Modified ribavirin analogues as antiviral agents against Junín virus

In this work, several ribavirin analogues were synthesized and incorporated into a multivalent arrangement. Both were subsequently modified by the addition of polyhydroxylated residues.Their antiviral activity was tested against Junín virus, etiological agent responsible of Argentine hemorrhagic fever.Some compounds inhibited Junín virus in the range of 13.2–389.1?µM. Two modified ribavirin analogues presented an effective concentration comparable to ribavirin but with a higher selectivity index.     

A novel zinc-binding domain is essential for formation of the functional Junín virus envelope glycoprotein complex

The envelope glycoprotein of the Junín arenavirus (GP-C) mediates entry into target cells through a pH-dependent membrane fusion mechanism. Unlike other class I viral fusion proteins, the mature GP-C complex retains a cleaved, 58-amino-acid signal peptide (SSP) as an essential subunit, required both for trafficking of GP-C to the cell surface and for the activation of membrane fusion. SSP has been shown to associate noncovalently in GP-C via the cytoplasmic domain (CTD) of the transmembrane fusion subunit G2. In this report we investigate the molecular basis for this intersubunit interaction. We identify an invariant series of six cysteine and histidine residues in the CTD of G2 that is essential for incorporation of SSP in the GP-C complex. Moreover, we show that a CTD peptide fragment containing His-447, His-449, and Cys-455 specifically binds Zn(2+) at subnanomolar concentrations. Together, these results suggest a zinc finger-like domain structure in the CTD of G2. We propose that the remaining residues in the series (His-459, Cys-467, and Cys-469) form an intersubunit zinc-binding center that incorporates Cys-57 of SSP. This unusual motif may act to retain SSP in the GP-C complex and position the ectodomain loop of SSP for its role in modulating membrane fusion activity. The unique tripartite organization of GP-C could provide novel molecular targets for therapeutic intervention in arenaviral disease.     

Junín virus infection activates the type I interferon pathway in a RIG-I-dependent manner

Junín virus (JUNV), an arenavirus, is the causative agent of Argentine hemorrhagic fever, an infectious human disease with 15-30% case fatality. The pathogenesis of AHF is still not well understood. Elevated levels of interferon and cytokines are reported in AHF patients, which might be correlated to the severity of the disease. However the innate immune response to JUNV infection has not been well evaluated. Previous studies have suggested that the virulent strain of JUNV does not induce IFN in human macrophages and monocytes, whereas the attenuated strain of JUNV was found to induce IFN response in murine macrophages via the TLR-2 signaling pathway. In this study, we investigated the interaction between JUNV and IFN pathway in human epithelial cells highly permissive to JUNV infection. We have determined the expression pattern of interferon-stimulated genes (ISGs) and IFN-β at both mRNA and protein levels during JUNV infection. Our results clearly indicate that JUNV infection activates the type I IFN response. STAT1 phosphorylation, a downstream marker of activation of IFN signaling pathway, was readily detected in JUNV infected IFN-competent cells. Our studies also demonstrated for the first time that RIG-I was required for IFN production during JUNV infection. IFN activation was detected during infection by either the virulent or attenuated vaccine strain of JUNV. Curiously, both virus strains were relatively insensitive to human IFN treatment. Our studies collectively indicated that JUNV infection could induce host type I IFN response and provided new insights into the interaction between JUNV and host innate immune system, which might be important in future studies on vaccine development and antiviral treatment.     

Bitopic membrane topology of the stable signal peptide in the tripartite Junín virus GP-C envelope glycoprotein complex

The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hphi1 and hphi2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hphi2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.     

Potent Inhibition of Junín Virus Infection by Interferon in Murine Cells

The new world arenavirus Junín virus (JUNV) is the causative agent of Argentine hemorrhagic fever, a lethal human infectious disease. Adult laboratory mice are generally resistant to peripheral infection by JUNV. The mechanism underlying the mouse resistance to JUNV infection is largely unknown. We have reported that interferon receptor knockout mice succumb to JUNV infection, indicating the critical role of interferon in restricting JUNV infection in mice. Here we report that the pathogenic and vaccine strains of JUNV were highly sensitive to interferon in murine primary cells. Treatment with low concentrations of interferon abrogated viral NP protein expression in murine cells. The replication of both JUNVs was enhanced in IRF3/IRF7 deficient cells. In addition, the vaccine strain of JUNV displayed impaired growth in primary murine cells. Our data suggested a direct and potent role of host interferon response in restricting JUNV replication in mice. The defect in viral growth for vaccine JUNV might also partially explain its attenuation in mice.     

