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1.  Sex-Dependent Influence of Endogenous Estrogen in Pulmonary Hypertension 
Rationale: The incidence of pulmonary arterial hypertension is greater in women, suggesting estrogens may play a role in the disease pathogenesis. Experimentally, in males, exogenously administered estrogen can protect against pulmonary hypertension (PH). However, in models that display female susceptibility, estrogens may play a causative role.
Objectives: To clarify the influence of endogenous estrogen and sex in PH and assess the therapeutic potential of a clinically available aromatase inhibitor.
Methods: We interrogated the effect of reduced endogenous estrogen in males and females using the aromatase inhibitor, anastrozole, in two models of PH: the hypoxic mouse and Sugen 5416/hypoxic rat. We also determined the effects of sex on pulmonary expression of aromatase in these models and in lungs from patients with pulmonary arterial hypertension.
Measurements and Main Results: Anastrozole attenuated PH in both models studied, but only in females. To verify this effect was caused by reduced estrogenic activity we confirmed that in hypoxic mice inhibition of estrogen receptor α also has a therapeutic effect specifically in females. Female rodent lung displays increased aromatase and decreased bone morphogenetic protein receptor 2 and Id1 expression compared with male. Anastrozole treatment reversed the impaired bone morphogenetic protein receptor 2 pathway in females. Increased aromatase expression was also detected in female human pulmonary artery smooth muscle cells compared with male.
Conclusions: The unique phenotype of female pulmonary arteries facilitates the therapeutic effects of anastrozole in experimental PH confirming a role for endogenous estrogen in the disease pathogenesis in females and suggests aromatase inhibitors may have therapeutic potential.
doi:10.1164/rccm.201403-0483OC
PMCID: PMC4214128  PMID: 24956156
pulmonary hypertension; estrogen; sex
2.  FMDV replicons encoding green fluorescent protein are replication competent 
Highlights
•FMDV replication can be studied outwith high disease secure facilities.•FMDV replicon genomes encoding GFP are replication competent.•These FMDV replicon systems can be used to study replication by live-cell imaging/image analyses.
The study of replication of viruses that require high bio-secure facilities can be accomplished with less stringent containment using non-infectious ‘replicon’ systems. The FMDV replicon system (pT7rep) reported by Mclnerney et al. (2000) was modified by the replacement of sequences encoding chloramphenicol acetyl-transferase (CAT) with those encoding a functional L proteinase (Lpro) linked to a bi-functional fluorescent/antibiotic resistance fusion protein (green fluorescent protein/puromycin resistance, [GFP-PAC]). Cells were transfected with replicon-derived transcript RNA and GFP fluorescence quantified. Replication of transcript RNAs was readily detected by fluorescence, whilst the signal from replication-incompetent forms of the genome was >2-fold lower. Surprisingly, a form of the replicon lacking the Lpro showed a significantly stronger fluorescence signal, but appeared with slightly delayed kinetics. Replication can, therefore, be quantified simply by live-cell imaging and image analyses, providing a rapid and facile alternative to RT-qPCR or CAT assays.
doi:10.1016/j.jviromet.2014.08.020
PMCID: PMC4201441  PMID: 25194890
FMDV; Replicon; Fluorescence; Replication
3.  Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers 
The Journal of General Virology  2014;95(Pt 12):2649-2657.
We have previously documented the inhibitory activity of RNA aptamers to the RNA-dependent RNA polymerase of foot-and-mouth disease virus (3Dpol). Here we report their modification and use with a subgenomic replicon incorporating GFP (pGFP-PAC replicon), allowing replication to be monitored and quantified in real-time. GFP expression in transfected BHK-21 cells reached a maximum at approximately 8 h post-transfection, at which time change in morphology of the cells was consistent with a virus-induced cytopathic effect. However, transfection of replicon-bearing cells with a 3Dpol aptamer RNA resulted in inhibition of GFP expression and maintenance of normal cell morphology, whereas a control aptamer RNA had little effect. The inhibition was correlated with a reduction in 3Dpol (detected by immunoblotting) and shown to be dose dependent. The 3Dpol aptamers appeared to be more effective than 2′-C-methylcytidine (2′CMC). Aptamers to components of the replication complex are therefore useful molecular tools for studying viral replication and also have potential as diagnostic molecules in the future.
doi:10.1099/vir.0.067751-0
PMCID: PMC4233629  PMID: 25096816
4.  NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system 
The Journal of General Virology  2014;95(Pt 11):2427-2441.
Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.
doi:10.1099/vir.0.068932-0
PMCID: PMC4202265  PMID: 25024280
5.  Capsid Protein VP4 of Human Rhinovirus Induces Membrane Permeability by the Formation of a Size-Selective Multimeric Pore 
PLoS Pathogens  2014;10(8):e1004294.
Non-enveloped viruses must deliver their viral genome across a cell membrane without the advantage of membrane fusion. The mechanisms used to achieve this remain poorly understood. Human rhinovirus, a frequent cause of the common cold, is a non-enveloped virus of the picornavirus family, which includes other significant pathogens such as poliovirus and foot-and-mouth disease virus. During picornavirus cell entry, the small myristoylated capsid protein VP4 is released from the virus, interacts with the cell membrane and is implicated in the delivery of the viral RNA genome into the cytoplasm to initiate replication. In this study, we have produced recombinant C-terminal histidine-tagged human rhinovirus VP4 and shown it can induce membrane permeability in liposome model membranes. Dextran size-exclusion studies, chemical crosslinking and electron microscopy demonstrated that VP4 forms a multimeric membrane pore, with a channel size consistent with transfer of the single-stranded RNA genome. The membrane permeability induced by recombinant VP4 was influenced by pH and was comparable to permeability induced by infectious virions. These findings present a molecular mechanism for the involvement of VP4 in cell entry and provide a model system which will facilitate exploration of VP4 as a novel antiviral target for the picornavirus family.
Author Summary
Human rhinovirus (HRV) is a non-enveloped virus of the picornavirus family and is responsible for respiratory infections (common colds) costing billions of dollars ($) annually. There remains no vaccine or licensed drug to prevent or reduce infection. Related members of the picornavirus family include significant pathogens such as poliovirus, enterovirus 71 and foot-and-mouth disease virus, for which improved control measures are also required. A fundamental step in virus infection is the delivery of the viral genetic material through the barrier of the cellular membrane. Viruses such as HIV and influenza are enveloped in an outer membrane which can fuse with the host cell membrane to allow the viral genome to penetrate into the cytoplasm. However, non-enveloped viruses such as picornaviruses lack a membrane and the mechanisms for penetration of the membrane by these viruses remain poorly understood. The capsid protein VP4 has previously been implicated in the delivery of the picornavirus genome. In this study we demonstrate that HRV VP4 interacts with membranes to make them permeable by the formation of multimeric, size-selective membrane pores with properties consistent with the transport of viral genome through the membrane. This function of VP4 provides a novel antiviral target for this family of viruses.
doi:10.1371/journal.ppat.1004294
PMCID: PMC4125281  PMID: 25102288
6.  Limits of Structural Plasticity in a Picornavirus Capsid Revealed by a Massively Expanded Equine Rhinitis A Virus Particle 
Journal of Virology  2014;88(11):6093-6099.
ABSTRACT
The Picornaviridae family of small, nonenveloped viruses includes major pathogens of humans and animals. They have positive-sense, single-stranded RNA genomes, and the mechanism(s) by which these genomes are introduced into cells to initiate infection remains poorly understood. The structures of presumed uncoating intermediate particles of several picornaviruses show limited expansion and some increased porosity compared to the mature virions. Here, we present the cryo-electron microscopy structure of native equine rhinitis A virus (ERAV), together with the structure of a massively expanded ERAV particle, each at ∼17–Å resolution. The expanded structure has large pores on the particle 3-fold axes and has lost the RNA genome and the capsid protein VP4. The expanded structure thus illustrates both the limits of structural plasticity in such capsids and a plausible route by which genomic RNA might exit.
