scholarly journals MARCH8 Restricts Ebola Virus Replication by Blocking the Viral Glycoprotein Processing and Glycosylation

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 123
Author(s):  
Changqing Yu ◽  
Sunan Li ◽  
Omid Madadgar ◽  
Iqbal Ahmad ◽  
Xianfeng Zhang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Ebola Virus ◽  
Class I ◽  
Class Iii ◽  
Viral Glycoprotein ◽  
E3 Ligases ◽  
Glycosylation Sites ◽  
Hiv 1

Ebola virus (EBOV) glycoprotein (GP) is a class I fusion protein whose maturation is dependent on furin-mediated processing. EBOV-GP is heavily glycosylated, with glycans constituting ~50% of its molecular mass. Compared with 15 N-linked glycosylation sites, EBOV-GP1 has ~80 potential O-linked glycosylation sites in the mucin-like domain (MLD), suggesting that O-linked glycans are dominated. The membrane-associated RING-CH (MARCH) family consists of 11 members that are RING-finger ubiquitin E3 ligases. Recently, human MARCH1, MARCH2, and MARCH8 were reported to inhibit HIV-1 replication by targeting its Env. Here, we show that human MARCH8 also inhibits EBOV replication by blocking GP incorporation into virions via downregulating its cell surface expression. To understand how the downregulation occurs, we investigated EBOV-GP subcellular localization, processing, glycosylation, and intracellular trafficking in the presence of human MARCH8. We find that MARCH8 interacts with GP and retains GP in the Golgi. MARCH8 also interacts with the homoB domain of furin that blocks its convertase activity. In consequence, MARCH8 blocks GP processing in an MLD-independent manner. Consistently, MARCH8 also blocks the O-linked, but not the N-linked glycosylation of GP. Importantly, in the presence of MARCH8, the shedding of GP1 but not the secreted GP (sGP) is blocked, suggesting that MARCH8 targets the GP1 C-terminal region. The MARCH8 activity is extended to its orthologs from Bos taurus and mice, and its paralogs MARCH1 and MARCH2. In addition, MARCH8 inhibits the processing of two other class I fusion proteins, including HIV-1 Env and IAV HA, and it triggers the degradation of the class III fusion protein VSV-G. We conclude that MARCH8 exerts a very broad and conserved antiviral activity by inhibiting the maturation of class I fusion proteins, which blocks their secretion to the cell surface and incorporation into virions. It should also target class III fusion proteins by triggering their degradation.

scholarly journals Trim-Cyclophilin A Fusion Proteins Can Restrict Human Immunodeficiency Virus Type 1 Infection at Two Distinct Phases in the Viral Life Cycle

2006 ◽  
Vol 80 (8) ◽  
pp. 4061-4067 ◽  
Author(s):  
Melvyn W. Yap ◽  
Mark P. Dodding ◽  
Jonathan P. Stoye
Keyword(s):  
Human Immunodeficiency Virus ◽  
Life Cycle ◽  
Fusion Protein ◽  
Human Immunodeficiency Virus Type ◽  
Fusion Proteins ◽  
Cyclophilin A ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
B30.2 Domain

ABSTRACT The Trim5α protein from several primates restricts retroviruses in a capsid (CA)-dependent manner. In owl monkeys, the B30.2 domain of Trim5 has been replaced by cyclophilin A (CypA) following a retrotransposition. Restriction of human immunodeficiency virus type 1 (HIV-1) by the resulting Trim5-CypA fusion protein depends on CA binding to CypA, suggesting both that the B30.2 domain might be involved in CA binding and that the tripartite RING motif, B-BOX, and coiled coil (RBCC) motif domain can function independently of the B30.2 domain in restriction. To investigate the potential of RBCCs from other Trims to participate in restricting HIV-1, CypA was fused to the RBCC of Trim1, Trim18, and Trim19 and tested for restriction. Despite low identity within the RBCC domain, all fusion proteins were found to restrict HIV-1 but not the nonbinding G89V mutant, indicating that the overall structure of RBCC and not its primary sequence was important for the restriction function. The critical interaction between CA and Trim-CypA appears to take place soon after viral entry. Quantitative PCR analysis of viral reverse transcriptase products revealed that the different fusion proteins block HIV-1 at two distinct stages of its life cycle, either prior to reverse transcription or just before integration. With Trim1 and Trim18, this timing is dependent on the length of the Trim component of the fusion protein. These observations suggest that restriction factor binding can have different mechanistic consequences.

