Lassa Virus Glycoprotein Research Articles

Novel Circular, Cyclic and Acyclic ψ(CH2O) Containing Peptide Inhibitors of SKI- 1/S1P: Synthesis, Kinetic and Biochemical Evaluations

Subtilisin Kexin Isozyme-1 (SKI-1)/Site1Protease (S1P) is a Ca(+2)-dependent membrane bound pyrolysin-type serine protease of mammalian subtilase super family Proprotein Convertases (PCs)/Proprotein Convertase Subtilisin Kexins (PCSKs). It cleaves precursor proteins at the carboxy terminus of a non basic amino acid characterized by the sequence Arg/Lys-θ-φ-Leu/Ser/Thr↓, where θ = any amino acid except Cys, φ = the alkyl side chain containing hydrophobic amino acid. SKI-1 cleaves pro-BDNF, pro-SREBP2, pro-ATF6, pro-somatostatin and viral glycoproteins to generate their active forms. As a result SKI-1 plays important roles in cartilage development, bone mineralization, cholesterol metabolism, fatty acid synthesis and infections caused by Arina viruses of hemorrhagic type. Interest has grown to develop inhibitors of SKI-1 that may find useful therapeutic and biochemical applications. Our objective is to develop small molecule inhibitors of SKI-1/S1P and study their kinetic and biochemical properties. Peptide analogs were designed by inserting a protease resistant methylene-oxy (-CH(2)-O-) pseudoamide function at the cleavage site of (251)Asp-Ile-Tyr-Ile-Ser-Arg-Arg-Leu-Leu↓Gly-Thr-Phe-Thr(263), derived from SKI-1 processing site of Lassa virus glycoprotein. The synthesis was conducted by substituting Leu-Gly with previously made Leu-CH(2)-O-Gly. Flexible linear and conformationally constrained circular and disulphide bridged cyclic peptides were prepared by solid phase method. Circular and cyclic peptides inhibited SKI-1 more potently (K(i)~14-20 µM) than the corresponding acyclic peptide (K(i)~51 µM). They also blocked SKI-1-mediated processing of pro-h(human)SREBP2 into its mature form in HepG2 cells. Circular pseudopeptides designed from hATF6 and hSREBP2 also inhibited SKI-1. This is the first report of circular and cyclic Ψ(CH(2)-O) containing peptides as SKI-1 inhibitors with potential therapeutic applications in cholesterol synthesis.

Molecular evolutionary studies of Lassa virus nucleoprotein 2

Objective To study the virulence diversity through molecular evolution, and to provide insight on circulating antibodies. Methods The nucleotide sequences of 18 Lassa virus genomic RNA encoding Lassa virus nucleoprotein isolates collected from different parts of the world since the identification of the Josiah strain were obtained from the GenBank and nucleotide substitution among them studied using the computer program MEGA 4. The genetic distances among strains were predicted by pairwise nucleotide differences. Results The rate of synonymous substitution was high 5.889 per nucleotide per year and nonsynonymous was higher at 49.664. The average predicted rate of synonymous and nonsynonymous using modified Nei-Gojobori (assuming transition/transversion bias=2) was 27.9 which was taken as the genetic distance between strains. The average number of synonymous sites is 150.741 while the average number of nonsynonymous sites is 392.259. The phylogenetic tree was inferred by unweighted pairwise grouping in MEGA4 and using neighbour-joining method. The time of emergence of Lassa virus was predicted to be around January 1920. However, the first human appearance of the virus was predicted to be around May (1 959±24) months. In synonymous substitution the rate of (G-T) rare was high. The nucleotide frequencies were 0.314 (A), 0.246 (T/U), 0.204 (C) and 0.235 (G). The transition/transversion ratio k 1=14.991 (purines) and k 2=69.916 (pyrimidines). The overall transition/transversion bias R=16.662 with a total of 620 position in the final data set. These figures are far higher than an earlier study using Lassa virus glycoprotein. The nucleotide diversity were also very high using the Taijima's model in MEGA 4. Conclusions The divergence within strains always coincides with the period of epidemic which goes to confirm that the cause of epidemic outbreak should be the emergence of new strain and also why the infection remains endemic despite circulating antibodies. A comparison with a similar study with the viral glycoprotein concludes that the glycoprotein is more suited for vaccine development.

