Human monoclonal antibodies against chikungunya virus target multiple distinct epitopes in the E1 and E2 glycoproteins.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes persistent arthritis in a subset of human patients. We report the isolation and functional characterization of monoclonal antibodies (mAbs) from two patients infected with CHIKV in the Dominican Republic. Single B cell sorting yielded a panel of 46 human mAbs of diverse germline lineages that targeted epitopes within the E1 or E2 glycoproteins. MAbs that recognized either E1 or E2 proteins exhibited neutralizing activity. Viral escape mutations localized the binding epitopes for two E1 mAbs to sites within domain I or the linker between domains I and III; and for two E2 mAbs between the ß-connector region and the B-domain. Two of the E2-specific mAbs conferred protection in vivo in a stringent lethal challenge mouse model of CHIKV infection, whereas the E1 mAbs did not. These results provide insight into human antibody response to CHIKV and identify candidate mAbs for therapeutic intervention.

Torsins: not your typical AAA+ ATPases.

Torsin ATPases (Torsins) belong to the widespread AAA+ (ATPases associated with a variety of cellular activities) family of ATPases, which share structural similarity but have diverse cellular functions. Torsins are outliers in this family because they lack many characteristics of typical AAA+ proteins, and they are the only members of the AAA+ family located in the endoplasmic reticulum and contiguous perinuclear space. While it is clear that Torsins have essential roles in many, if not all metazoans, their precise cellular functions remain elusive. Studying Torsins has significant medical relevance since mutations in Torsins or Torsin-associated proteins result in a variety of congenital human disorders, the most frequent of which is early-onset torsion (DYT1) dystonia, a severe movement disorder. A better understanding of the Torsin system is needed to define the molecular etiology of these diseases, potentially enabling corrective therapy. Here, we provide a comprehensive overview of the Torsin system in metazoans, discuss functional clues obtained from various model systems and organisms and provide a phylogenetic and structural analysis of Torsins and their regulatory cofactors in relation to disease-causative mutations. Moreover, we review recent data that have led to a dramatically improved understanding of these machines at a molecular level, providing a foundation for investigating the molecular defects underlying the associated movement disorders. Lastly, we discuss our ideas on how recent progress may be utilized to inform future studies aimed at determining the cellular role(s) of these atypical molecular machines and their implications for dystonia treatment options.

Site-specific Proteolysis Mobilizes TorsinA from the Membrane of the Endoplasmic Reticulum (ER) in Response to ER Stress and B Cell Stimulation.

Torsin ATPases are the only representatives of the AAA+ ATPase family that reside in the lumen of the endoplasmic reticulum (ER) and nuclear envelope. Two of these, TorsinA and TorsinB, are anchored to the ER membrane by virtue of an N-terminal hydrophobic domain. Here we demonstrate that the imposition of ER stress leads to a proteolytic cleavage event that selectively removes the hydrophobic domain from the AAA+ domain of TorsinA, which retains catalytic activity. Both the pharmacological inhibition profile and the identified cleavage site between two juxtaposed cysteine residues are distinct from those of presently known proteases, suggesting that a hitherto uncharacterized, membrane-associated protease accounts for TorsinA processing. This processing occurs not only in stress-exposed cell lines but also in primary cells from distinct organisms including stimulated B cells, indicating that Torsin conversion in response to physiologically relevant stimuli is an evolutionarily conserved process. By establishing 5-nitroisatin as a cell-permeable inhibitor for Torsin processing, we provide the methodological framework for interfering with Torsin processing in a wide range of primary cells without the need for genetic manipulation.

The mechanism of Torsin ATPase activation.

