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.     

Site-specific antibodies define a cleavage site conserved among arenavirus GP-C glycoproteins.

Arenaviruses share a common strategy for glycoprotein synthesis and processing in which a mannose-rich precursor glycoprotein, termed GP-C in lymphocytic choriomeningitis virus (LCMV), is posttranslationally processed by oligosaccharide trimming and proteolytic cleavage to yield two structural glycoproteins, GP-1 and GP-2. Mapping the orientation and proteolytic cleavage site(s) in such polyproteins has traditionally required direct protein sequencing of one or more of the cleaved products. This technique requires rigorous purification of the products for sequencing and may be complicated by amino-terminal modifications which interfere with sequence analysis. We used an alternative approach in which synthetic peptides corresponding to sequences bracketing a potential protease cleavage site were used to raise antisera which define the boundaries of the cleaved products. We found that cleavage of LCMV GP-C to yield GP-1 and GP-2 occurs within a 9-amino-acid stretch of GP-C which contains a paired basic amino acid group -Arg-Arg-, corresponding to amino acids 262 to 263 in the LCMV GP-C sequence. By comparison with the predicted amino acid sequences of a second LCMV strain, LCMV-WE, as well as with the deduced amino acid sequences of the New World arenavirus Pichinde and the Old World virus Lassa, we observed similar conservation of paired basic and flanking amino acid sequences among these viruses.     

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.     

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.     

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.     

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.     

Lassa virus Z protein is a matrix protein and sufficient for the release of virus-like particles [corrected

Lassa virus is an enveloped virus with glycoprotein spikes on its surface. It contains an RNA ambisense genome that encodes the glycoprotein precursor GP-C, the nucleoprotein NP, the polymerase L, and the Z protein. Here we demonstrate that the Lassa virus Z protein (i). is abundant in viral particles, (ii). is strongly membrane associated, (iii). is sufficient in the absence of all other viral proteins to release enveloped particles, and (iv). contains two late domains, PTAP and PPXY, necessary for the release of virus-like particles. Our data provide evidence that Z is the Lassa virus matrix protein that is the driving force for virus particle release.     

Changes in human immunodeficiency virus type 1 Gag at positions L449 and P453 are linked to I50V protease mutants in vivo and cause reduction of sensitivity to amprenavir and improved viral fitness in vitro

Human immunodeficiency virus type 1 (HIV-1) Gag protease cleavage sites (CS) undergo sequence changes during the development of resistance to several protease inhibitors (PIs). We have analyzed the association of sequence variation at the p7/p1 and p1/p6 CS in conjunction with amprenavir (APV)-specific protease mutations following PI combination therapy with APV. Querying a central resistance data repository resulted in the detection of significant associations (P < 0.001) between the presence of APV protease signature mutations and Gag L449F (p1/p6 LP1'F) and P453L (p1/p6 PP5'L) CS changes. In population-based sequence analyses the I50V mutant was invariably linked to either L449F or P453L. Clonal analysis revealed that both CS mutations were never present in the same genome. Sequential plasma samples from one patient revealed a transition from I50V M46L P453L viruses at early time points to I50V M46I L449F viruses in later samples. Various combinations of the protease and Gag mutations were introduced into the HXB2 laboratory strain of HIV-1. In both single- and multiple-cycle assay systems and in the context of I50V, the L449F and P453L changes consistently increased the 50% inhibitory concentration of APV, while the CS changes alone had no measurable effect on inhibitor sensitivity. The decreased in vitro fitness of the I50V mutant was only partially improved by addition of either CS change (I50V M46I L449F mutant replicative capacity approximately 16% of that of wild-type virus). Western blot analysis of Pr55 Gag precursor cleavage products from infected-cell cultures indicated accumulation of uncleaved Gag p1-p6 in all I50V viruses without coexisting CS changes. Purified I50V protease catalyzed cleavage of decapeptides incorporating the L449F or P453L change 10-fold and 22-fold more efficiently than cleavage of the wild-type substrate, respectively. HIV-1 protease CS changes are selected during PI therapy and can have effects on both viral fitness and phenotypic resistance to PIs.     