Ultrastructure of Junín Virus in Mouse Whole Brain and Mouse Brain Tissue Cultures

Comparative ultrastructural studies were performed on the development of Junín virus in mouse brain and in cerebellum explants and brain monolayers of the same animal. In mouse brain, neurons and astrocytes released virus particles by a budding mechanism identical to that previously described for this virus. In the neurons, the viral multiplication took place in the perikarion as well as in the cytoplasmic processes, including areas near synapses. Viral particles were observed emerging from pericapillary neurons and astrocytes. In the explants, the budding also occurred in neurons and astrocytes. In the monolayers, however, the virus originated in astrocytes and cells of fibroblastic appearance, which were the two cell types that developed in this substrate. These results indicate that the characteristics of the development of Junín virus in mouse brain are faithfully reproduced in cerebellum explants from the same animal, thus allowing some extrapolation of data from one system to the other. The explant proved to be a better model than the monolayer, not only because it reproduced the structural complexity of nervous tissue better, but also because it contains neurons and astrocytes, i.e., the two cell types that release the virus in the in vivo system.     

Role of the stable signal peptide and cytoplasmic domain of G2 in regulating intracellular transport of the Junín virus envelope glycoprotein complex

Enveloped viruses utilize the membranous compartments of the host cell for the assembly and budding of new virion particles. In this report, we have investigated the biogenesis and trafficking of the envelope glycoprotein (GP-C) of the Junín arenavirus. The mature GP-C complex is unusual in that it retains a stable signal peptide (SSP) as an essential component in association with the typical receptor-binding (G1) and transmembrane fusion (G2) subunits. We demonstrate that, in the absence of SSP, the G1-G2 precursor is restricted to the endoplasmic reticulum (ER). This constraint is relieved by coexpression of SSP in trans, allowing transit of the assembled GP-C complex through the Golgi and to the cell surface, the site of arenavirus budding. Transport of a chimeric CD4 glycoprotein bearing the transmembrane and cytoplasmic domains of G2 is similarly regulated by SSP association. Truncations to the cytoplasmic domain of G2 abrogate SSP association yet now permit transport of the G1-G2 precursor to the cell surface. Thus, the cytoplasmic domain of G2 is an important determinant for both ER localization and its control through SSP binding. Alanine mutations to either of two dibasic amino acid motifs in the G2 cytoplasmic domain can also mobilize the G1-G2 precursor for transit through the Golgi. Taken together, our results suggest that SSP binding masks endogenous ER localization signals in the cytoplasmic domain of G2 to ensure that only the fully assembled, tripartite GP-C complex is transported for virion assembly. This quality control process points to an important role of SSP in the structure and function of the arenavirus envelope glycoprotein.     

A specific interaction of small molecule entry inhibitors with the envelope glycoprotein complex of the Junín hemorrhagic fever arenavirus

Arenaviruses are responsible for acute hemorrhagic fevers worldwide and are recognized to pose significant threats to public health and biodefense. Small molecule compounds have recently been discovered that inhibit arenavirus entry and protect against lethal infection in animal models. These chemically distinct inhibitors act on the tripartite envelope glycoprotein (GPC) through its unusual stable signal peptide subunit to stabilize the complex against pH-induced activation of membrane fusion in the endosome. Here, we report the production and characterization of the intact transmembrane GPC complex of Junín arenavirus and its interaction with these inhibitors. The solubilized GPC is antigenically indistinguishable from the native protein and forms a homogeneous trimer in solution. When reconstituted into a lipid bilayer, the purified complex interacts specifically with its cell-surface receptor transferrin receptor-1. We show that small molecule entry inhibitors specific to New World or Old World arenaviruses bind to the membrane-associated GPC complex in accordance with their respective species selectivities and with dissociation constants comparable with concentrations that inhibit GPC-mediated membrane fusion. Furthermore, competitive binding studies reveal that these chemically distinct inhibitors share a common binding pocket on GPC. In conjunction with previous genetic studies, these findings identify the pH-sensing interface of GPC as a highly vulnerable target for antiviral intervention. This work expands our mechanistic understanding of arenavirus entry and provides a foundation to guide the development of small molecule compounds for the treatment of arenavirus hemorrhagic fevers.     