scholarly journals A combined EM and proteomic analysis places HIV-1 Vpu at the crossroads of retromer and ESCRT complexes: PTPN23 is a Vpu-cofactor

2021 ◽  
Vol 17 (11) ◽  
pp. e1009409
Author(s):  
Charlotte A. Stoneham ◽  
Simon Langer ◽  
Paul D. De Jesus ◽  
Jacob M. Wozniak ◽  
John Lapek ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Trafficking ◽  
Fusion Proteins ◽  
Immune Surveillance ◽  
Accessory Protein ◽  
Wild Type ◽  
Membrane Systems ◽  
Membrane Protein Trafficking ◽  
Cellular Pathways ◽  
Hiv 1

The HIV-1 accessory protein Vpu modulates membrane protein trafficking and degradation to provide evasion of immune surveillance. Targets of Vpu include CD4, HLAs, and BST-2. Several cellular pathways co-opted by Vpu have been identified, but the picture of Vpu’s itinerary and activities within membrane systems remains incomplete. Here, we used fusion proteins of Vpu and the enzyme ascorbate peroxidase (APEX2) to compare the ultrastructural locations and the proximal proteomes of wild type Vpu and Vpu-mutants. The proximity-omes of the proteins correlated with their ultrastructural locations and placed wild type Vpu near both retromer and ESCRT-0 complexes. Hierarchical clustering of protein abundances across the mutants was essential to interpreting the data and identified Vpu degradation-targets including CD4, HLA-C, and SEC12 as well as Vpu-cofactors including HGS, STAM, clathrin, and PTPN23, an ALIX-like protein. The Vpu-directed degradation of BST-2 was supported by STAM and PTPN23 and to a much lesser extent by the retromer subunits Vps35 and SNX3. PTPN23 also supported the Vpu-directed decrease in CD4 at the cell surface. These data suggest that Vpu directs targets from sorting endosomes to degradation at multi-vesicular bodies via ESCRT-0 and PTPN23.

scholarly journals The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

1994 ◽  
Vol 127 (6) ◽  
pp. 1843-1857 ◽  
Author(s):  
K C Hart ◽  
Y F Xu ◽  
A N Meyer ◽  
B A Lee ◽  
D J Donoghue
Keyword(s):  
Cell Surface ◽  
Fusion Protein ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Proteolytic Processing ◽  
Nih3t3 Cells ◽  
Transforming Activity ◽  
Retention Signal

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

scholarly journals Purification and Crystallization Reveal Two Types of Interactions of the Fusion Protein Homotrimer of Semliki Forest Virus

2004 ◽  
Vol 78 (7) ◽  
pp. 3514-3523 ◽  
Author(s):  
Don L. Gibbons ◽  
Brigid Reilly ◽  
Anna Ahn ◽  
Marie-Christine Vaney ◽  
Armelle Vigouroux ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Semliki Forest Virus ◽  
Three Dimensional ◽  
Class Ii ◽  
Class I ◽  
Structural Basis ◽  
Viral Fusion ◽  
Purification Method ◽  
Viral Fusion Proteins