Contribution of the conservative cleavage motif to posttranslational processing of the carboxyl terminal domain of rodent Muc3

In previous works, we showed by transient expression studies in COS-1 cells that the C-terminal domain of rat intestinal membrane mucin (rMuc3) that was cloned in the pSecTag2 plasmid (named as p20) is posttranslationally cleaved twice. One location is between the glycine and the serine within a LS1KGS2IV1V2 motif, and the other is in the 49 kDa membrane-tethered fragment at an undefined site. The sea-urchin sperm protein, enterokinase and agrin module of rMuc3 is responsible for the cleavage and association of the cleaved fragments. The present study demonstrates how the conservative cleavage motif LS1KGS2IV1V2 contributes to posttranslational processing through mutagenesis of each residue in the LS(1)KGS2IV1V2 motif. Mutation of S2 to alanine (p20s2/a) completely prevented cleavage. While p20k/a (in this construct the K is replaced by A) and p20s1/a (in this construct the S1 is replaced by A) (6 and 3%) showed almost the same result as the wild-type p20 transfectant (4%), 79, 39, 22, 17, and 14% of the products from p20g/a (in this construct the G is replaced by A), p20i/a (in this construct the I is replaced by A), p20l/a (in this construct the L is replaced by A), p20v2/a (in this construct the V2 is replaced by A), and p20v1/a (in this construct the V1 is replaced by A) remained uncleaved. The cleaved N-terminal fragment of the p20s1/a transfectant was 26 kDa, but the N-terminal fragments from p20, p20g/a, p20l/a, p20k/a, p20i/a, p20v1/a, and p20v2/a were 30 kDa. The S1 residue was possibly O-glycosylated, which was supported by deglycosylation with O-cocktail (a mixture of glycosidases). The N-terminal fragment of p20s1/a transfected cells was present at high levels in the spent media. Thus, the S2, G, I, L, V2, and V1 residues within the conserved cleavage motif, LS1KGS2IV1V2, are important for cleavage and contribute to the structural formation and conformational stress of the small loop between the beta2 and the beta3 strands. The S1 residue is possibly O-glycosylated, and mutation of S1 residue to alanine does not affect the cleavage of the LS1KGS2IV1V2 motif, but it is important for the dissociation and further release of the cleaved N-terminal fragment from the cell surface.     

Identification of an N-terminal trimeric coiled-coil core within arenavirus glycoprotein 2 permits assignment to class I viral fusion proteins

The lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) consists of the transmembrane subunit GP-2 and the receptor binding subunit GP-1. Both are synthesized as one precursor protein and stay noncovalently attached after cleavage. In this study, we determined the oligomeric state of the LCMV GP and expressed it in two different conformations suitable for structural analysis. Sequence analysis of GP-2 identified a trimeric heptad repeat pattern containing an N-terminal alpha-helix. An alpha-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second alpha-helical peptide corresponding to a predicted C-terminal alpha-helix within GP-2 did not oligomerize. Refolding of the complete GP-2 ectodomain revealed trimeric all-alpha complexes probably representing the six-helix bundle state that is considered a hallmark of class I viral fusion proteins. Based on these results, we generated a construct consisting of the complete uncleavable LCMV GP ectodomain fused C-terminally to the trimeric motif of fibritin. Gel filtration analysis of the secreted fusion protein identified two complexes of approximately 230 and approximately 440 kDa. Both complexes bound to a set of conformational and linear antibodies. Cross-linking confirmed the 230-kDa complex to be a trimer. The 440-kDa complexes were found to represent disulfide-linked pairs of trimers, since partial reduction converted them to a complex species migrating at 250 kDa. By electron microscopy, the 230-kDa complexes appeared as single spherical particles and showed no signs of rosette formation. Our results clearly demonstrate that the arenavirus GP is a trimer and must be considered a member of the class I viral fusion protein family.     

Functional correlation between a novel amino acid insertion at codon 19 in the protease of human immunodeficiency virus type 1 and polymorphism in the p1/p6 Gag cleavage site in drug resistance and replication fitness

Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1' position (Leu to Pro) and the P5' position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.     