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa.

Use of Single-Cycle Infectious Lymphocytic Choriomeningitis Virus To Study Hemorrhagic Fever Arenaviruses

Several arenaviruses, chiefly Lassa virus (LASV) and Junin virus in West Africa and Argentina, respectively, cause hemorrhagic fever (HF) disease in humans that is associated with high morbidity and significant mortality. The investigation of antiviral strategies to combat HF arenaviruses is hampered by the requirement of biosafety level 4 (BSL-4) facilities to work with these viruses. These biosafety hurdles could be overcome by the use of recombinant single-cycle infectious arenaviruses. To explore this concept, we have developed a recombinant lymphocytic choriomeningitis virus (LCMV) (rLCMVΔGP/GFP) where we replaced the viral glycoprotein (GP) with the green fluorescent protein (GFP). We generated high titers of GP-pseudotyped rLCMVΔGP/GFP via genetic trans complementation using stable cell lines that constitutively express LCMV or LASV GPs. Replication of these GP-pseudotyped rLCMVΔGP/GFP viruses was restricted to GP-expressing cell lines. This system allowed us to rapidly and reliably characterize and quantify the neutralization activities of serum antibodies against LCMV and LASV within a BSL-2 facility. The sensitivity of the GFP-based microneutralization assay we developed was similar to that obtained with a conventionally used focus reduction neutralization (FRNT) assay. Using GP-pseudotyped rLCMVΔGP/GFP, we have also obtained evidence supporting the feasibility of this approach to identify and evaluate candidate antiviral drugs against HF arenaviruses without the need of BSL-4 laboratories.

Maturation cleavage within the ectodomain of Lassa virus glycoprotein relies on stabilization by the cytoplasmic tail

The Lassa virus glycoprotein consists of an ectodomain, a transmembrane anchor, and a cytoplasmic domain. It is synthesized as an inactive precursor and cleaved within the ectodomain to yield the mature form. Here, we show that this maturation cleavage can be abolished by mutation of single conserved amino acids within the cytoplasmic domain at the carboxy-terminus of the glycoprotein. Moreover, substitutions and deletions of multiple amino acids result in destabilization of the glycoprotein oligomers. These results indicate that conformation changes in the cytoplasmic domain travel across the membrane and subsequently abolish the maturation cleavage. Therefore, we postulate that the cytoplasmic domain is an important maturation factor stabilizing the overall conformation of the glycoprotein. Structured summary MINT- 7997004: LASV GP (uniprotkb: P08669) and LASV GP (uniprotkb: P08669) physically interact (MI: 0915) by cosedimentation through density gradient (MI: 0029)

Characterization of Lassa Virus Glycoprotein Oligomerization and Influence of Cholesterol on Virus Replication

Mature glycoprotein spikes are inserted in the Lassa virus envelope and consist of the distal subunit GP-1, the transmembrane-spanning subunit GP-2, and the signal peptide, which originate from the precursor glycoprotein pre-GP-C by proteolytic processing. In this study, we analyzed the oligomeric structure of the viral surface glycoprotein. Chemical cross-linking studies of mature glycoprotein spikes from purified virus revealed the formation of trimers. Interestingly, sucrose density gradient analysis of cellularly expressed glycoprotein showed that in contrast to trimeric mature glycoprotein complexes, the noncleaved glycoprotein forms monomers and oligomers spanning a wide size range, indicating that maturation cleavage of GP by the cellular subtilase SKI-1/S1P is critical for formation of the correct oligomeric state. To shed light on a potential relation between cholesterol and GP trimer stability, we performed cholesterol depletion experiments. Although depletion of cholesterol had no effect on trimerization of the glycoprotein spike complex, our studies revealed that the cholesterol content of the viral envelope is important for the infectivity of Lassa virus. Analyses of the distribution of viral proteins in cholesterol-rich detergent-resistant membrane areas showed that Lassa virus buds from membrane areas other than those responsible for impaired infectivity due to cholesterol depletion of lipid rafts. Thus, derivation of the viral envelope from cholesterol-rich membrane areas is not a prerequisite for the impact of cholesterol on virus infectivity.

