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.     

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 a novel consensus sequence at the cleavage site of the Lassa virus glycoprotein

The Lassa virus glycoprotein consists of an amino-terminal and a carboxy-terminal cleavage fragment designated GP-1 and GP-2, respectively, that are derived by proteolysis from the precursor GP-C. The membrane-anchored GP-2 obtained from purified virions of the Josiah strain revealed the N-terminal tripeptide GTF(262) when analyzed by Edman degradation. Upstream of this site, GP-C contains the tetrapeptide sequence RRLL(259), which is conserved in all Lassa virus isolates published to date. Systematic mutational analysis of vector-expressed GP-C revealed that the motif R-X (L/I/V)-L(259) (where X stands for L, I, or V) is essential for cleavage of the peptide bond between leucine(259) and glycine(260). This cleavage motif is homologous to the consensus sequence recognized by a novel class of cellular endoproteases which have so far not been implicated in the processing of viral glycoproteins.     

cDNA sequence analysis confirms that the etiologic agent of callitrichid hepatitis is lymphocytic choriomeningitis virus.

Callitrichid hepatitis is an infection of New World primates caused by an arenavirus, currently referred to as callitrichid hepatitis virus, that is closely related to lymphocytic choriomeningitis virus (LCMV). We have cloned and sequenced the GP-C gene of callitrichid hepatitis virus and found that the cDNA sequence is 84 to 86% identical to those of the GP-C genes of LCMV strains Armstrong and WE, while the deduced amino acid sequence is 95 to 96% identical to those of the GP-C gene products of the same strains. This high degree of similarity indicates that the etiologic agent of callitrichid hepatitis is in fact LCMV. The wide geographic distribution of callitrichid hepatitis outbreaks in the United States serves as a reminder that LCMV is also a human pathogen whose public health implications are not well understood.     

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.     

Viral proteins and RNAs in BHK cells persistently infected by lymphocytic choriomeningitis virus

Some Syrian hamster cell lines persistently infected with lymphocytic choriomeningitis virus (LCMV) do not produce extracellular virus particles but do contain intracytoplasmic infectious material. The proteins of these cells were labeled with [35S]methionine or with [3H]glucosamine and [3H]mannose, and immunoprecipitates were prepared with anti-LCMV sera. A substantial amount of the LCMV nucleocapsid protein (molecular weight about 58,000) was detected, along with GP-C, the precursor of the virion glycoproteins GP-1 and GP-2. GP-1 and GP-2 themselves were not detected. A new method of transferring proteins electrophoretically from sodium dodecyl sulfate-polyacrylamide gels to diazotized paper in high yield revealed several additional LCMV proteins present specifically in the persistently infected cells, at apparent molecular weights (X10(3] of 112, 107, 103, 89, 71 (probably GP-C), 58 (nucleocapsid protein), 42 to 47 (probably GP-1), and 40 (possibly GP-2). By iodinating intact cells with I3, GP-1 but not GP-2 or GP-C was revealed on the surfaces of the persistently infected cells, whereas both GP-1 and GP-C were found on the surfaces of acutely infected cells. The absence of GP-C from the plasma membrane of the persistently infected cells might be related to defective maturation of the virus in these cells. Cytoplasmic viral nucleoprotein complexes were labeled with [3H]uridine in the presence or absence of actinomycin D, purified partially by sedimentation in D2O-sucrose gradients, and adsorbed to fixed Staphylococus aureus cells in the presence of anti-LCMV immunoglobulin G. Several discrete species of viral RNA were released from the immune complexes with sodium dodecyl sulfate. Some were appreciably smaller than the 31S and 23S species of standard LCMV virions, indicating that defective interfering viral RNAs are probably present in the persistently infected cells. Ribosomal 28S and 18S RNAs, labeled only in the absence of actinomycin D, were coprecipitated with anti-LCMV serum but not with control serum, indicating their association with LCMV nucleoproteins in the cells.     

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

Background

Proteolytic 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 Finding

We 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/Significance

Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.     

Molecular characterization of gag proteins from simian immunodeficiency virus (SIVMne).