Role of the stable signal peptide of Junín arenavirus envelope glycoprotein in pH-dependent membrane fusion

The envelope glycoprotein of the arenaviruses (GP-C) is unusual in that the mature complex retains the cleaved, 58-amino-acid signal peptide. Association of this stable signal peptide (SSP) has been shown to be essential for intracellular trafficking and proteolytic maturation of the GP-C complex. We identify here a specific and previously unrecognized role of SSP in pH-dependent membrane fusion. Amino acid substitutions that alter the positive charge at lysine K33 in SSP affect the ability of GP-C to mediate cell-cell fusion and the threshold pH at which membrane fusion is triggered. Based on the presumed location of K33 at or near the luminal domain of SSP, we postulate that SSP interacts with the membrane-proximal or transmembrane regions of the G2 fusion protein. This unique organization of the GP-C complex may suggest novel strategies for intervention in arenavirus infection.     

An Antibody Directed against the Fusion Peptide of Junín Virus Envelope Glycoprotein GPC Inhibits pH-Induced Membrane Fusion

The arenavirus envelope glycoprotein (GPC) initiates infection in the host cell through pH-induced fusion of the viral and endosomal membranes. As in other class I viral fusion proteins, this process proceeds through a structural reorganization in GPC in which the ectodomain of the transmembrane fusion subunit (G2) engages the host cell membrane and subsequently refolds to form a highly stable six-helix bundle structure that brings the two membranes into apposition for fusion. Here, we describe a G2-directed monoclonal antibody, F100G5, that prevents membrane fusion by binding to an intermediate form of the protein on the fusion pathway. Inhibition of syncytium formation requires that F100G5 be present concomitant with exposure of GPC to acidic pH. We show that F100G5 recognizes neither the six-helix bundle nor the larger trimer-of-hairpins structure in the postfusion form of G2. Rather, Western blot analysis using recombinant proteins and a panel of alanine-scanning GPC mutants revealed that F100G5 binding is dependent on an invariant lysine residue (K283) near the N terminus of G2, in the so-called fusion peptide that inserts into the host cell membrane during the fusion process. The F100G5 epitope is located in the internal segment of the bipartite GPC fusion peptide, which also contains four conserved cysteine residues, raising the possibility that this fusion peptide may be highly structured. Collectively, our studies indicate that F100G5 identifies an on-path intermediate form of GPC. Binding to the transiently exposed fusion peptide may interfere with G2 insertion into the host cell membrane. Strategies to effectively target fusion peptide function in the endosome may lead to novel classes of antiviral agents.Enveloped viruses enter their target cells through fusion of the virus and cell membranes, in a process promoted by the viral envelope glycoprotein. For some viruses, such as human immunodeficiency virus (HIV), entry is initiated by interaction of the envelope glycoprotein with cell surface receptor proteins. Other viruses, such as influenza virus, are endocytosed and membrane fusion is triggered by exposure to acidic pH in the maturing endosome. The subsequent merger of the viral and cell membranes is accomplished through a major structural reorganization of the envelope glycoprotein. Antiviral strategies that target virus entry by using neutralizing antibodies or small-molecule fusion inhibitors can, in many cases, prevent virus infection and disease.The Arenaviridae comprise a diverse group of rodent-borne viruses, some of which are responsible for severe hemorrhagic fevers in humans. Lassa fever virus (LASV) is endemic in western Africa (59), and at least five New World species are recognized to cause fatal disease in the Americas, including the Argentine hemorrhagic fever virus Junín (JUNV) (63). New pathogenic arenavirus species continue to emerge from their distinct animal reservoirs (1, 11, 24). At present, there are no licensed vaccines or effective therapies to address the threat of arenavirus infection.Arenaviruses are enveloped, negative-strand RNA viruses whose bipartite genome encodes ambisense expression of four viral proteins (12, 22). The arenavirus envelope glycoprotein, GPC, is a member of the class I virus fusion proteins (33, 40, 75), a group that includes HIV Env, influenza virus hemagglutinin (HA), and paramyxovirus F protein. These envelope glycoproteins share several salient features. The precursor glycoproteins assemble as trimeric complexes and are subsequently rendered competent for membrane fusion by a proteolytic cleavage that results in the formation of the mature receptor-binding and transmembrane fusion subunits. The GPC precursor glycoprotein is cleaved by the cellular SKI-1/S1P protease (6, 51, 54) to generate the respective G1 and G2 subunits, which remain noncovalently associated. The ectodomain of the class I fusion subunit is distinguished by the presence of two 4-3 heptad repeat (HR1 and HR2) sequences that, in the course of membrane fusion, refold to form the now-classical six-helix bundle structure, which defines this class of envelope glycoproteins. Unlike other class I fusion proteins, GPC also contains a cleaved and stable signal peptide (SSP) as a third and essential subunit in the mature complex (2, 32, 69, 77, 81).Arenavirus infection is initiated by G1 binding to a cell surface receptor. The pathogenic clade B New World arenaviruses utilize transferrin receptor 1 (TfR1) for entry (1, 64, 65), whereas those in clades A and C, as well as the Old World viruses, bind α-dystroglycan and/or an unknown receptor (15, 34, 71). The virion particle is subsequently endocytosed (9), and membrane fusion is initiated by acidification in the maturing endosome (17, 28, 29). pH-dependent activation of GPC is modulated through a unique interaction between SSP and G2 (79, 80) and can be targeted by small-molecule inhibitors that block membrane fusion (76) and protect against arenavirus infection (8, 52).A generally accepted model for membrane fusion by the class I envelope glycoproteins (reviewed in references 45 and 73) posits that the native complex exists in a metastable state that is established on proteolytic maturation of the biosynthetic precursor. Upon activation, whether by acidic pH in the endosome or receptor binding at the plasma membrane, the fusion subunit that was sequestered in the prefusion state is exposed and undergoes a series of dramatic conformational changes leading to membrane fusion. In this process, a hydrophobic region at or near the N terminus of the fusion subunit (the fusion peptide) inserts into the host cell membrane, thus allowing the protein to bridge the two membranes. This so-called prehairpin intermediate subsequently collapses upon itself to form the highly stable six-helix bundle structure, in which the three HR2 helices pack into hydrophobic grooves on the trimeric HR1 coiled-coil in an antiparallel manner, bringing the virus and cell membranes into apposition. Free energy made available in the formation of this stable structure is thought to drive fusion of the lipid bilayers. Peptides that correspond in sequence to HR2 (C-peptides) bind to the putative prehairpin intermediate and interfere with its refolding, thereby preventing membrane fusion (18, 57, 74). While the structure of the six-helix bundle core has been elucidated in atomic detail (45, 73), information regarding the molecular pathway leading to this postfusion state is largely indirect. Indeed, the prehairpin intermediate is conceptualized through the activity of C-peptide fusion inhibitors (57, 74).In this report, we describe a G2-directed monoclonal antibody (MAb), F100G5, that recognizes a pH-induced intermediate of JUNV GPC and prevents GPC-mediated membrane fusion. This MAb binds at or near the internal fusion peptide of G2 and may act by interfering with its penetration into the host cell membrane. These studies highlight the feasibility of targeting short-lived GPC intermediates for inhibition of membrane fusion.     