Torsins are membrane-associated ATPases whose activity is dependent on two activating cofactors, lamina-associated polypeptide 1 (LAP1) and luminal domain-like LAP1 (LULL1). The mechanism by which these cofactors regulate Torsin activity has so far remained elusive. In this study, we identify a conserved domain in these activators that is predicted to adopt a fold resembling an AAA+ (ATPase associated with a variety of cellular activities) domain. Within these domains, a strictly conserved Arg residue present in both activating cofactors, but notably missing in Torsins, aligns with a key catalytic Arg found in AAA+ proteins. We demonstrate that cofactors and Torsins associate to form heterooligomeric assemblies with a defined Torsin-activator interface. In this arrangement, the highly conserved Arg residue present in either cofactor comes into close proximity with the nucleotide bound in the neighboring Torsin subunit. Because this invariant Arg is strictly required to stimulate Torsin ATPase activity but is dispensable for Torsin binding, we propose that LAP1 and LULL1 regulate Torsin ATPase activity through an active site complementation mechanism.

The Torsin Activator LULL1 Is Required for Efficient Growth of Herpes Simplex Virus 1.

UNLABELLED: TorsinA is a membrane-tethered AAA+ ATPase implicated in nuclear envelope dynamics as well as the nuclear egress of herpes simplex virus 1 (HSV-1). The activity of TorsinA and the related ATPase TorsinB strictly depends on LAP1 and LULL1, type II transmembrane proteins that are integral parts of the Torsin/cofactor AAA ring, forming a composite, membrane-spanning assembly. Here, we use CRISPR/Cas9-mediated genome engineering to create single- and double knockout (KO) cell lines of TorA and TorB as well as their activators, LAP1 and LULL1, to investigate the effect on HSV-1 production. Consistent with LULL1 being the more potent Torsin activator, a LULL1 KO reduces HSV-1 growth by one order of magnitude, while the deletion of other components of the Torsin system in combination causes subtle defects. Notably, LULL1 deficiency leads to a 10-fold decrease in the number of viral genomes per host cell without affecting viral protein production, allowing us to tentatively assign LULL1 to an unexpected role that precedes HSV-1 nuclear egress. IMPORTANCE: In this study, we conduct the first comprehensive genetic and phenotypic analysis of the Torsin/cofactor system in the context of HSV-1 infection, establishing LULL1 as the most important component of the Torsin system with respect to viral production.

Regulation of Torsin ATPases by LAP1 and LULL1.

TorsinA is a membrane-associated AAA+ (ATPases associated with a variety of cellular activities) ATPase implicated in primary dystonia, an autosomal-dominant movement disorder. We reconstituted TorsinA and its cofactors in vitro and show that TorsinA does not display ATPase activity in isolation; ATP hydrolysis is induced upon association with LAP1 and LULL1, type II transmembrane proteins residing in the nuclear envelope and endoplasmic reticulum. This interaction requires TorsinA to be in the ATP-bound state, and can be attributed to the luminal domains of LAP1 and LULL1. This ATPase activator function controls the activities of other members of the Torsin family in distinct fashion, leading to an acceleration of the hydrolysis step by up to two orders of magnitude. The dystonia-causing mutant of TorsinA is defective in this activation mechanism, suggesting a loss-of-function mechanism for this congenital disorder.

A designed repeat protein as an affinity capture reagent.

Repeat proteins are an attractive target for protein engineering and design. We have focused our attention on the design and engineering of one particular class: tetratricopeptide repeat (TPR) proteins. In previous work, we have shown that the structure and stability of TPR proteins can be manipulated in a rational fashion [Cortajarena (2011) Prot. Sci. 20: , 1042-1047; Main (2003) Structure 11: , 497-508]. Building on those studies, we have designed and characterized a number of different peptide-binding TPR modules and we have also assembled these modules into supramolecular arrays [Cortajarena (2009) ACS Chem. Biol. 5: , 545-552; Cortajarena (2008) ACS Chem. Biol. 3: , 161-166; Jackrel (2009) Prot. Sci. 18: , 762-774; Kajander (2007) Acta Crystallogr. D Biol. Crystallogr. 63: , 800-811]. Here we focus on the development of one such TPR-peptide interaction for a practical application, affinity purification. We illustrate the general utility of our designed protein interaction. Furthermore, this example highlights how basic research on protein-peptide interactions can lead to the development of novel reagents with important practical applications.

