John R. Gallagher

Herpes Virus Fusion and Entry: A Story with Many Characters

Herpesviridae comprise a large family of enveloped DNA viruses all of whom employ orthologs of the same three glycoproteins, gB, gH and gL. Additionally, herpesviruses often employ accessory proteins to bind receptors and/or bind the heterodimer gH/gL or even to determine cell tropism. Sorting out how these proteins function has been resolved to a large extent by structural biology coupled with supporting biochemical and biologic evidence. Together with the G protein of vesicular stomatitis virus, gB is a charter member of the Class III fusion proteins. Unlike VSV G, gB only functions when partnered with gH/gL. However, gH/gL does not resemble any known viral fusion protein and there is evidence that its function is to upregulate the fusogenic activity of gB. In the case of herpes simplex virus, gH/gL itself is upregulated into an active state by the conformational change that occurs when gD, the receptor binding protein, binds one of its receptors. In this review we focus primarily on prototypes of the three subfamilies of herpesviruses. We will present our model for how herpes simplex virus (HSV) regulates fusion in series of highly regulated steps. Our model highlights what is known and also provides a framework to address mechanistic questions about fusion by HSV and herpesviruses in general.

A critical analysis of on-scene helicopter transport on survival in a statewide trauma system

BACKGROUND Recently, questions have been raised regarding the effectiveness of helicopters in trauma care. We conducted a retrospective study to evaluate the effect of on-scene helicopter transport on survival after trauma in a statewide trauma system. METHODS Data were obtained from a statewide trauma registry of 162,730 patients treated at 28 accredited trauma centers. Patients transported from the scene by helicopter (15,938) were compared with those transported by ground with advanced life support (ALS) (6,473). Interhospital transfers and transports without ALS were excluded. Statistical analysis was performed using one-way analysis of variance and logistic regression. RESULTS Patients transported by helicopter were significantly (p < 0.01) younger, were more seriously injured, and had lower blood pressure. They were also more likely to be male and to have systolic blood pressure < 90 mm Hg. Logistic regression analysis revealed that when adjusting for other risk factors, transportation by helicopter did not affect the estimated odds of survival. CONCLUSION A reappraisal of the cost-effectiveness of helicopter triage and transport criteria, when access to ground ALS squads is available, may be warranted.

Classifying errors in preventable and potentially preventable trauma deaths: a 9-year review using the Joint Commission's standardized methodology

BACKGROUND Benchmarking and classification of avoidable errors in trauma care are difficult as most reports classify errors using variable locally derived schemes. We sought to classify errors in a large trauma population using standardized Joint Commission taxonomy. METHODS All preventable/potentially preventable deaths identified at an urban, level-1 trauma center (January 2002 to December 2010) were abstracted from the trauma registry. Errors deemed avoidable were classified within the 5-node (impact, type, domain, cause, and prevention) Joint Commission taxonomy. RESULTS Of the 377 deaths in 11,100 trauma contacts, 106 (7.7%) were preventable/potentially preventable deaths related to 142 avoidable errors. Most common error types were in clinical performance (inaccurate diagnosis). Error domain involved primarily the emergency department (therapeutic interventions), caused mostly by knowledge deficits. Communication improvement was the most common mitigation strategy. CONCLUSION Standardized classification of errors in preventable trauma deaths most often involve clinical performance in the early phases of care and can be mitigated with universal strategies.

Dual Split Protein-Based Fusion Assay Reveals that Mutations to Herpes Simplex Virus (HSV) Glycoprotein gB Alter the Kinetics of Cell-Cell Fusion Induced by HSV Entry Glycoproteins