A simian immunodeficiency virus (SIV) designated SIVMne was isolated from a pig-tailed macaque with lymphoma housed at the University of Washington Regional Primate Research Center, Seattle. To better establish the relationship of SIVMne to other immunodeficiency viruses, we purified and determined the partial amino acid sequences of six structural proteins (p1, p2, p6, p8, p16, and p28) from SIVMne and compared these amino acid sequences to the translated nucleotide sequences of SIVMac and human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). A total of 125 residues of SIVMne amino acid sequence were compared to the predicted amino acid sequences of the gag precursors of SIV and HIVs. In the compared regions 92% of the SIVMne amino acids were identical to predicted residues of SIVMac, 83% were identical to predicted residues of HIV-2, and 41% were identical to predicted residues of HIV-1. These data reveal that the six SIVMne proteins are proteolytic cleavage products of the gag precursor (Pr60gag) and that their order in the structure of Pr60gag is p16-p28-p2-p8-p1-p6. Rabbit antisera prepared against purified p28 and p16 were shown to cross-react with proteins of 60, 54, and 47 kilodaltons present in the viral preparation and believed to be SIVMne Pr60gag and intermediate cleavage products, respectively. SIVMne p16 was shown to contain covalently bound myristic acid, and p8 was identified as a nucleic acid-binding protein. The high degree of amino acid sequence homology between SIVs and HIV-2 around proven proteolytic cleavage sites in SIV Pr60gag suggests that proteolytic processing of the HIV-2 gag precursor is probably very similar to processing of the SIV gag precursor. Peptide bonds cleaved during proteolytic processing of the SIV gag precursor were similar to bonds cleaved during processing of HIV-1 gag precursors, suggesting that the SIV and HIV viral proteases have similar cleavage site specificities.     

Identification of simian immunodeficiency virus SIVMAC env gene products.

A monoclonal antibody recognizing an antigenic determinant on the env transmembrane protein, gp32 of simian immunodeficiency virus SIVMAC has been developed and designated SF8/5E11. The reactivity of this antibody was found to be type specific, since it did not cross-react with either SIVSMM or SIVMNe transmembrane proteins. The availability of both this antibody and the complete nucleotide sequence of SIVMAC allowed us to define the organization of the env gene products of this virus. Radiolabel sequencing of the amino termini of both gp160 and gp32 confirmed the positions of both cleavage sites predicted by alignment of the inferred amino acid sequences of the SIVMAC and human immunodeficiency virus type 1 env genes. The cleavage site between the signal peptide and the external env glycoprotein resides between the cysteine residue at position 21 and the threonine residue at position 22, starting from the first residue after the env gene initiator methionine. The env precursor polyprotein gp160 is cleaved between arginine 526 and glycine 527 to give rise to the external glycoprotein and the transmembrane of SIVMAC.     

Cross-protection against lymphocytic choriomeningitis virus mediated by a CD4+ T-cell clone specific for an envelope glycoprotein epitope of Lassa virus.

Recombinant vaccinia virus expressing the Lassa virus (LV) envelope glycoprotein precursor, V-LSGPC, was used to study the basis of LV-induced cross-protective immunity against the closely related arenavirus lymphocytic choriomeningitis virus (LCMV). C3H/HeJ mice primed with V-LSGPC developed neither circulating antibodies nor CD8+ cytotoxic T cells specific for LCMV, yet they resisted a normally lethal LCMV challenge. Spleen cells from such mice gave a proliferative response to LCMV in vitro that was inhibitable by anti-CD4 antibody. Synthetic peptides corresponding to predicted T-cell sites common to the envelope glycoprotein precursor (GP-C) of LV and that of LCMV were used to map the specificity of the proliferative response to an epitope located between amino acids 403 and 417 of LV GP-C. Several CD4+ T-cell clones specific for the 403-417 peptide were isolated and found to produce gamma interferon in response to both the peptide and LCMV. One of these clones, C9, was selected for further study. C9 lysed I-AK-bearing target cells, and when adoptively transferred to C3H/HeJ mice, it was capable of mediating both a peptide-specific delayed hypersensitivity reaction and resistance to lethal LCMV challenge. These collective findings demonstrate, for the first time, that CD4+ T cells can play a major role in arenavirus-specific cross-protective immunity.     

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.     

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.     

Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus.