ABSTRACT The fusion proteins of the alphaviruses and flaviviruses have a similar native structure and convert to a highly stable homotrimer conformation during the fusion of the viral and target membranes. The properties of the alpha- and flavivirus fusion proteins distinguish them from the class I viral fusion proteins, such as influenza virus hemagglutinin, and establish them as the first members of the class II fusion proteins. Understanding how this new class carries out membrane fusion will require analysis of the structural basis for both the interaction of the protein subunits within the homotrimer and their interaction with the viral and target membranes. To this end we report a purification method for the E1 ectodomain homotrimer from the alphavirus Semliki Forest virus. The purified protein is trimeric, detergent soluble, retains the characteristic stability of the starting homotrimer, and is free of lipid and other contaminants. In contrast to the postfusion structures that have been determined for the class I proteins, the E1 homotrimer contains the fusion peptide region responsible for interaction with target membranes. This E1 trimer preparation is an excellent candidate for structural studies of the class II viral fusion proteins, and we report conditions that generate three-dimensional crystals suitable for analysis by X-ray diffraction. Determination of the structure will provide our first high-resolution views of both the low-pH-induced trimeric conformation and the target membrane-interacting region of the alphavirus fusion protein.

scholarly journals Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits.

1987 ◽  
Vol 105 (4) ◽  
pp. 1873-1884 ◽  
Author(s):  
E A Wayner ◽  
W G Carter
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Cell Surface ◽  
Surface Protein ◽  
Class Ii ◽  
Class I ◽  
Beta Subunits ◽  
Type I ◽  
Class Iii ◽  
Alpha Subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of Recombinant HIV-1 p24-Nef Protein in Two Forms as Potential Candidate Vaccines in Three Vehicles

2020 ◽  
Vol 17 (5) ◽  
pp. 387-395
Author(s):  
Mona Sadat Larijani ◽  
Mohammad Hassan Pouriayevali ◽  
Seyed Mehdi Sadat ◽  
Amitis Ramezani
Keyword(s):  
Fusion Protein ◽  
Western Blotting ◽  
Highly Active Antiretroviral Therapy ◽  
Fusion Proteins ◽  
Therapeutic Vaccine ◽  
Potential Candidate ◽  
Conserved Regions ◽  
Bioinformatics Tools ◽  
Hiv 1

Background: Different approaches have been investigated to develop a preventive or therapeutic vaccine, although none of them has been fully practical. Therapeutic vaccines against HIV-1 have been studied with the aim of eliminating the virus from reservoir cells with or without HAART (Highly Active Antiretroviral Therapy). Fusion proteins with the most immunogenic features among conserved regions can facilitate this achievement in such a variable virus. To achieve the most immunogenic and also conserved regions, bioinformatics tools are widely used to predict antigens’ features before applying them. Objective: This study aimed at the in vitro evaluation of p24 -Nef fusion protein based on the previous in silico design to achieve a potential therapeutic subunit vaccine against HIV-1. Methods: The truncated form of p24-Nef using AAY flexible linker and the full protein were expressed and evaluated in the prokaryotic system and confirmed by western blotting. We also used pcDNA3.1 to transfect Lenti-X 293T cells. Moreover, lentiviral vectors were applied to produce recombinant virions harboring the genes of interest and cell transduction. Results: Both fusion proteins in a truncated and a full form were expressed and confirmed by Anti Nef polyclonal antibody in western blotting. Recombinant virions were generated and transduced Lenti-X 293T cells confirming by immunofluorescence microscope and p24 ELISA assay kit. Transduced cells were analyzed by SDS-PAGE and western blotting, which resulted in approved protein expression. Conclusion: Fusion protein of p24 and Nef is well expressed in eukaryotic cell lines according to its pre-evaluated features by bioinformatics tools.

Virion-Targeted Viral Inactivation of Human Immunodeficiency Virus Type 1 by Using Vpr Fusion Proteins

