Analysis of Envelope Glycoprotein Complex of Arenaviruses

Arenaviruses, the causative agents of severe hemorrhagic fevers, are endemic in rodent populations and can be transmitted to humans by contact. Without effective treatment or licensed vaccines, these viruses pose serious public health and biodefense concern. The sole treatment option is the off-label use of the nucleoside analog ribavirin, which is effective only when given at an early stage of infection and shows significant toxicity in humans. Hence, there exists a clear need for developing better therapies. Arenavirus entry into the cell is initiated by the virus envelope glycoprotein complex (GPC), primed to undergo conformational changes triggered by the acidic pH of the maturing endosome, leading to virus and endosomal membrane fusion. Thus, GPC represents as an important molecular target for therapeutic intervention. Recently, several chemically diverse small-molecule fusion inhibitors were identified that block virus entry by stabilizing the prefusion form of GPC against pH-activation. Improved structural and mechanistic understanding of pH-dependent membrane fusion will advance the design and development of potent inhibitors. Here we report that recombinant native-like GPC can be expressed and purified from insect cells, and mediate pH-dependent membrane fusion when reconstituted into proteoliposomes. This fusion reaction is inhibited by small-molecule fusion inhibitors. Further, I show the first physical evidence of binding of small-molecule inhibitors to the pH-sensitive SSP-GP2 interface using photoreactive inhibitors. In addition, I explored mechanism of pH-induced activation of membrane fusion in Old World Lassa virus (LASV). Although the pH-induced activation mechanism is similar to the well-studied New World Junφn virus (JUNV), the differences lie in the usage of an additional secondary (LAMP1) receptor for LASV entry. Another antiviral strategy is to block the packaging and release of virus particles from an infected host cell. Studies have shown that the viral matrix protein Z plays a critical role in virus assembly and budding. Additionally, accumulation of Z at the plasma membrane and interaction with GPC and nucleoprotein (NP) is thought to orchestrate the assembly and budding events. The assembly process involving interaction of GPC with Z has not been clearly understood. I explored the use of confocal microscopy approach to study the association of GPC and Z at the plasma membrane during assembly of the virus

Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein

Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1–Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern

Vaccine-elicited receptor-binding site antibodies neutralize two New World hemorrhagic fever arenaviruses

While five arenaviruses cause human hemorrhagic fevers in the Western Hemisphere, only Junin virus (JUNV) has a vaccine. The GP1 subunit of their envelope glycoprotein binds transferrin receptor 1 (TfR1) using a surface that substantially varies in sequence among the viruses. As such, receptor-mimicking antibodies described to date are type-specific and lack the usual breadth associated with this mode of neutralization. Here we isolate, from the blood of a recipient of the live attenuated JUNV vaccine, two antibodies that cross-neutralize Machupo virus with varying efficiency. Structures of GP1–Fab complexes explain the basis for efficient cross-neutralization, which involves avoiding receptor mimicry and targeting a conserved epitope within the receptor-binding site (RBS). The viral RBS, despite its extensive sequence diversity, is therefore a target for cross-reactive antibodies with activity against New World arenaviruses of public health concern

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Purification and proteolytic cleavage of rGPC^fur.

<p><b>A.</b> Affinity-purified rGPC^fur was subjected to size-exclusion chromatography (SEC) and the peak fraction was analyzed by SDS-PAGE. Molecular weight markers used in SEC and SDS-PAGE are shown (in kilodaltons) and G1G2 precursor, G1 and G2 subunits and SSP are indicated. <b>B.</b> rGPC^fur precursor was incubated with sFurin (+sFurin) and examined by SDS-PAGE (top panel). MALDI mass spectrometry was used to determine the molecular weights of the deglycosylated (degly) G1 and G2 subunits. The calculated mass is based on the assumption that all potential glycosylation sites are used and subsequently deglycosylated.</p

SPR studies of interactions with lipid-reconstituted rGPC^fur.

<p>The rGPC^fur precursor was immobilized onto a Biacore L1 chip and reconstituted in a lipid bilayer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051114#s2" target="_blank">Material and Methods</a>. Two or three concentration-dependent data sets were analyzed and sensorgram figures were generated using a five-point smoothing procedure and ORIGIN graphing software. Labels to the right are drawn to coincide with the maximum RUs achieved by the respective ligand. <b>A.</b> Binding of G1-directed MAbs (BF11, BF09, BE08, and AG02; 0.5 µM), sTfR (1.5 µM), and a nucleoprotein-directed MAb (aN; BG12). <b>B.</b> Binding of SIGA (ST-294, ST-375, ST-193, and ST-761; 150 µM) and TSRI (17C8; 100 µM) small-molecule fusion inhibitors. ST-161 and TSRI 8C1 are specific to the OW LASV and do not inhibit the NW arenavirus JUNV.</p

pH-induced membrane fusion by rGPC^fur proteoliposomes: lipid mixing.

<p>rGPC^fur was incorporated into POPG-POPC liposomes and mixed with target POPG-POPC liposomes doped with 1% Rhodamine-PE. In most cases, the rGPC^fur proteoliposomes were first treated with sFurin (as indicated by+in the first position of the labels, at right). Exposure to acidic pH at the start of the experiment (time = 0) is indicated by+in the second position of the labels. 15 µM of ST-294 or ST-161 was present prior to and during exposure to acidic pH, where indicated. Lipid mixing and the resulting dequenching of the rhodamine fluorophore were measured at 600 nm (excitation 508 nm). Complete dequenching (100%) was determined by subsequent solubilization in Triton X-100 nonionic detergent.</p

SPR studies of ST-294 binding to rGPC^fur following cleavage and exposure to acidic pH. A.

<p>ST-294 (100 µM) was bound to lipid-reconstituted rGPC^fur on a Biacore L1 chip prior to (black) and after (blue) sFurin cleavage, and following exposure of the latter to acidic pH (red). Bound ST-294 was allowed to dissociate from the complex between subsequent injections. <b>B.</b> In a similar study using immobilized cleavage-defective rGPC^CD, ST-294 was bound prior to (blue) and following one and two sequential exposures to acidic pH (red and green, respectively).</p