Cellular factors required for Lassa virus budding.
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It is known that Lassa virus Z protein is sufficient for the release of virus-like particles (VLPs) and that it has two L domains, PTAP and PPPY, in its C terminus. However, little is known about the cellular factor for Lassa virus budding. We examined which cellular factors are used in Lassa virus Z budding. We demonstrated that Lassa Z protein efficiently produces VLPs and uses cellular factors, Vps4A, Vps4B, and Tsg101, in budding, suggesting that Lassa virus budding uses the multivesicular body pathway functionally. Our data may provide a clue to develop an effective antiviral strategy for Lassa virus. (+info)
Historical Lassa fever reports and 30-year clinical update.
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Five cases of Lassa fever have been imported from West Africa to the United States since 1969. We report symptoms of the patient with the second imported case and the symptoms and long-term follow-up on the patient with the third case. Vertigo in this patient has persisted for 30 years. (+info)
The role of single N-glycans in proteolytic processing and cell surface transport of the Lassa virus glycoprotein GP-C.
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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. (+info)
The proprotein convertase SKI-1/S1P. In vitro analysis of Lassa virus glycoprotein-derived substrates and ex vivo validation of irreversible peptide inhibitors.
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Herein we designed, synthesized, tested, and validated fluorogenic methylcoumarinamide (MCA) and chloromethylketone-peptides spanning the Lassa virus GPC cleavage site as substrates and inhibitors for the proprotein convertase SKI-1/S1P. The 7-mer MCA (YISRRLL-MCA) and 8-mer MCA (IYISRRLL-MCA) are very efficiently cleaved with respect to both the 6-mer MCA (ISRRLL-MCA) and point mutated fluorogenic analogues, except for the 7-mer mutant Y253F. The importance of the P7 phenylic residue was confirmed by digestions of two 16-mer non-fluorogenic peptidyl substrates that differ by a single point mutation (Y253A). Because NMR analysis of these 16-mer peptides did not reveal significant structural differences at recognition motif RRLL, the P7 Tyr residue is likely important in establishing key interactions within the catalytic pocket of SKI-1. Based on these data, we established through analysis of pro-ATF6 and pro-SREBP-2 cellular processing that decanoylated chloromethylketone 7-mer, 6-mer, and 4-mer peptides containing the core RRLL sequence are irreversible and potent ex vivo SKI-1 inhibitors. Although caution must be exercised in using these inhibitors in in vitro reactions, as they can also inhibit the basic amino acid-specific convertase furin, within cells and when used at concentrations < or = 100 microM these inhibitors are relatively specific for inhibition of SKI-1 processing events, as opposed to those performed by furin-like convertases. (+info)
Identification of protective Lassa virus epitopes that are restricted by HLA-A2.
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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. (+info)
Mutational analysis of the lassa virus promoter.
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The promoter sequences directing viral gene expression and genome replication of arenaviruses reside within the 3' and 5' termini of each RNA segment. The terminal 19 nucleotides at both ends are highly conserved among all arenavirus species and are almost completely complementary to each other. This study aimed at characterizing the Lassa virus promoter in detail. The relevance of each position in the promoter was studied by site-directed mutagenesis using the Lassa virus minireplicon system. The data indicate that the Lassa virus promoter functions as a duplex, regulates transcription and replication in a coordinated manner, and is composed of two functional elements, a sequence-specific region from residue 1 to 12 and a variable complementary region from residue 13 to 19. The first region appears to interact with the replication complex mainly via base-specific interactions, while in the second region solely base pairing between 3' and 5' promoter ends is important for promoter function. (+info)
Antigenic relatedness between arenaviruses defined at the epitope level by monoclonal antibodies.
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Monoclonal antibodies (MAbs) were produced against two African arenaviruses, Lassa virus and Mopeia virus. Competitive binding analysis of MAbs identified four antigenic sites on the nucleoprotein (NP), two on glycoprotein 1 (GP1) and six on glycoprotein 2 (GP2) of the Josiah strain of Lassa virus. 64 virus isolates from western, central and southern Africa were all consistently distinguishable by MAbs to certain epitopic sites on GP1, GP2 and NP viral proteins. Furthermore, MAbs to Lassa virus GP1 and NP uniformly distinguished viruses from the West African countries of Sierra Leone, Liberia and Guinea from those of Nigeria. GP2-directed MAbs to two African arenaviruses reacted broadly with South American arenaviruses demonstrating that an epitopic site on GP2 may be the most highly conserved antigen in the arenavirus group. (+info)
The role of myristoylation in the membrane association of the Lassa virus matrix protein Z.
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The Z protein is the matrix protein of arenaviruses and has been identified as the main driving force for budding. Both LCMV and Lassa virus Z proteins bud from cells in the absence of other viral proteins as enveloped virus-like particles. Z accumulates near the inner surface of the plasma membrane where budding takes place. Furthermore, biochemical data have shown that Z is strongly membrane associated. The primary sequence of Z lacks a typical transmembrane domain and until now it is not understood by which mechanism Z is able to interact with cellular membranes. In this report, we analyzed the role of N-terminal myristoylation for the membrane binding of Lassa virus Z. We show that disruption of the N-terminal myristoylation signal by substituting the N-terminal glycine with alanine (Z-G2A mutant) resulted in a significant reduction of Z protein association with cellular membranes. Furthermore, removal of the myristoylation site resulted in a relocalization of Z from a punctuate distribution to a more diffuse cellular distribution pattern. Finally, treatment of Lassa virus-infected cells with various myristoylation inhibitors drastically reduced efficient Lassa virus replication. Our data indicate that myristoylation of Z is critical for its binding ability to lipid membranes and thus, for effective virus budding. (+info)