Maturation of the Gag core decreases the stability of retroviral lipid membranes

Davidoff, Candice; Payne, Riley; Willis, Sharon H.; Doranz, Benjamin J.; Rucker, Joseph

doi:10.1016/j.virol.2012.08.023

Cited by 9 publications

(7 citation statements)

References 69 publications

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“…2B). These observations confirm the criticality of detergent concentration on viral inactivation, and are consistent with results from a Triton X-100 study that also demonstrates the detergent concentration needs to be at or above the CMC for robust anti-viral activity (Davidoff et al, 2012).…”

Section: Optimization Of the Ldao Viral Inactivation Stepsupporting

confidence: 88%

Evaluation of eco‐friendly zwitterionic detergents for enveloped virus inactivation

Conley

Tao

Henry

et al. 2016

Biotech & Bioengineering

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Inclusion of a detergent in protein biotherapeutic purification processes is a simple and very robust method for inactivating enveloped viruses. The detergent Triton X-100 has been used for many years and is part of the production process of several commercial therapeutic proteins. However, recent ecological studies have suggested that Triton X-100 and its break-down products can potentially behave as endocrine disrupters in aquatic organisms, raising concerns from an environmental impact perspective. As such, discharge of Triton X-100 into the waste water treatment plants is regulated in some jurisdictions, and alternative detergents for viral inactivation are required. In this work, we report on the identification and evaluation of more eco-friendly detergents as viable replacements for Triton X-100. Five detergent candidates with low to moderate environmental impact were initially identified and evaluated with respect to protein stability, followed by proof-of-concept virus inactivation studies using a model enveloped virus. From the set of candidates lauryldimethylamine N-oxide (LDAO) was identified as the most promising detergent due to its low ecotoxicity, robust anti-viral activity (LRV >4 at validation set-point conditions with X-MuLX), and absence of any negative impact on protein function. This detergent exhibited effective and robust virus inactivation in a broad range of protein concentrations, solution conductivities, pHs, and in several different cell culture fluid matrices. The only process parameter which correlated with reduced virus inactivation potency was LDAO concentration, and then only when the concentration was reduced to below the detergent's critical micelle concentration (CMC). Additionally, this work also demonstrated that LDAO was cleared to below detectable levels after Protein A affinity chromatography, making it suitable for use in a platform process that utilizes this chromatographic mode for protein capture. All these findings suggest that LDAO may be a practical alternative to Triton X-100 for use in protein therapeutic production processes for inactivating enveloped viruses. Biotechnol. Bioeng. 2017;114: 813-820. © 2016 Wiley Periodicals, Inc.

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Section: Optimization Of the Ldao Viral Inactivation Stepsupporting

confidence: 88%

Evaluation of eco‐friendly zwitterionic detergents for enveloped virus inactivation

Conley

Tao

Henry

et al. 2016

Biotech & Bioengineering

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“…M2-VLPs contain a noninfectious retroviral (MLV) Gag core surrounded by a host cell-derived lipid membrane containing high concentrations of incorporated M2 protein. M2-VLPs, produced and purified as described previously (14,15,19), showed high purity and homogeneity, low polydispersity (4.9%), and a hydrodynamic diameter of ϳ250 nm, similar to other purified VLPs (Fig. 1a).…”

Section: Incorporation Of M2 Ion Channels Into Vlpssupporting

confidence: 63%

“…The M2-VLP assay described here provides a safe, rapid, and robust biochemical format for discovering novel influenza virus inhibitors using cell-free HTS campaigns. Our work also provides, to the best of our knowledge, the first measurements of membrane potential directly across viral membranes and the first evidence that viral membranes are impermeable to ions and so may enable a better understanding of the electrochemical and biophysical properties of viral membranes (19,24,25,39), which have been underexplored areas of virology.…”

Section: Discussionmentioning

confidence: 83%

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Sulli

Banik

Schilling

et al. 2013

J Virol

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The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.T he M2 gene of influenza virus encodes a 97-amino-acid, homotetrameric, proton-selective ion channel necessary for influenza virus infectivity (1, 2). Activation of M2 is triggered by low pH in host cell endosomes, resulting in an influx of protons into the virus and acidification that releases the viral RNA into the host cell (2-4). M2 also helps to package viral RNA (5, 6) and regulate pH in the Golgi apparatus of infected cells during viral assembly (7). Two FDA-approved adamantane-based drugs, amantadine and rimantadine (2,8), are potent M2 inhibitors that can neutralize influenza virus in humans and other animals. However, the emergence of widespread highly virulent adamantane-resistant strains such as avian and swine influenza virus strains (9, 10) has prompted the need for newer antivirals. Drug discovery efforts targeting M2 have been difficult due to the toxicity of M2 in cells (11), the difficulty of reconstituting M2 in liposomes for largescale application (12, 13), and the labor-intensive, low-throughput electrophysiological approaches typically used to study ion channels.To address these challenges, we developed a rapid, cell-free assay to measure the movement of proton ions directly across the lipid bilayer of virus-like particles (VLPs). High-throughput screening (HTS) of M2-VLPs using Ͼ100,000 compounds identified 19 M2-specific inhibitors, including two novel chemical scaffolds, that inhibited both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) protonophore activity also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane but that is not t...

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“…Three different Gag proteins were utilized for this study: wild‐type Gag (Gag‐only), fusion proteins containing either eGFP (Gag‐GFP), or TGP (Gag‐TGP). Fluorescent Gag constructs were cloned as in‐frame fusions to the Gag ORF to eliminate the pol gene as previously described . Forty‐eight hours after transfection, supernatants containing VLPs were harvested, passed through a 0.45 µm filter, centrifuged through a 20% sucrose cushion and resuspended in 10 m M HEPES, pH 7.5.…”

Section: Methodsmentioning

confidence: 99%

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure‐guided surface engineering

Paul

Langan

et al. 2015

Proteins

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In this paper we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction of high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.

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Maturation of the Gag core decreases the stability of retroviral lipid membranes

Cited by 9 publications

References 69 publications

Evaluation of eco‐friendly zwitterionic detergents for enveloped virus inactivation

Evaluation of eco‐friendly zwitterionic detergents for enveloped virus inactivation

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

Thermal green protein, an extremely stable, nonaggregating fluorescent protein created by structure‐guided surface engineering

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