Inhibition of Lassa Virus Glycoprotein Cleavage and Multicycle Replication by Site 1 Protease-Adapted α1-Antitrypsin Variants

BackgroundProteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P) is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.Methodology/Principal FindingWe demonstrate that stable cell lines inducibly expressing S1P-adapted α1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of α1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific α1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different α1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.Conclusions/SignificanceOur data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

Characterization of the Lassa virus GP1 ectodomain shedding: implications for improved diagnostic platforms.

BackgroundThere is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, detection of early markers of Lassa virus (LASV) infection may improve diagnosis and ultimately successful treatment with antivirals. Characterization of LASV GP1 ectodomain shedding is an important step toward developing sensitive diagnostics to detect circulating levels of this viral glycoprotein in infected patient sera.ResultsSecretion of GP1 from mammalian cells expressing a native LASV GPC gene was not mediated by proteolytic cleavage, as determined by treatment with a panel of matrix metalloprotease (MMP) inhibitors. The shedding of GP1 was also not the result of over-expression of GPC under the control of a strong intron-A containing CMV promoter, as the soluble component could be immunoprecipitated from supernatants of cells expressing low levels of GPC under the control of an intronless promoter. Cells transfected with GPC retained surface membrane-associated expression of GP1 as determined by immunofluorescence assay, in addition to secreting the glycoprotein.Secreted GP1 derived from GPC expression has a higher content of high mannose N-linked glycosylation than sGP1 expressed independently from the GP2 portion of the protein. Neither GP1 isoform contains sialylated N-glycans, O-linked carbohydrate chains, or galactose-β(1-4)-N-acetylglucosamine commonly present in complex and hybrid N-glycan structures.ConclusionThese results demonstrate the non-proteolytic secretory nature of GP1 shedding during expression of the arenaviral glycoprotein complex. This phenomenon parallels shedding of a secretory glycoprotein component in filovirus replication. The glycosylation pattern of soluble GP1 resulting from expression of GPC was different from that of a soluble GP1 construct (sGP1-RRAA-FLAG), highlighting the intricately orchestrated post translational processing of the LASV glycoprotein complex.

Uncoupling GP1 and GP2 expression in the Lassa virus glycoprotein complex: implications for GP1 ectodomain shedding.

BackgroundSera from convalescent Lassa fever patients often contains antibodies to Lassa virus (LASV) glycoprotein 1 (GP1), and glycoprotein 2 (GP2); Immunization of non-human primates with viral vectors expressing the arenaviral glycoprotein complex (GPC) confers full protective immunity against a lethal challenge with LASV. Thus, the development of native or quasi native recombinant LASV GP1 and GP2 as soluble, uncoupled proteins will improve current diagnostics, treatment, and prevention of Lassa fever. To this end, mammalian expression systems were engineered for production and purification of secreted forms of soluble LASV GP1 and GP2 proteins.ResultsDeterminants for mammalian cell expression of secreted uncoupled Lassa virus (LASV) glycoprotein 1 (GP1) and glycoprotein 2 (GP2) were established. Soluble GP1 was generated using either the native glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the intracellular domain (IC) was fused to the ectodomain, and the gene was co-expressed with a complete GP1 gene in cis; (2) the TM and IC domains were deleted and GP1 was co-expressed in cis; (3) expression of GP1 was driven by the native GPC SP. These data implicate GP1 as a chaperone for processing and shuttling GP2 to the cell surface. The soluble forms of GP1 and GP2 generated through these studies were secreted as homogeneously glycosylated proteins that contained high mannose glycans. Furthermore, observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC represents a novel aspect of arenaviral glycoprotein expression.ConclusionThese results implicate GP1 as a chaperone for the correct processing and shuttling of GP2 to the cell surface, and suggest that native GPC SP plays a role in this process. In the absence of GP1 and GPC SP the GP2 protein may be processed by an alternate pathway that produces heterogeneously glycosylated protein, or the polypeptide may not fully mature in the secretory cascade in mammalian cells. The expression constructs developed in these studies resulted in the generation and purification of soluble, uncoupled GP1 and GP2 proteins from mammalian cells with quasi-native properties. The observation of GP1 ectodomain shedding from cells expressing wild type LASV GPC establishes new correlates of disease progression and highlights potential opportunities for development of diagnostics targeting the early stages of Lassa fever.