IMPORTANCE Picornaviruses are important animal and human pathogens that protect their genomic RNAs within a protective protein capsid. Upon infection, this genomic RNA must be able to leave the capsid to initiate a new round of infection. We describe here the structure of a unique, massively expanded state of equine rhinitis A virus that provides insight into how this exit might occur.
doi:10.1128/JVI.01979-13
PMCID: PMC4093863  PMID: 24648455
7.  The Effects of Uniquely-Processed Titanium on Biological Systems: Implications for Human Health and Performance 
Titanium is biocompatible and widely utilized in a variety of applications. Recently, titanium in pico-nanometer scale and soluble form (Aqua Titan) has expanded its use to applied human health and performance. The purpose of this article is to review the current evidence associated with specific physiological responses to Aqua Titan-treated materials. In vitro studies have shown that application of Aqua Titan can modify membrane potential and long-term potentiation in isolated hippocampal neurons, suggesting reduced pain memory as a possible mechanism for reported analgesia. Proximal contact with Aqua Titan-treated titanium increased gene expression, protein synthesis, cell growth and adhesion in normal cultured muscle and bone cells, suggesting application for Aqua Titan in clinical implant procedures and wound healing. Evidence for beneficial effects on neuromuscular control of muscle-tendon function and improvements in running economy in human athletes was seen when Aqua Titan-treated tape was applied to the human triceps surae following fatigue induced by prior strenuous exercise. Finally, behavioral responses and effects on the autonomic nervous system to environmental exposure suggest Aqua Titan may promote a mild relaxant, or stress-suppressive response. Together, data suggest exposure to Aqua Titan-treated materials modulates aspects of growth and function in neuronal and other musculoskeletal cells with possible benefits to musculotendinous recovery from exercise and to the systemic response to stress.
doi:10.3390/jfb5010001
PMCID: PMC4030905  PMID: 24956353
Aqua Titan; action potential; long-term potentiation; tendon compliance; musculotendinous function; cell adhesion and growth; autonomic nervous system; pico-nanometer scale
8.  Effect of microtitanium impregnated tape on the recovery of triceps surae musculotendinous function following strenuous running 
SpringerPlus  2013;2:653.
We previously reported increased running economy and joint range of motion (ROM) during subsequent exercise performed 48-h following strenuous exercise while wearing garments containing micro-titanium particles generated from high-pressure aqueous processing of titanium (AQUA TITANTM). Here we utilised an isolated plantarflexion triceps surae model and AQUA TITAN-treated flexible tape to determine if dermal application of the micro-titanium could account for meaningful changes in functional properties of the musculotendinous unit. In a randomised double-blind crossover, 20 trained men day 1, baseline measures, AQUA TITAN or placebo tape covering the triceps surae, intermittent high-intensity treadmill running; day 2, rest; day 3, post-stress post-treatment outcome measures. Outcomes comprised: plantarflexion ROM via isokinetic dynamometry; short latency reflex from electromyography; Achilles tendon stiffness from isometric dynamometry, ultrasonography (Achilles-medial-gastrocnemius junction), motion analysis, and force-length modelling. High-intensity exercise with placebo tape reduced tendon stiffness (-16.5%; 95% confidence limits ±8.1%; small effect size), relative to non-taped baseline, but this effect was negligible (-5.9%; ±9.2%) with AQUA TITAN (AQUA TITAN-placebo difference -11.3%; ±11.6%). Change in latency relative to baseline was trivial with placebo (1.6%; ±3.8%) but large with AQUA TITAN (-11.3%; ±3.3%). The effects on ROM with AQUA TITAN (1.6%; ±2.0%) and placebo were trivial (-1.6% ±1.9%), but the small difference (3.1%; ±2.7%) possibly greater with AQUA TITAN. AQUA TITAN tape accelerated the reflex response and attenuated reduced Achilles tendon stiffness following fatiguing exercise. Altered neuromuscular control of tendon stiffness via dermal application of micro-titanium treated materials may facilitate restoration of musculotendinous contractile performance following prior strenuous exercise.
doi:10.1186/2193-1801-2-653
PMCID: PMC3863395  PMID: 24349956
Tendon compliance; Recovery; Stretch reflex; Tendon tap; Dynamometry
9.  Mitochondrial transfer from bone marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury 
Nature medicine  2012;18(5):759-765.