This study documents that the immunosuppressive lymphocytic choriomeningitis virus (LCMV) variant, clone 13, shows a specific predilection for enhanced infection of macrophages both in vitro and in vivo and that single amino acid changes in the viral polymerase and glycoprotein are responsible for macrophage tropism. The growth difference seen between variant clone 13 and the parental Armstrong strain was specific for macrophages, since both clone 13 and Armstrong grew equally well in fibroblasts and neither isolate infected lymphocytes efficiently. Complete sequencing of the clone 13 genome, along with genetic analysis, showed that a single amino acid change in the polymerase (K-->Q at position 1079) was the major determinant of virus yield in macrophages. This was proven unequivocally by comparing the sequences of parental and reassortant viruses, which were identical at all loci except for the single mutation in the polymerase gene. This finding was further strengthened by showing that reversion at this site back to lysine (Q-->K) resulted in loss of macrophage tropism. In addition, an independently derived macrophage-tropic variant of LCMV, clone 28b, had a K-->N mutation at the same position. Thus, these results show that substitution of the positively charged amino acid K with a neutral amino acid (either Q or N) at residue 1079 of the polymerase resulted in enhanced viral replication in macrophages. In addition to the polymerase change, a mutation in the glycoprotein was also associated with macrophage tropism. This single amino acid change in the glycoprotein (F-->L at position 260) did not affect virus yield per macrophage but was critical in determining the number of macrophages infected. Our previous studies have shown that the same two mutations in the polymerase and glycoprotein are essential for establishing a chronic infection in adult mice. Since the same mutations confer macrophage tropism and ability to persist in vivo, these studies provide compelling evidence that infection of macrophages is a critical determinant of viral persistence and immune suppression.     

Ribozymes which cleave arenavirus RNAs: identification of susceptible target sites and inhibition by target site secondary structure.

The development of safe and effective antiviral agents has been a slow process, largely because of the difficulty in distinguishing between virus and host functions; materials toxic to the virus are frequently harmful also to the host in which the agent resides. Recently, techniques which target nucleic acid sequences as a means of reducing gene expression have emerged. This antisense armamentarium includes ribozymes, RNA enzymes which cleave other RNA molecules in a sequence-specific manner. We wish to assess the ability of ribozymes to control animal virus infection. Reasoning that the viruses most vulnerable to ribozyme intervention will be those whose complete life cycle is based on RNA (with no DNA stage), we have begun to develop ribozymes directed toward lymphocytic choriomeningitis virus (LCMV), the prototype of the arenavirus family. Using ribozymes of the hammerhead variety, we have identified several sites on the LCMV genome which can be efficiently cleaved in trans. The efficiency of cleavage is site dependent, and we demonstrate that secondary structure at the target site can abolish ribozyme cleavage. Computer-assisted analysis indicates that much of the LCMV genome may be involved in base pairing, which may render it similarly resistant to ribozyme attack. The few remaining open regions of LCMV lack a GUC target site, on which most studies to date have relied. Here we show that AUC, CUC, and AUU are alternative sites which can be cleaved by trans-acting ribozymes. This finding is important given the aforementioned restriction of available sites, imposed by secondary structure.     

Maturation of Borna disease virus glycoprotein

The maturation of Borna disease virus (BDV) glycoprotein GP was studied in regard to intracellular compartmentalization, compartmentalization signal-domains, proteolytic processing, and packaging into virus particles. Our data show that BDV-GP is (i) predominantly located in the endoplasmic reticulum (ER), (ii) partially exists in the ER already as cleaved subunits GP-N and GP-C, (iii) is directed to the ER/cis-Golgi region by its transmembrane and/or cytoplasmic domains in CD8-BDV-GP hybrid constructs and (iv) is incorporated in the virus particles as authentic BDV glycoprotein exclusively in the cleaved form decorated with N-glycans of the complex type. Downregulation of BDV-glycoproteins on the cell surface, their limited proteolytic processing, and protection of antigenic epitopes on the viral glycoproteins by host-identical N-glycans are different strategies for persistent virus infections.     