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance.

BackgroundThere is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2).ResultsFull-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA).ConclusionThese results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination.

Different Mechanisms of Cell Entry by Human-Pathogenic Old World and New World Arenaviruses

The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.

Amino acids from both N-terminal hydrophobic regions of the Lassa virus envelope glycoprotein GP-2 are critical for pH-dependent membrane fusion and infectivity

Lassa virus glycoprotein 2 (LASV GP-2) belongs to the class I fusion protein family. Its N terminus contains two stretches of highly conserved hydrophobic amino acids (residues 260-266 and 276-298) that have been proposed as N-terminal or internal fusion peptide segments (N-FPS, I-FPS) by analogy with similar sequences of other viral glycoproteins or based on experimental data obtained with synthetic peptides, respectively. By using a pH-dependent, recombinant LASV glycoprotein mediated cell-cell fusion assay and a retroviral pseudotype infectivity assay, an alanine scan of all hydrophobic amino acids within both proposed FPSs was performed. Fusogenicity and infectivity were correlated, both requiring correct processing of the glycoprotein precursor. Most point mutations in either FPS accounted for reduced or abolished fusion or infection, respectively. Some mutations also had an effect on pre-fusion steps of virus entry, possibly by inducing structural changes in the glycoprotein. The data demonstrate that several amino acids from both hydrophobic regions of the N terminus, some of which (W264, G277, Y278 and L280) are 100 % conserved in all arenaviruses, are involved in fusogenicity and infectivity of LASV GP-2.

Identification of protective Lassa virus epitopes that are restricted by HLA-A2.

Recovery from Lassa virus (LASV) infection usually precedes the appearance of neutralizing antibodies, indicating that cellular immunity plays a primary role in viral clearance. To date, the role of LASV-specific CD8(+) T cells has not been evaluated in humans. To facilitate such studies, we utilized a predictive algorithm to identify candidate HLA-A2 supertype epitopes from the LASV nucleoprotein and glycoprotein precursor (GPC) genes. We identified three peptides (GPC(42-50), GLVGLVTFL; GPC(60-68), SLYKGVYEL; and GPC(441-449), YLISIFLHL) that displayed high-affinity binding (< or =98 nM) to HLA-A*0201, induced CD8(+) T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LASV GPC in human HLA-A*0201-positive target cells. HLA-A*0201 mice immunized with either GPC(42-50) or GPC(60-68) were protected against challenge with a recombinant vaccinia virus that expressed LASV GPC. The epitopes identified in this study represent potential diagnostic reagents and candidates for inclusion in epitope-based vaccine constructs. Our approach is applicable to any pathogen with existing sequence data, does not require manipulation of the actual pathogen or access to immune human donors, and should therefore be generally applicable to category A through C agents and other emerging pathogens.

The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C

Lassa virus glycoprotein is synthesised as a precursor (preGP-C) into the lumen of the endoplasmic reticulum. After cotranslational cleavage of the signal peptide, the immature GP-C is posttranslationally processed into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by the host cell subtilase SKI-1/S1P. The glycoprotein precursor contains eleven potential N-glycosylation sites. In this report, we investigated the effect of each N-glycan on proteolytic cleavage and cell surface transport by disrupting the consensus sequences of eleven potential N-glycan attachment sites individually. Five glycoprotein mutants with disrupted N-glycosylation sites were still proteolytically processed, whereas the remaining N-glycosylation sites are necessary for GP-C cleavage. Despite the lack of proteolytic processing, all cleavage-defective mutants were transported to the cell surface and remained completely endo H-sensitive. The findings indicate that N-glycans are needed for correct conformation of GP-C in order to be cleaved by SKI-1/S1P.