Bone marrow-derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in the protection, we airway-instilled mice first with lipopolysaccharide (LPS), then with mouse BMSCs (mBMSCs). Live optical studies revealed that mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelium, releasing mitochondria-containing microvesicles that the epithelium engulfed. The presence of BMSC mitochondria in the epithelium was evident optically, as also by the presence of human mitochondrial DNA in mouse lungs in which we instilled human BMSCs (hBMSCs). The mitochondrial transfer increased alveolar ATP. LPS-induced ALI, indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by wild type mBMSCs, but not by mutant, GJC-incompetent mBMSCs, or by mBMSCs with dysfunctional mitochondria. This is the first evidence that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.
doi:10.1038/nm.2736
PMCID: PMC3727429  PMID: 22504485
10.  A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71 
Enterovirus 71 (EV71), a major agent of hand-foot-and-mouth disease in children, can cause severe central nervous system disease and mortality. At present no vaccine or antiviral therapy is available. We have determined high-resolution structures for the mature virus and natural empty particles. The structure of the mature virus is similar to that of other enteroviruses, whilst the empty particles are dramatically expanded, with notable fissures, resembling elusive enterovirus uncoating intermediates not previously characterized in atomic detail. Hydrophobic capsid pockets within the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. The results provide a paradigm for enterovirus uncoating, in which the VP1 GH loop acts as an adaptor-sensor for the attachment of cellular receptors, converting heterologous inputs to a generic uncoating mechanism, spotlighting novel points for therapeutic intervention.
doi:10.1038/nsmb.2255
PMCID: PMC3378640  PMID: 22388738
11.  A plate-based high-throughput assay for virus stability and vaccine formulation 
Journal of Virological Methods  2012;185(1):166-170.
Highlights
► A plate-based assay for virus measuring virus stability. ► Two fluorescent dyes measure independently but simultaneously capsid stability and capsid protein stability. ► A fast and efficient high-throughput method to optimise vaccine formulation. ► Facilitates the dissection of virus uncoating.
Standard methods for assessing the thermal stability of viruses can be time consuming and rather qualitative yet such data is a necessary requisite for vaccine formulation. In this study a novel plate-based thermal scanning assay for virus particle stability has been developed (PaSTRy: Particle Stability Thermal Release Assay). Two environment-sensitive fluorescent dyes, with non-overlapping emission spectra and different affinities, are used to accrue simultaneously independent data for the overall stability of the virus capsid, as judged by the exposure of the genome, and for capsid protein stability according to the exposure of hydrophobic side chains which are normally buried. This offers a fast and efficient high-throughput method to optimise vaccine formulation and to investigate the processes of virus uncoating.
doi:10.1016/j.jviromet.2012.06.014
PMCID: PMC3470038  PMID: 22744000
High-throughput; Virus stability; Vaccine formulation
12.  Exercise intervention in New Zealand Polynesian peoples with type 2 diabetes: Cultural considerations and clinical trial recommendations 
The Australasian Medical Journal  2012;5(8):429-435.
The Maori and Pacific Islands peoples of New Zealand suffer a greater burden of type 2 diabetes mellitus (T2DM) and associated comorbidities than their European counterparts. Empirical evidence supports the clinical application of aerobic and resistance training for effective diabetes management and potential remission, but few studies have investigated the effectiveness of these interventions in specific ethnic cohorts. We recently conducted the first trial to investigate the effect of prescribed exercise training in Polynesian people with T2DM. This article presents the cultural considerations undertaken to successfully implement the study. The research procedures were accepted and approved by cultural liaisons and potential participants. The approved methodology involved a trial evaluating and comparing the effects of two, 16-week exercise regimens (i.e. aerobic training and resistance training) on glycosylated haemoglobin (HbA1c), related diabetes markers (i.e. insulin resistance, blood lipids, relevant cytokines and anthropometric and hemodynamic indices) and health-related quality of life. Future exercise-related research or implementation strategies in this cohort should focus on cultural awareness and techniques to enhance participation and compliance. Our approach to cultural consultation could be considered by researchers undertaking trials in this and other ethnic populations suffering an extreme burden of T2DM, including indigenous Australians and Americans.
doi:10.4066/AMJ.2012.1311
PMCID: PMC3442187  PMID: 23024717
Resistance; Aerobic; Obesity; Maori; Pacific Islands; Polynesia; Ethnic; High-Risk
13.  Formation of Higher-Order Foot-and-Mouth Disease Virus 3Dpol Complexes Is Dependent on Elongation Activity 
Journal of Virology  2012;86(4):2371-2374.