Anticoagulación en población anciana con fibrilación auricular no valvular. Artículo de revisión

Aging is an important risk factor for patients with atrial fibrillation. The estimated prevalence of atrial fibrillation in patients aged ≥80 years is 9–10%, and is associated with a four to five fold increased risk of embolic stroke, and with an estimated increased stroke risk of 1.45-fold per decade in aging. Older age is also associated with an increased risk of major bleeding with oral anticoagulant therapy. This review will focus on the role of oral anticoagulation with new oral anticoagulants, non-vitamin K antagonist in populations with common comorbid conditions, including age, chronic kidney disease, coronary artery disease, on multiple medication, and frailty. In patients 75 years and older, randomised trials have shown new oral anticoagulants to be as effective as warfarin, or in some cases superior, with an overall better safety profile, consistently reducing rates of intracranial haemorrhages. Prior to considering oral anticoagulant therapy in an elderly frail patient, a comprehensive assessment should be performed to include the risks and benefits, stroke risk, baseline kidney function, cognitive status, mobility and fall risk, multiple medication, nutritional status assessment, and life expectancy.     

Bartolius pierrei n. g., n. sp. (Digenea: Gymnophallidae) from the Península Valdés, Argentina

Bartolius pierrei n. g., n. sp. (Digenea: Gymnophallidae) is described from metacercariae and naturally and cultivated obtained adults from southern Argentina. The second intermediate host is Darina solenoides (King) (Bivalvia: Mactridae) and the definitive host is Larus dominicanus Lichtenstein (Aves: Laridae). The diagnostic characters are as follows: Body small, oval. Oral sucker without lateral projections, twice size of ventral sucker (except in young metacercariae). Caeca short (in adults), without dorsal diverticula. Ventral sucker in posterior third of body. Ventral pit absent. Seminal vesicle bipartite. Ovary post-testicular. Vitelline glands paired, compact, close to ventral sucker. Uterus in fore- and hindbody. Genital atrium tubular. Genital pore inconspicuous, close to anterior margin of ventral sucker. Excretory vesicle Y-shaped with very short stem. Excretory formula: 2[(2+2)+(2+2)]=16. Bartolius is distinguished from other genera of the Gymnophallidae by the post-testicular position of the ovary.