strain. Interestingly, one of the two CTL clones that failed to lyse LCMV Pasteur-infected target cells nevertheless efficiently and specifically killed uninfected target cells coated with the appropriate LCMV Pasteur peptide, while the other clone failed to do so. This indicated a dichotomy between processing of the synthesized protein initiated by infection and a peptide exogenously applied. Dose-response studies utilizing several peptides with substitutions in GP amino acid 278 indicate that CTL recognition occurs at the level of a single amino acid and suggest that this difference is likely recognized at the level of the T-cell receptor.