The replication of many viruses involves the formation of higher-order structures or replication “factories.” We show that the key replication enzyme of foot-and-mouth disease virus (FMDV), the RNA-dependent RNA polymerase, forms fibrils in vitro. Although there are similarities with previously characterized poliovirus polymerase fibrils, FMDV fibrils are narrower, are composed of both protein and RNA, and, importantly, are seen only when all components of an elongation assay are present. Furthermore, an inhibitory RNA aptamer prevents fibril formation.
doi:10.1128/JVI.05696-11
PMCID: PMC3302376  PMID: 22156531
14.  Mouse models of rhinovirus-induced disease and exacerbation of allergic airway inflammation 
Nature medicine  2008;14(2):199-204.
Rhinoviruses cause serious morbidity and mortality as the major etiological agents of asthma exacerbations and the common cold. A major obstacle to understanding disease pathogenesis and to the development of effective therapies has been the lack of a small-animal model for rhinovirus infection. Of the 100 known rhinovirus serotypes, 90% (the major group) use human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor and do not bind mouse ICAM-1; the remaining 10% (the minor group) use a member of the low-density lipoprotein receptor family and can bind the mouse counterpart. Here we describe three novel mouse models of rhinovirus infection: minor-group rhinovirus infection of BALB/c mice, major-group rhinovirus infection of transgenic BALB/c mice expressing a mouse-human ICAM-1 chimera and rhinovirus-induced exacerbation of allergic airway inflammation. These models have features similar to those observed in rhinovirus infection in humans, including augmentation of allergic airway inflammation, and will be useful in the development of future therapies for colds and asthma exacerbations.
doi:10.1038/nm1713
PMCID: PMC3384678  PMID: 18246079
15.  Equol-Stimulated Mitochondrial Reactive Oxygen Species Activate Endothelial Nitric Oxide Synthase and Redox Signaling in Endothelial Cells 
Hypertension  2011;57(4):833-840.
We reported previously that dietary isoflavones modulate arterial blood pressure in vivo and that the daidzein metabolite equol rapidly activates endothelial NO synthase (eNOS) via Akt and extracellular signal–regulated kinase 1/2– dependent signaling. In this study, we report the first evidence in human endothelial cells that acute stimulation of mitochondrial superoxide generation by equol (100 nmol/L) is required for eNOS activation. Scavengers of superoxide (superoxide dismutase and manganese [III] tetrakis[1-methyl-4-pyridyl]porphyrin) abrogated equol stimulated Akt and eNOS phosphorylation, and the mitochondrial complex I inhibitor rotenone inhibited Akt, extracellular signal–regulated kinase 1/2, and eNOS phosphorylation, as well as NO-mediated increases in intracellular cGMP. Equol also induced rapid alterations in F-actin fiber distribution, with depolymerization of F-actin with cytochalasin D abrogating equol-stimulated mitochondrial superoxide generation. Treatment of cells with pertussis toxin or inhibition of GPR30/epidermal growth factor receptor kinase transactivation prevented equol-induced activation of extracellular signal–regulated kinase 1/2 via c-Src, Akt, and eNOS. Moreover, inhibition of epidermal growth factor receptor kinase activation with AG-1478 abrogated equol-stimulated mitochondrial reactive oxygen species generation and subsequent kinase and eNOS activation. Our findings suggest that equol-stimulated mitochondrial reactive oxygen species modulate endothelial redox signaling and NO release involving transactivation of epidermal growth factor receptor kinase and reorganization of the F-actin cytoskeleton. Identification of these novel actions of equol may provide valuable insights for therapeutic strategies to restore endothelial function in cardiovascular disease.