A recombinant Yellow Fever 17D vaccine expressing Lassa virus glycoproteins

The Yellow Fever Vaccine 17D (YFV17D) has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) resulting in construction of YFV17D/LASV-GPC recombinant virus. The virus was replication-competent and processed the LASV-GPC in cell cultures. The recombinant replicated poorly in guinea pigs but still elicited specific antibodies against LASV and YFV17D antigens. A single subcutaneous injection of the recombinant vaccine protected strain 13 guinea pigs against fatal Lassa Fever. This study demonstrates the potential to develop an YFV17D-based bivalent vaccine against two viruses that are endemic in the same area of Africa.

Lassa Virus Glycoprotein Signal Peptide Displays a Novel Topology with an Extended Endoplasmic Reticulum Luminal Region

Lassa virus glycoprotein C (GP-C) is translated as a precursor (preGP-C) into the lumen of the endoplasmic reticulum (ER) and cotranslationally cleaved into the signal peptide and immature GP-C before GP-C is proteolytically processed into its subunits, GP-1 and GP-2, which form the mature virion spikes. The signal peptide of preGP-C comprises 58 amino acids and contains two distinct hydrophobic domains. Here, we show that each hydrophobic domain alone can insert preGP-C into the ER membrane. Furthermore, we demonstrate that the native signal peptide only uses the N-terminal hydrophobic domain for membrane insertion, exhibiting a novel type of a topology for signal peptides with an extended ER luminal part, which is essential for proteolytic processing of GP-C into GP-1 and GP-2.

Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor.

Lassa virus glycoprotein is translated as a precursor (pre-GP-C) into the lumen of the endoplasmic reticulum and is cotranslationally cleaved into the signal peptide and GP-C, before GP-C is proteolytically processed into its subunits GP1 and GP2. The signal peptide of pre-GP-C comprises 58 amino acids. The substitution of Lassa virus pre-GP-C signal peptide with another signal peptide still mediates translocation and the release of signal peptide but abolishes the proteolytic cleavage of GP-C into GP1 and GP2. Remarkably, cleavage of GP-C from these hybrid pre-GP-C substrates was restored on coexpression of the wild-type pre-GP-C signal peptide, indicating that the signal peptide functions as a trans-acting factor to promote Lassa virus GP-C processing. To our knowledge, this is the first report on a signal peptide that is essential for proteolytic processing of a secretory pathway protein.

Identification of Lassa virus glycoprotein signal peptide as a trans‐acting maturation factor

Lassa virus glycoprotein is translated as a precursor (pre‐GP‐C) into the lumen of the endoplasmic reticulum and is cotranslationally cleaved into the signal peptide and GP‐C, before GP‐C is proteolytically processed into its subunits GP1 and GP2. The signal peptide of pre‐GP‐C comprises 58 amino acids. The substitution of Lassa virus pre‐GP‐C signal peptide with another signal peptide still mediates translocation and the release of signal peptide but abolishes the proteolytic cleavage of GP‐C into GP1 and GP2. Remarkably, cleavage of GP‐C from these hybrid pre‐GP‐C substrates was restored on coexpression of the wild‐type pre‐GP‐C signal peptide, indicating that the signal peptide functions as a trans‐acting factor to promote Lassa virus GP‐C processing. To our knowledge, this is the first report on a signal peptide that is essential for proteolytic processing of a secretory pathway protein.

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.

Signal peptide of Lassa virus glycoprotein GP-C exhibits an unusual length

Lassa virus glycoprotein is synthesized as precursor GP-C into the lumen of the endoplasmic reticulum and cleaved posttranslationally into the N-terminal subunit GP-1 and the C-terminal subunit GP-2 by subtilase SKI-1/S1P. The N-terminal portion of the primary translation product preGP-C contains a signal peptide of unknown length. In order to demonstrate the signal peptide cleavage site, purified viral GP-1 isolated from Lassa virus particles was N-terminally sequenced as TSLYKGV, identical to amino acids 59–65 of GP-C. Mutational analysis of the amino acid residues flanking the putative cleavage site led to non-cleavable preGP-C indicating that no other signal peptide cleavage site exists. Interestingly, GP-C mutants with a non-cleavable signal peptide were not further processed by SKI-1/S1P. This observation suggests that the signal peptide cleavage is necessary for GP-C maturation and hence for Lassa virus replication.