ABSTRACT Herpes simplex virus (HSV) entry and cell-cell fusion require glycoproteins gD, gH/gL, and gB. We propose that receptor-activated changes to gD cause it to activate gH/gL, which then triggers gB into an active form. We employed a dual split-protein (DSP) assay to monitor the kinetics of HSV glycoprotein-induced cell-cell fusion. This assay measures content mixing between two cells, i.e., fusion, within the same cell population in real time (minutes to hours). Titration experiments suggest that both gD and gH/gL act in a catalytic fashion to trigger gB. In fact, fusion rates are governed by the amount of gB on the cell surface. We then used the DSP assay to focus on mutants in two functional regions (FRs) of gB, FR1 and FR3. FR1 contains the fusion loops (FL1 and FL2), and FR3 encompasses the crown at the trimer top. All FL mutants initiated fusion very slowly, if at all. However, the fusion rates caused by some FL2 mutants increased over time, so that total fusion by 8 h looked much like that of the WT. Two distinct kinetic patterns, “slow and fast,” emerged for mutants in the crown of gB (FR3), again showing differences in initiation and ongoing fusion. Of note are the fusion kinetics of the gB syn mutant (LL871/872AA). Although this mutant was originally included as an ongoing high-rate-of-fusion control, its initiation of fusion is so rapid that it appears to be on a “hair trigger.” Thus, the DSP assay affords a unique way to examine the dynamics of HSV glycoprotein-induced cell fusion.

Functional Fluorescent Protein Insertions in Herpes Simplex Virus gB Report on gB Conformation before and after Execution of Membrane Fusion

Entry of herpes simplex virus (HSV) into a target cell requires complex interactions and conformational changes by viral glycoproteins gD, gH/gL, and gB. During viral entry, gB transitions from a prefusion to a postfusion conformation, driving fusion of the viral envelope with the host cell membrane. While the structure of postfusion gB is known, the prefusion conformation of gB remains elusive. As the prefusion conformation of gB is a critical target for neutralizing antibodies, we set out to describe its structure by making genetic insertions of fluorescent proteins (FP) throughout the gB ectodomain. We created gB constructs with FP insertions in each of the three globular domains of gB. Among 21 FP insertion constructs, we found 8 that allowed gB to remain membrane fusion competent. Due to the size of an FP, regions in gB that tolerate FP insertion must be solvent exposed. Two FP insertion mutants were cell-surface expressed but non-functional, while FP insertions located in the crown were not surface expressed. This is the first report of placing a fluorescent protein insertion within a structural domain of a functional viral fusion protein, and our results are consistent with a model of prefusion HSV gB constructed from the prefusion VSV G crystal structure. Additionally, we found that functional FP insertions from two different structural domains could be combined to create a functional form of gB labeled with both CFP and YFP. FRET was measured with this construct, and we found that when co-expressed with gH/gL, the FRET signal from gB was significantly different from the construct containing CFP alone, as well as gB found in syncytia, indicating that this construct and others of similar design are likely to be powerful tools to monitor the conformation of gB in any model system accessible to light microscopy.

Repertoire of Epitopes Recognized by Serum IgG from Humans Vaccinated with Herpes Simplex Virus 2 Glycoprotein D