doi:10.1161/HYPERTENSIONAHA.110.162198
PMCID: PMC3086276  PMID: 21300668
equol; isoflavones; endothelium; eNOS; mitochondria; cytoskeleton; redox signaling
16.  Transplantation Pneumonia 
A clinically distinct pneumonitis occurred in six renal transplant recipients receiving azathioprine and prednisone immunosuppressive therapy. The patients ranged in age from 3 to 20 years. The onset was 42 to 102 days postoperatively, coinciding with decrease in prednisone dosage below approximately 1 mg per kilogram of body weight per day. Mild nonproductive cough, fever, and cynanosis were present. Chest x-rays demonstrated extensive hazy to nodular infiltrates usually involving both hila and lower lung fields. Cold agglutinins were present in five patients. Pulmonary function studies demonstrated an alveolar capillary block. The duration of illness was 12 to 34 days and was not influenced by antibiotic therapy. Autopsy of the single case in which death occurred revealed Pneumocystis carinii pneumonia and disseminated cytomegalic inclusion-body disease.
PMCID: PMC3095840  PMID: 14172276
17.  SPLENIC HOMOTRANSPLANTATION* 
During the past 12 months, five clinical whole-organ splenic homotransplantations have been carried out with the objective of providing active immunologic tissue for the recipient patients. In one case with hypogammaglobulinemia, it was hoped that the transplanted tissue would alleviate a state of immunologic deficiency. In the other four, all of whom had terminal malignancies, the purpose was to superimpose a state of altered immunologic reactivity upon the host in the hope of thereby suppressing the inexorable growth of the neoplasms.
As will be described, these procedures can now be judged in each instance to have been without benefit. Nevertheless, full documentation of the cases seems justified not only because of the many implications of transplantation of immunologically competent tissue, but also because of the potentially important observations made during the care of these patients.
In addition, a full account will be presented of the supporting canine studies of splenic homotransplantation, inasmuch as many of the principles of clinical therapy and investigation derived from prior observations in the dog. The fact that it is possible to obtain viable splenic homografts in the dog for as long as two-thirds of a year without the production of runt disease or other harmful effects may have application in future research on bone marrow, other lymphoid, or hepatic homografts.
PMCID: PMC3095853  PMID: 14235278
18.  Activation of TNFR1 ectodomain shedding by mitochondrial Ca2+ determines the severity of inflammation in mouse lung microvessels  
The Journal of Clinical Investigation  2011;121(5):1986-1999.
Shedding of the extracellular domain of cytokine receptors allows the diffusion of soluble receptors into the extracellular space; these then bind and neutralize their cytokine ligands, thus dampening inflammatory responses. The molecular mechanisms that control this process, and the extent to which shedding regulates cytokine-induced microvascular inflammation, are not well defined. Here, we used real-time confocal microscopy of mouse lung microvascular endothelium to demonstrate that mitochondria are key regulators of this process. The proinflammatory cytokine soluble TNF-α (sTNF-α) increased mitochondrial Ca2+, and the purinergic receptor P2Y2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor–1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca2+ increase blocked the shedding and augmented inflammation, as denoted by increases in endothelial expression of the leukocyte adhesion receptor E-selectin and in microvascular leukocyte recruitment. The shedding was also blocked in microvessels after knockdown of a complex III component and after mitochondria-targeted catalase overexpression. Endothelial deletion of the TNF-α converting enzyme (TACE) prevented the TNF-α receptor shedding response, which suggests that exposure of microvascular endothelium to sTNF-α induced a Ca2+-dependent increase of mitochondrial H2O2 that caused TNFR1 shedding through TACE activation. These findings provide what we believe to be the first evidence that endothelial mitochondria regulate TNFR1 shedding and thereby determine the severity of sTNF-α–induced microvascular inflammation.