A Novel Cytotoxic Sequence Contributes to Influenza A Viral Protein PB1-F2 Pathogenicity and Predisposition to Secondary Bacterial Infection

Enhancement of cell death is a distinguishing feature of H1N1 influenza virus A/Puerto Rico/8/34 protein PB1-F2. Comparing the sequences (amino acids [aa] 61 to 87 using PB1-F2 amino acid numbering) of the PB1-F2-derived C-terminal peptides from influenza A viruses inducing high or low levels of cell death, we identified a unique I68, L69, and V70 motif in A/Puerto Rico/8/34 PB1-F2 responsible for promotion of the peptide''s cytotoxicity and permeabilization of the mitochondrial membrane. When administered to mice, a 27-mer PB1-F2-derived C-terminal peptide with this amino acid motif caused significantly greater weight loss and pulmonary inflammation than the peptide without it (due to I68T, L69Q, and V70G mutations). Similar to the wild-type peptide, A/Puerto Rico/8/34 elicited significantly higher levels of macrophages, neutrophils, and cytokines in the bronchoalveolar lavage fluid of mice than its mutant counterpart 7 days after infection. Additionally, infection of mice with A/Puerto Rico/8/34 significantly enhanced the levels of morphologically transformed epithelial and immune mononuclear cells recruited in the airways compared with the mutant virus. In the mouse bacterial superinfection model, both peptide and virus with the I68, L69, and V70 sequence accelerated development of pneumococcal pneumonia, as reflected by increased levels of viral and bacterial lung titers and by greater mortality. Here we provide evidence suggesting that the newly identified cytotoxic sequence I68, L69, and V70 of A/Puerto Rico/8/34 PB1-F2 contributes to the pathogenesis of both primary viral and secondary bacterial infections.     

A mutational analysis of the PD...D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli

McrBC is a unique restriction enzyme which binds specifically to the bipartite recognition sequence R(m)CN( approximately )(30)(-)( approximately )(2000)R(m)C and in the presence of GTP translocates the DNA and cleaves both strands at multiple positions within the two R(m)C "half-sites". It is known that McrBC is composed of two subunits: McrB which binds and hydrolyzes GTP and specifically interacts with DNA and McrC whose function is not clear but which has been suspected to harbor the catalytic center for DNA cleavage. A multiple-sequence alignment of the amino acid sequence of Escherichia coli McrC and of six presumably homologous open reading frames from various bacterial species shows that a sequence motif found in many restriction enzymes, but also in other nucleases, the PD.D/EXK motif, is conserved among these sequences. A mutational analysis, in which the carboxylates (aspartic acid in McrC) of this motif were substituted with alanine or asparagine and lysine was substituted with alanine or arginine, strongly suggests that Asp244, Asp257, and Lys259 represent the catalytic center of E. coli McrC. Whereas the variants D244A (or -N), D257A (or -N), and K259A are inactive in DNA cleavage (K259R has residual DNA cleavage activity), they interact with McrB like wild-type McrC, as can be deduced from the finding that they stimulate the McrB-catalyzed GTP hydrolysis to the same extent as wild-type McrC. Thus, whereas McrC variants defective in DNA cleavage can stimulate the GTPase activity of McrB, the DNase activity of McrC is not supported by McrB variants defective in GTP hydrolysis.     

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.     