Comprehensive investigation of microRNAs enhanced by analysis of sequence variants, expression patterns, ARGONAUTE loading, and target cleavage.

MicroRNAs (miRNAs) are a class of small RNAs that typically function by guiding the cleavage of target messenger RNAs. They have been shown to play major roles in a variety of plant processes, including development, and responses to pathogens and environmental stresses. To identify new miRNAs and regulation in Arabidopsis (Arabidopsis thaliana), 27 small RNA libraries were constructed and sequenced from various tissues, stresses, and small RNA biogenesis mutants, resulting in 95 million genome-matched sequences. The use of rdr2 to enrich the miRNA population greatly enhanced this analysis and led to the discovery of new miRNAs arising from both known and new precursors, increasing the total number of Arabidopsis miRNAs by about 10%. Parallel Analysis of RNA Ends data provide evidence that the majority guide target cleavage. Many libraries represented novel stress/tissue conditions, such as submergence-stressed flowers, which enabled the identification of new stress regulation of both miRNAs and their targets, all of which were validated in wild-type plants. By combining small RNA expression analysis with ARGONAUTE immunoprecipitation data and global target cleavage data from Parallel Analysis of RNA Ends, a much more complete picture of Arabidopsis miRNAs was obtained. In particular, the discovery of ARGONAUTE loading and target cleavage biases gave important insights into tissue-specific expression patterns, pathogen responses, and the role of sequence variation among closely related miRNA family members that would not be evident without this combinatorial approach.

Pediatric Trauma and Posttraumatic Symptom Screening at Well-child Visits.

Adverse childhood experiences (ACEs), including abuse or neglect, parental substance abuse, mental illness, or separation, are public health crises that require identification and response. We aimed to increase annual rates of trauma screening during well-child visits from 0% to 70%, post-traumatic stress disorder (PTSD) symptom screening for children with identified trauma from 0% to 30%, and connection to behavioral health for children with symptoms from 0% to 60%. Methods: Our interdisciplinary behavioral and medical health team implemented 3 plan-do-study-act cycles to improve screening and response to pediatric traumatic experiences. Automated reports and chart reviews measured progress toward goals as we changed screening methods and provider training. Results: During plan-do-study-act cycle 1, a chart review of patients with positive trauma screenings identified various trauma types. During cycle 2, a comparison of screening methods demonstrated that written screening identified trauma among more children than verbal screening (8.3% versus 1.7%). During cycle 3, practices completed trauma screenings at 25,287 (89.8%) well-child visits. Among screenings, 2,441 (9.7%) identified trauma. The abbreviated Post Traumatic Stress Disorder Reaction Index was conducted at 907 (37.2%) encounters and identified 520 children (57.3%) with PTSD symptoms. Among a sample of 250, 26.4% were referred to behavioral health, 43.2% were already connected, and 30.4% had no connection. Conclusions: It is feasible to screen and respond to trauma during well-child visits. Screening method and training implementation changes can improve screening and response to pediatric trauma and PTSD. Further work is needed to increase rates of PTSD symptomology screening and connection to behavioral health.

Specific Recognition of a Stem-Loop RNA Structure by the Alphavirus Capsid Protein.

Alphaviruses are small enveloped viruses with positive-sense RNA genomes. During infection, the alphavirus capsid protein (Cp) selectively packages and assembles with the viral genomic RNA to form the nucleocapsid core, a process critical to the production of infectious virus. Prior studies of the alphavirus Semliki Forest virus (SFV) showed that packaging and assembly are promoted by Cp binding to multiple high affinity sites on the genomic RNA. Here, we developed an in vitro Cp binding assay based on fluorescently labeled RNA oligos. We used this assay to explore the RNA sequence and structure requirements for Cp binding to site #1, the top binding site identified on the genomic RNA during all stages of virus assembly. Our results identify a stem-loop structure that promotes specific binding of the SFV Cp to site #1 RNA. This structure is also recognized by the Cps of the related alphaviruses chikungunya virus and Ross River virus.