1998 ◽  
Vol 72 (7) ◽  
pp. 5441-5448 ◽  
Author(s):  
Gary P. Kobinger ◽  
Alessandra Borsetti ◽  
Zilin Nie ◽  
Johanne Mercier ◽  
Nesrine Daniel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Fusion Protein ◽  
Fusion Proteins ◽  
Wild Type Virus ◽  
Progeny Virus ◽  
Hiv Replication ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Inactivation of progeny virions with chimeric virion-associated proteins represents a novel therapeutic approach against human immunodeficiency virus (HIV) replication. The HIV type 1 (HIV-1) Vpr gene product, which is packaged into virions, is an attractive candidate for such a strategy. In this study, we developed Vpr-based fusion proteins that could be specifically targeted into mature HIV-1 virions to affect their structural organization and/or functional integrity. Two Vpr fusion proteins were constructed by fusing to the first 88 amino acids of HIV-1 Vpr the chloramphenicol acetyltransferase enzyme (VprCAT) or the last 18 C-terminal amino acids of the HIV-1 Vpu protein (VprIE). These Vpr fusion proteins were initially designed to quantify their efficiency of incorporation into HIV-1 virions when produced in cis from the provirus. Subsequently, CD4+ Jurkat T-cell lines constitutively expressing the VprCAT or the VprIE fusion protein were generated with retroviral vectors. Expression of the VprCAT or the VprIE fusion protein in CD4+ Jurkat T cells did not interfere with cellular viability or growth but conferred substantial resistance to HIV replication. The resistance to HIV replication was more pronounced in Jurkat-VprIE cells than in Jurkat-VprCAT cells. Moreover, the antiviral effect mediated by VprIE was dependent on an intact p6 gag domain, indicating that the impairment of HIV-1 replication required the specific incorporation of Vpr fusion protein into virions. Gene expression, assembly, or release was not affected upon expression of these Vpr fusion proteins. Indeed, the VprIE and VprCAT fusion proteins were shown to affect the infectivity of progeny virus, since HIV virions containing the VprCAT or the VprIE fusion proteins were, respectively, 2 to 3 times and 10 to 30 times less infectious than the wild-type virus. Overall, this study demonstrated the successful transfer of resistance to HIV replication in tissue cultures by use of Vpr-based antiviral genes.

scholarly journals Conserved Leucines in N-Terminal Heptad Repeat HR1 of Envelope Fusion Protein F of Group II Nucleopolyhedroviruses Are Important for Correct Processing and Essential for Fusogenicity

2007 ◽  
Vol 82 (5) ◽  
pp. 2437-2447 ◽  
Author(s):  
Gang Long ◽  
Xiaoyu Pan ◽  
Just M. Vlak
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Leucine Zipper ◽  
Fusion Peptide ◽  
Class I ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
F Protein ◽  
Viral Fusion Proteins ◽  
Budded Virus

ABSTRACT The heptad repeat (HR), a conserved structural motif of class I viral fusion proteins, is responsible for the formation of a six-helix bundle structure during the envelope fusion process. The insect baculovirus F protein is a newly found budded virus envelope fusion protein which possesses common features to class I fusion proteins, such as proteolytic cleavage and the presence of an N-terminal open fusion peptide and multiple HR domains on the transmembrane subunit F1. Similar to many vertebrate viral fusion proteins, a conserved leucine zipper motif is predicted in this HR region proximal to the fusion peptide in baculovirus F proteins. To facilitate our understanding of the functional role of this leucine zipper-like HR1 domain in baculovirus F protein synthesis, processing, and viral infectivity, key leucine residues (Leu209, Leu216, and Leu223) were replaced by alanine (A) or arginine (R), respectively. By using Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) as a pseudotype expression system, we demonstrated that all mutant F proteins incorporated into budded virus, indicating that leucine substitutions did not affect intercellular trafficking of F. Furin-like protease cleavage was not affected by any of the leucine substitutions; however, the disulfide bridging and N-linked glycosylation patterns were partly altered. Single substitutions in HR1 showed that the three leucine residues were critical for F fusogenicity and the rescue of AcMNPV infectivity. Our results support the view that the leucine zipper-like HR1 domain is important to safeguard the proper folding, glycosylation, and fusogenicity of baculovirus F proteins.

scholarly journals Two Functional Variants of AP-1 Complexes Composed of either γ2 or γ1 Subunits Are Independently Required for Major Histocompatibility Complex Class I Downregulation by HIV-1 Nef