Inhibition of Lassa and Marburg Virus Production by Tetherin

Recently, tetherin has been identified as an effective cellular factor that prevents the release of human immunodeficiency virus type 1. Here, we show that the production of virus-like particles induced by viral matrix proteins of Lassa virus or Marburg virus was markedly inhibited by tetherin and that N-linked glycosylation of tetherin was dispensable for this antiviral activity. Our data also suggest that viral matrix proteins or one or more components that originate from host cells are targets of tetherin but that viral surface glycoproteins are not. These results suggest that tetherin inhibits the release of a wide variety of enveloped viruses from host cells by a common mechanism.There are a number of innate host defense systems against virus infection, including interferon (IFN) and toll-like receptor signaling pathways. Cellular factors that inhibit viral replication through interactions with viral components at various steps have also been identified.Recently, tetherin (also known as BST2, CD317, or HM1.24) was identified as a cellular factor that inhibits the release of human immunodeficiency virus type 1 (HIV-1) from infected cells (6). Tetherin is a membrane-associated protein with an N-terminal transmembrane domain, a central extracellular domain with two potential N-linked glycosylation sites, and a C-terminal glycosylphosphatidylinositol (GPI) anchor (Fig. ​(Fig.1A)1A) (3, 4), which appears to prevent HIV-1 release by retaining fully formed progeny virions on the surfaces of infected cells (6, 11). Tetherin is constitutively present on the surfaces of HeLa and CEM cells, while its cell surface expression is induced by alpha IFN (IFN-α) in HEK293, 293T, HOS, HT1080, and COS-7 cells. Tetherin expression has also been reported to be stimulated by IFN in various tissues, including those of the liver, lung, placenta, heart, pancreas, kidney, skeletal muscle, and brain (1, 3), suggesting that it may function as part of IFN-induced innate immunity against enveloped viruses in vivo.Open in a separate windowFIG. 1.Inhibitory effects of tetherin and its mutants against Lassa VLP release. (A) Tetherin (WT) contains an N-terminal intracellular domain (ID), a transmembrane domain (TM), a central extracellular domain (ED), and a C-terminal GPI anchor (GPI). Arrowheads indicate the predicted sites of cleavage prior to the addition of the GPI anchor. Tetherin possesses two potential N-linked glycosylation sites at positions 65 and 92 in the ED. N65A and N92A are mutants with the loss of a glycosylation site by an Asn-to-Ala substitution at positions 65 and 92, respectively. N65A/N92A is a nonglycosylated mutant with the loss of both glycosylation sites. (B and D) The Lassa virus Z and GP-C expression plasmids were cotransfected with the expression plasmid for WT or mutant tetherin or an empty vector (Control) into COS-7 cells (B) or 293T cells (D). Extracellular VLPs induced by Lassa virus Z/GP-C were pelleted from the culture fluids. Cell- or VLP-associated Z and GP-C (GP-2) were detected by Western blotting using rabbit anti-Z antiserum and mouse anti-GP-2 monoclonal antibody. WB using anti-FLAG antibody was also performed to examine the expression of WT and mutant tetherin in cells. WB for actin was done as the internal control. (C) The intensities of the bands for VLP-associated Z or GP-2 in panel B were quantified using a LAS3000 imaging system (Fujifilm). The level of Z or GP-2 in VLPs released from cells cotransfected with control vector was set to 100%. The data are shown as averages and standard deviations for three independent experiments. (E) COS-7 cells were cotransfected with the Lassa virus Z expression plasmid and the expression plasmid for tetherin (WT) or the empty vector (Control). VLPs induced by Z alone were examined by WB as described above. (F) 293T cells were cotransfected with pCLV-Z and the empty vector (left) or the expression plasmid for tetherin (right). At 48 h posttransfection, cells were observed by electron microscopy, which was performed as described previously (9). Mock, mock infected; Teth, tetherin. Bars, 500 nm.The antiviral activity of tetherin is antagonized by HIV-1 Vpu due to the downregulation of cell surface expression of tetherin by Vpu (6, 11). Previously, the IFN-α-induced cell surface retention of virus-like particles (VLPs) induced by Ebola virus matrix protein VP40 was shown to be overcome by Vpu expression (5). Thus, the release of enveloped viruses other than HIV-1 may also be inhibited by tetherin.Lassa and Marburg viruses are emerging viruses belonging to the families Arenaviridae and Filoviridae, respectively, that cause hemorrhagic fever with high mortality rates. No approved vaccines or antiviral drugs are available to prevent or treat these viral diseases. Similar to HIV-1, both are enveloped viruses that exit the host cells by membrane extrusion, known as budding, from the plasma membrane. Therefore, having an antiviral effect against Lassa and Marburg viruses would make tetherin a potent tool for novel antiviral strategies against a wide variety of enveloped viruses.We examined the antiviral activities of tetherin against Lassa and Marburg viruses and analyzed the characteristics required for its antiviral activity in order to gain insight into its antiviral mechanism of action.     

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.     