doi:10.1172/JCI43839
PMCID: PMC3083796  PMID: 21519143
19.  Picornaviruses 
The picornavirus family consists of a large number of small RNA viruses, many of which are significant pathogens of humans and livestock. They are amongst the simplest of vertebrate viruses comprising a single stranded positive sense RNA genome within a T = 1 (quasi T = 3) icosahedral protein capsid of approximately 30 nm diameter. The structures of a number of picornaviruses have been determined at close to atomic resolution by X-ray crystallography. The structures of cell entry intermediate particles and complexes of virus particles with receptor molecules or antibodies have also been obtained by X-ray crystallography or at a lower resolution by cryo-electron microscopy. Many of the receptors used by different picornaviruses have been identified, and it is becoming increasingly apparent that many use co-receptors and alternative receptors to bind to and infect cells. However, the mechanisms by which these viruses release their genomes and transport them across a cellular membrane to gain access to the cytoplasm are still poorly understood. Indeed, detailed studies of cell entry mechanisms have been made only on a few members of the family, and it is yet to be established how broadly the results of these are applicable across the full spectrum of picornaviruses. Working models of the cell entry process are being developed for the best studied picornaviruses, the enteroviruses. These viruses maintain particle integrity throughout the infection process and function as genome delivery modules. However, there is currently no model to explain how viruses such as cardio- and aphthoviruses that appear to simply dissociate into subunits during uncoating deliver their genomes into the cytoplasm.
doi:10.1007/82_2010_37
PMCID: PMC3018333  PMID: 20397067
21.  Cell Entry of the Aphthovirus Equine Rhinitis A Virus Is Dependent on Endosome Acidification▿  
Journal of Virology  2010;84(12):6235-6240.
Equine rhinitis A virus (ERAV) is genetically closely related to foot-and-mouth disease virus (FMDV), and both are now classified within the genus Aphthovirus of the family Picornaviridae. For disease security reasons, FMDV can be handled only in high-containment facilities, but these constraints do not apply to ERAV, making it an attractive alternative for the study of aphthovirus biology. Here, we show, using immunofluorescence, pharmacological agents, and dominant negative inhibitors, that ERAV entry occurs (as for FMDV) via clathrin-mediated endocytosis and acidification of early endosomes. This validates the use of ERAV as a model system to study the mechanism of cell entry by FMDV.
doi:10.1128/JVI.02375-09
PMCID: PMC2876639  PMID: 20375159
22.  Intracellular Proton Conductance of the Hepatitis C Virus p7 Protein and Its Contribution to Infectious Virus Production 
PLoS Pathogens  2010;6(9):e1001087.
The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process.
Author Summary
The hepatitis C virus (HCV) is the most common cause of chronic liver disease. Current therapy is only partially effective and fraught with side effects. A greater understanding of viral replication and new virus particle formation is thus important for developing new therapeutic targets. The HCV p7 protein is a virally encoded protein that is absolutely required for the production of new virus particles. It behaves as an ion channel when reconstituted into artificial lipid membranes but its function in infected cells is unknown. We have examined the possibility that p7 functions as an intracellular ion channel, preventing pH gradients from developing inside the cells. We have shown that p7 serves this function and it causes a loss of acidity in multiple intracellular compartments. We demonstrate that this alkalinization is required for successful virus production. Either direct inhibition of intracellular ATPases or replacement of p7 with an alternative ion channel is able to compensate for a defect in p7 and allow active virus to be produced. Therefore, HCV uses p7 to prevent cellular acidification processes. This understanding will allow for the targeting of this mechanism with novel therapeutic agents, and offers new insights into the mechanisms of liver pathogenesis during infection.
doi:10.1371/journal.ppat.1001087
PMCID: PMC2932723  PMID: 20824094
23.  Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner▿  
Journal of Virology  2009;83(21):11275-11282.