Thyroid cell surface glycoproteins. Nature and disposition of carbohydrate units and evaluation of their blood group I activity

The distribution along the polypeptide of the carbohydrate units of two major calf thyroid cell surface glycoproteins, GP-1 and GP-3, was obtained from a study of their glycopeptides obtained after Pronase digestion. The GP-3 molecule (Mr = 20,000) yielded two large glycopeptides (Mr = 9,500 and 7,000) in equimolar amounts which each consisted of one N-linked (Mr = 5,400) and several small O-linked oligosaccharides accounting for a total of nine carbohydrate attachment sites in a 27-amino acid residue segment of the peptide chain. The Pronase treatment of GP-1 (Mr = 100,000) revealed the presence of a large protease-resistant fragment (Mr = 50,000) which contained 34 carbohydrate units (eight N-linked and 26 O-linked) in a segment of 105 amino acids. In addition to these densely glycosylated peptides (one glycosylation site/3 amino acid residues), small glycopeptides with polymannose saccharide units were found in the digests of both proteins. The occurrence of repeating N-acetyllactosamine sequences in the N-linked carbohydrate units of GP-1 and GP-3 was suggested by the composition and size of the oligosaccharides released by hydrazinolysis and was demonstrated by endo-beta-galactosidase treatment. The cleavage products from digestion with this enzyme were identified as NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----3Gal, Gal alpha 1----3Gal beta 1----4GlcNAc beta 1----3Gal, Gal beta 1----4GlcNAc beta 1----3Gal, and GlcNAc beta 1----3Gal with the tetrasaccharides constituting the predominant species. The terminal alpha-D-Gal residues accounted for the binding of GP-1 and GP-3 glycopeptides to Bandeiraea simplicifolia I-agarose; concanavalin A-Sepharose affinity chromatography indicated that most of the N-linked carbohydrate units of both glycoproteins contained more than two branches. Difference in the branching on the poly-N-acetyllactosamine sequences of GP-1 and GP-3 was suggested by the finding that only the latter glycoprotein, as well as its glycopeptides, reacted with anti-blood group I antibodies; neither glycoprotein demonstrated blood group i antigenicity. Examination of cultured thyroid follicular cells revealed that both I and i determinants were present at the cell surface.     

Early antibodies specific for the neutralizing epitope on the receptor binding subunit of the lymphocytic choriomeningitis virus glycoprotein fail to neutralize the virus

Lymphocytic choriomeningitis virus (LCMV) is a murine arenavirus whose glycoprotein consists of a transmembrane subunit (GP-2) and a receptor-binding subunit (GP-1). LCMV-neutralizing antibodies (nAbs) are directed against a single site on GP-1 and occur 1 month after the infection of cytotoxic-T-lymphocyte (CTL) deficient mice. In wild-type mice, however, CTLs control early infection, and weak nAb titers emerge very late (after 70 to 150 days) if at all. Production of recombinant GP-1 in native conformation enabled us to study the emergence of GP-1-binding antibodies directed against the neutralizing epitope. By combining binding and neutralization assays, we correlated the development of binding antibodies versus nAbs in wild-type and CTL-deficient mice after infection with different LCMV doses. We found that wild-type mice developed GP-1-specific antibodies already by day 8 after exposure to high but not low doses, demonstrating that naive GP-1-specific B cells were infrequent. Furthermore, the induced antibodies bound to the neutralizing GP-1 epitope but failed to neutralize the virus and therefore were of low affinity. In CTL-deficient mice, where massive viremia quickly levels initial differences in viral load, low and high doses induced low-affinity non-neutralizing GP-1-binding antibodies with kinetics similar to high-dose-infected wild-type mice. Only in CTL-deficient mice, however, the GP-1-specific antibodies developed into nAbs within 1 month. We conclude that LCMV uses a dual strategy to evade nAb responses in wild-type mice. First, LCMV exploits a "hole" in the murine B-cell repertoire, which provides only a small and narrow initial pool of low-affinity GP-1-specific B cells. Second, affinity maturation of the available low-affinity non-neutralizing antibodies is impaired.     

Endoproteolytic processing of the lymphocytic choriomeningitis virus glycoprotein by the subtilase SKI-1/S1P

The envelope glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) is posttranslationally cleaved into two subunits. We show here that this endoproteolytic processing is not required for transport to the cell surface but is essential for LCMV GP to mediate infectivity of pseudotyped retroviral vectors. By systematic mutational analysis of the LCMV GP cleavage site, we determined that the consensus motif R-(R/K/H)-L-(A/L/S/T/F)(265) is essential for the endoproteolytic processing. In agreement with the identified consensus motif, we show that the cellular subtilase SKI-1/S1P cleaves LCMV GP.