Structural rationale for the recognition of arginine at P₃ in PEXEL motif containing proteins of Plasmodium falciparum by plasmepsin V

The virulent form of malaria is caused by Plasmodium falciparum that infects red blood cells. In order to survive inside the host, the parasite remodels the infected erythrocytes by exporting more than 300 effector proteins outside the parasitophorous vacuole membrane into the cytosol. The main feature of all the export proteins is the presence of a pentapeptide sequence motif; RxLxE/Q/D. This sequence motif is hydrolysed between L-x and the proteins with the acetylated new N-terminus xE/Q/D are exported. The enzyme responsible for this hydrolysis is plasmepsin V which is one of the ten aspartic proteases in P. falciparum. In order to understand the structural rationale for the specificity of this protease towards cleavage of the above motif, we generated three-dimensional models of seven plasmepsins (I, V to X) for which experimental structures are not available and compared these along with the crystal structures of three P. falciparum plasmepsins (II to IV). The structure comparisons revealed the importance of Tyr13, Glu77 and Ala117 specific to plasmepsin V that facilitates the accommodation of arginine at P? in the RxLxE/Q/D motif. Our analysis correlates the structure-function relationship of plasmepsin V.     

Inhibition of different Lassa virus strains by alpha and gamma interferons and comparison with a less pathogenic arenavirus

The high pathogenicity of Lassa virus is assumed to involve resistance to the effects of interferon (IFN). We have analyzed the effects of alpha IFN (IFN-alpha), IFN-gamma, and tumor necrosis factor alpha (TNF-alpha) on replication of Lassa virus compared to the related, but less pathogenic, lymphocytic choriomeningitis virus (LCMV). Three low-passage Lassa virus strains (AV, NL, and CSF), isolated from humans with mild to fulminant Lassa fever, were tested. Lassa virus replication was inhibited by IFN-alpha and IFN-gamma, but not TNF-alpha, in Huh7 and Vero cells. The degree of IFN sensitivity of a Lassa virus isolate did not correlate with disease severity in human patients. Furthermore, cytokine effects observed for Lassa virus and LCMV (strains CH-5692, Armstrong, and WE) were similar. To address the mechanisms involved in the IFN effect, we used cell lines in which overexpression of IFN-stimulated proteins promyelocytic leukemia protein (PML) and Sp100 could be induced. Both proteins reside in PML bodies, a cellular target of the LCMV and Lassa virus Z proteins. Overexpression of PML or Sp100 did not affect replication of either virus. This, together with the previous finding that PML knockout facilitates LCMV replication in vitro and in vivo (M. Djavani, J. Rodas, I. S. Lukashevich, D. Horejsh, P. P. Pandolfi, K. L. Borden, and M. S. Salvato, J. Virol. 75:6204-6208, 2001; W. V. Bonilla, D. D. Pinschewer, P. Klenerman, V. Rousson, M. Gaboli, P. P. Pandolfi, R. M. Zinkernagel, M. S. Salvato, and H. Hengartner, J. Virol. 76:3810-3818, 2002), describes PML as a mediator within the antiviral pathway rather than as a direct effector protein. In conclusion, the high pathogenicity of Lassa virus compared to LCMV is probably not due to increased resistance to the effects of IFN-alpha or IFN-gamma. Both cytokines inhibit replication which is relevant for the design of antiviral strategies against Lassa fever with the aim of enhancing the IFN response.     

In vivo and in vitro characterization of TEV protease mutants

Tobacco etch virus protease (TEVp) is frequently applied in the cleavage of fusion protein. However, production of TEV protease in Escherichia coli is hampered by low yield and poor solubility, and auto-cleavage of wild type TEVp gives rise to the loss-of-function. Previously it was reported that TEVp S219V displayed more stability, and TEVp variant containing T17S/N68D/I77V and double mutant L56V/S135G resulted in the enhanced production and solubility, respectively. Here, we introduced T17S/N68D/I77V in TEVp S219V to generate TEVpM1 and combined five amino acid mutations (T17S/L56V/N68D/I77V/S135G) in TEVp S219V to create TEVpM2. Among TEVp S219V, and two constructed variants, TEVpM2 displayed highest solubility and catalytic activity in vivo, using EmGFP as the solubility reporter, and the designed fusion protein as in vivo substrate containing an N-terminal hexahistidine tagged GST, a peptide sequence for thrombin and TEV cut and E. coli diaminopropionate ammonia-lyase. The purified TEVp mutants fused with double hexahistidine-tag at N and C terminus showed highest yield, solubility and cleavage efficiency. Mutations of five amino acid residues in TEVpM2 slightly altered protein secondary structure conformed by circular dichroism assay.