2020 ◽  
Vol 94 (7) ◽  
Author(s):  
Lucas A. Tavares ◽  
Julianne V. de Carvalho ◽  
Cristina S. Costa ◽  
Roberta M. Silveira ◽  
Andreia N. de Carvalho ◽  
...  
Keyword(s):  
T Cells ◽  
Major Histocompatibility Complex ◽  
Cell Surface ◽  
Major Histocompatibility Complex Class ◽  
Adaptor Protein ◽  
Class I ◽  
Complex Class ◽  
Mhc I ◽  
Histocompatibility Complex ◽  
Hiv 1

ABSTRACT The HIV-1 accessory protein Nef downregulates the cell surface expression of major histocompatibility complex class I (MHC-I) molecules to facilitate virus spreading. The Nef-induced downregulation of MHC-I molecules such as HLA-A requires the clathrin adaptor protein 1 (AP-1) complex. The cooperative interaction of Nef, AP-1, and the cytosolic tail (CT) of HLA-A leads to a redirection of HLA-A targeting from the trans-Golgi network (TGN) to lysosomes for degradation. Although the γ-adaptin subunit of AP-1 has two distinct isoforms (γ1 and γ2), which may form two AP-1 complex variants, so far, only the importance of AP-1γ1 in MHC-I downregulation by Nef has been investigated. Here, we report that the AP-1γ2 isoform also participates in this process. We found that AP-1γ2 forms a complex with Nef and HLA-A2_CT and that this interaction depends on the Y320 residue in HLA-A2_CT and Nef expression. Moreover, Nef targets AP-1γ1 and AP-1γ2 to different compartments in T cells, and the depletion of either AP-1 variant impairs the Nef-mediated reduction of total endogenous HLA-A levels and rescues HLA-A levels on the cell surface. Finally, immunofluorescence and immunoelectron microscopy analyses reveal that the depletion of γ2 in T cells compromises both the Nef-mediated retention of HLA-A molecules in the TGN and targeting to multivesicular bodies/late endosomes. Altogether, these results show that in addition to AP-1γ1, Nef also requires the AP-1γ2 variant for efficient MHC-I downregulation. IMPORTANCE HIV-1 Nef mediates evasion of the host immune system by inhibiting MHC-I surface presentation of viral antigens. To achieve this goal, Nef modifies the intracellular trafficking of MHC-I molecules in several ways. Despite being the subject of intense study, the molecular details underlying these modifications are not yet fully understood. Adaptor protein 1 (AP-1) plays an essential role in the Nef-mediated downregulation of MHC-I molecules such as HLA-A in different cell types. However, AP-1 has two functionally distinct variants composed of either γ1 or γ2 subunit isoforms. Because previous studies on the role of AP-1 in MHC-I downregulation by Nef focused on AP-1γ1, an important open question is the participation of AP-1γ2 in this process. Here, we show that AP-1γ2 is also essential for Nef-mediated depletion of surface HLA-A molecules in T cells. Our results indicate that Nef hijacks AP-1γ2 to modify HLA-A intracellular transport, redirecting these proteins to lysosomes for degradation.

scholarly journals Identification of an N-Terminal Trimeric Coiled-Coil Core within Arenavirus Glycoprotein 2 Permits Assignment to Class I Viral Fusion Proteins

2006 ◽  
Vol 80 (12) ◽  
pp. 5897-5907 ◽  
Author(s):  
Bruno Eschli ◽  
Katharina Quirin ◽  
Alexander Wepf ◽  
Jacqueline Weber ◽  
Rolf Zinkernagel ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Gel Filtration ◽  
Class I ◽  
Spherical Particles ◽  
Viral Fusion ◽  
Viral Fusion Protein ◽  
Helical Peptide ◽  
Viral Fusion Proteins ◽  
Α Helix

ABSTRACT 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 α-helix. An α-helical peptide matching this region formed a stable oligomer as revealed by gel filtration chromatography and dynamic light scattering. In contrast, a second α-helical peptide corresponding to a predicted C-terminal α-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 ∼230 and ∼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.

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