ABSTRACT The results of a clinical trial of a subunit vaccine against genital herpes were recently reported (R. B. Belshe, P. A. Leone, D. I. Bernstein, A. Wald, M. J. Levin, J. T. Stapleton, I. Gorfinkel, R. L. Morrow, M. G. Ewell, A. Stokes-Riner, G. Dubin, T. C. Heineman, J. M. Schulte, C. D. Deal, N. Engl. J. Med. 366:34–43, 2012, doi:10.1056/NEJMoa1103151). The vaccine consisted of a soluble form of herpes simplex virus 2 (HSV-2) glycoprotein D (gD2) with adjuvant. The goal of the current study was to examine the composition of the humoral response to gD2 within a selected subset of vaccinated individuals. Serum samples from 30 vaccine recipients were selected based upon relative enzyme-linked immunosorbent assay (ELISA) titers against gD2; 10 samples had high titers, 10 had medium titers, and the remaining 10 had low ELISA titers. We employed a novel, biosensor-based monoclonal antibody (MAb)-blocking assay to determine whether gD2 vaccination elicited IgG responses against epitopes overlapping those of well-characterized MAbs. Importantly, IgGs from the majority of gD2-immunized subjects competed for gD binding with four antigenically distinct virus-neutralizing MAbs (MC2, MC5, MC23, and DL11). Screening of patient IgGs against overlapping peptides spanning the gD2 ectodomain revealed that about half of the samples contained antibodies against linear epitopes within the N and C termini of gD2. We found that the virus-neutralizing abilities of the 10 most potent samples correlated with overall gD-binding activity and to an even greater extent with the combined content of IgGs against the epitopes of MAbs MC2, MC5, MC23, and DL11. This suggests that optimal virus-neutralizing activity is achieved by strong and balanced responses to the four major discontinuous neutralizing epitopes of gD2. IMPORTANCE Several herpes simplex virus 2 (HSV-2) subunit vaccine studies have been conducted in human subjects using a recombinant form of HSV-2 glycoprotein D (gD2). Although several distinct, well-characterized virus-neutralizing epitopes on gD2 are targeted by murine monoclonal antibodies, it is not known whether the same epitopes are targeted by the humoral response to gD2 in humans. We have developed a novel, biosensor-based competition assay to directly address this important question. Using this approach, we identified epitopes that elicit strong humoral responses in humans, as well as other epitopes that elicit much weaker responses. These data provide new insight into the human response to known neutralizing gD2 epitopes and reveal characteristics of this response that may guide future vaccine development.

Displacement of the C Terminus of Herpes Simplex Virus gD Is Sufficient To Expose the Fusion-Activating Interfaces on gD

ABSTRACT Viral entry by herpes simplex virus (HSV) is executed and tightly regulated by four glycoproteins. While several viral glycoproteins can mediate viral adhesion to host cells, only binding of gD to cellular receptor can activate core fusion proteins gB and gH/gL to execute membrane fusion and viral entry. Atomic structures of gD bound to receptor indicate that the C terminus of the gD ectodomain must be displaced before receptor can bind to gD, but it is unclear which conformational changes in gD activate membrane fusion. We rationally designed mutations in gD to displace the C terminus and observe if fusion could be activated without receptor binding. Using a cell-based fusion assay, we found that gD V231W induced cell-cell fusion in the absence of receptor. Using recombinant gD V231W protein, we observed binding to conformationally sensitive antibodies or HSV receptor and concluded that there were changes proximal to the receptor binding interface, while the tertiary structure of gD V231W was similar to that of wild-type gD. We used a biosensor to analyze the kinetics of receptor binding and the extent to which the C terminus blocks binding to receptor. We found that the C terminus of gD V231W was enriched in the open or displaced conformation, indicating a mechanism for its function. We conclude that gD V231W triggers fusion through displacement of its C terminus and that this motion is indicative of how gD links receptor binding to exposure of interfaces on gD that activate fusion via gH/gL and gB.

Bundles of care for resuscitation from hemorrhagic shock and severe brain injury in trauma patients - Translating knowledge into practice

I njuries are a leading cause of death, disability, years of productive lives lost, and health care costs in the United States. Approximately 190,000 people die each year from injuries, and the total medical and work loss costs of injuries exceed

The Fusion Loops of the Initial Prefusion Conformation of Herpes Simplex Virus 1 Fusion Protein Point Toward the Membrane

600 billion per year. The 2 leading causes of death after injury are hemorrhagic shock and traumatic brain injury (TBI), in both military and civilian settings, with mortality rates of 30% to 50%. Injured patients treated at designated trauma centers are more likely to survive than those treated at hospitals that are not trauma centers; however, risk-adjustedmortality rates are nearly 50% higher at some trauma centers than at others. Given that designated trauma centers have similar structures and resources, these variations in risk-adjusted patient outcomes are likely explained by variations in clinical practices. Although clinical practice guidelines for the management of hemorrhagic shock and TBI have been developed and disseminated by multiple organizations, adoption of evidence-based practices at trauma centers remains suboptimal. In a study of 5 Level I trauma centers, compliance with 22 commonly