The assembly of foot-and-mouth disease virus (FMDV) particles is poorly understood. In addition, there are important differences in the antigenic and receptor binding properties of virus assembly and dissociation intermediates, and these also remain unexplained. We have established an experimental model in which the antigenicity, receptor binding characteristics, and in vitro assembly of capsid precursor can be studied entirely from purified components. Recombinant capsid precursor protein (P1 region) was expressed in Escherichia coli as myristoylated or unmyristoylated protein. The protein sedimented in sucrose gradients at 5S and reacted with monoclonal antibodies which recognize conformational or linear antigen determinants on the virion surface. In addition, it bound the integrin αvβ6, a cellular receptor for FMDV, indicating that unprocessed recombinant capsid precursor is both structurally and antigenically similar to native virus capsid. These characteristics were not dependent on the presence of 2A at the C terminus but were altered by N-terminal myristoylation and in mutant precursors which lacked VP4. Proteolytic processing of myristoylated precursor by recombinant FMDV 3Cpro in vitro induced a shift in sedimentation from 5S to 12S, indicating assembly into pentameric capsid subunits. Nonmyristoylated precursor still assembled into higher-order structures after processing with 3Cpro, but these particles sedimented in sucrose gradients at approximately 17S. In contrast, mutant precursors lacking VP4 were antigenically distinct, were unable to form pentamers, and had reduced capacity for binding integrin receptor. These studies demonstrate the utility of recombinant capsid precursor protein for investigating the initial stages of assembly of FMDV and other picornaviruses.
doi:10.1128/JVI.01263-09
PMCID: PMC2772760  PMID: 19710148
24.  Determinants of Hepatitis C Virus p7 Ion Channel Function and Drug Sensitivity Identified In Vitro▿  
Journal of Virology  2009;83(16):7970-7981.
Hepatitis C virus (HCV) chronically infects 170 million individuals, causing severe liver disease. Although antiviral chemotherapy exists, the current regimen is ineffective in 50% of cases due to high levels of innate virus resistance. New, virus-specific therapies are forthcoming although their development has been slow and they are few in number, driving the search for new drug targets. The HCV p7 protein forms an ion channel in vitro and is critical for the secretion of infectious virus. p7 displays sensitivity to several classes of compounds, making it an attractive drug target. We recently demonstrated that p7 compound sensitivity varies according to viral genotype, yet little is known of the residues within p7 responsible for channel activity or drug interactions. Here, we have employed a liposome-based assay for p7 channel function to investigate the genetic basis for compound sensitivity. We demonstrate using chimeric p7 proteins that neither the two trans-membrane helices nor the p7 basic loop individually determines compound sensitivity. Using point mutation analysis, we identify amino acids important for channel function and demonstrate that null mutants exert a dominant negative effect over wild-type protein. We show that, of the three hydrophilic regions within the amino-terminal trans-membrane helix, only the conserved histidine at position 17 is important for genotype 1b p7 channel activity. Mutations predicted to play a structural role affect both channel function and oligomerization kinetics. Lastly, we identify a region at the p7 carboxy terminus which may act as a specific sensitivity determinant for the drug amantadine.
doi:10.1128/JVI.00521-09
PMCID: PMC2715780  PMID: 19493992
25.  Equine Rhinitis A Virus and Its Low pH Empty Particle: Clues Towards an Aphthovirus Entry Mechanism? 
PLoS Pathogens  2009;5(10):e1000620.
Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form ‘altered’ particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.
Author Summary
Picornaviruses are small animal viruses comprising an RNA genome protected by a roughly spherical protein shell with icosahedral symmetry. How the RNA is introduced into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. Instead, they become internalized into endocytic vesicles whence the viral genome must be delivered through the vesicle membrane, into the cytoplasm. In some picornaviruses (enteroviruses), genome delivery is proposed to be coordinated by an intact particle inducing pore formation in the membrane through which the genome can be transferred directly without exposure to the hostile vesicle environment. In contrast, other picornaviruses (aphthoviruses e.g. ERAV, FMDV) present a dilemma by appearing to simply fall apart in acidified vesicles. Here we show that acid treatment results in the formation of an intact but transient aphthovirus empty particle from which the genome has been released. We have determined the crystal structures of the ERAV particle at native and acidic pH. The acid induced structure is consistent with a destabilized particle en-route to disassembly. We propose that the entry process for this group of viruses involves externalisation of the RNA from a novel capsid intermediate and unifies in principle the entry process for all picornaviruses.
doi:10.1371/journal.ppat.1000620
PMCID: PMC2752993  PMID: 19816570

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