Atovaquone and Berberine Chloride Reduce SARS-CoV-2 Replication In Vitro.

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.

Berberine Chloride is an Alphavirus Inhibitor That Targets Nucleocapsid Assembly.

Alphaviruses are enveloped positive-sense RNA viruses that can cause serious human illnesses such as polyarthritis and encephalitis. Despite their widespread distribution and medical importance, there are no licensed vaccines or antivirals to combat alphavirus infections. Berberine chloride (BBC) is a pan-alphavirus inhibitor that was previously identified in a replicon-based small-molecule screen. This work showed that BBC inhibits alphavirus replication but also suggested that BBC might have additional effects later in the viral life cycle. Here, we show that BBC has late effects that target the virus nucleocapsid (NC) core. Infected cells treated with BBC late in infection were unable to form stable cytoplasmic NCs or assembly intermediates, as assayed by gradient sedimentation. In vitro studies with recombinant capsid protein (Cp) and purified genomic RNA (gRNA) showed that BBC perturbs core-like particle formation and potentially traps the assembly process in intermediate states. Particles produced from BBC-treated cells were less infectious, despite efficient particle production and only minor decreases in genome packaging. In addition, BBC treatment of free virus particles strongly decreased alphavirus infectivity. In contrast, the infectivity of the negative-sense RNA virus vesicular stomatitis virus was resistant to BBC treatment of infected cells or free virus. Together, our data indicate that BBC alters alphavirus Cp-gRNA interactions and oligomerization and suggest that this may cause defects in NC assembly and in disassembly during subsequent virus entry. Thus, BBC may be considered a novel alphavirus NC assembly inhibitor.IMPORTANCE The alphavirus chikungunya virus (CHIKV) is an example of an emerging human pathogen with increased and rapid global spread. Although an acute CHIKV infection is rarely fatal, many patients suffer from debilitating chronic arthralgia for years. Antivirals against chikungunya and other alphaviruses have been identified in vitro, but to date none have been shown to be efficacious and have been licensed for human use. Here, we investigated a small molecule, berberine chloride (BBC), and showed that it inhibited infectious virus production by several alphaviruses including CHIKV. BBC acted on a late step in the alphavirus exit pathway, namely the formation of the nucleocapsid containing the infectious viral RNA. Better understanding of nucleocapsid formation and its inhibition by BBC will provide important information on the mechanisms of infectious alphavirus production and may enable their future targeting in antiviral strategies.

Multiple capsid protein binding sites mediate selective packaging of the alphavirus genomic RNA.

The alphavirus capsid protein (Cp) selectively packages genomic RNA (gRNA) into the viral nucleocapsid to produce infectious virus. Using photoactivatable ribonucleoside crosslinking and an innovative biotinylated Cp retrieval method, here we comprehensively define binding sites for Semliki Forest virus (SFV) Cp on the gRNA. While data in infected cells demonstrate Cp binding to the proposed genome packaging signal (PS), mutagenesis experiments show that PS is not required for production of infectious SFV or Chikungunya virus. Instead, we identify multiple Cp binding sites that are enriched on gRNA-specific regions and promote infectious SFV production and gRNA packaging. Comparisons of binding sites in cytoplasmic vs. viral nucleocapsids demonstrate that budding causes discrete changes in Cp-gRNA interactions. Notably, Cp's top binding site is maintained throughout virus assembly, and specifically binds and assembles with Cp into core-like particles in vitro. Together our data suggest a model for selective alphavirus genome recognition and assembly.

The Alphavirus Exit Pathway: What We Know and What We Wish We Knew.

Alphaviruses are enveloped positive sense RNA viruses and include serious human pathogens, such as the encephalitic alphaviruses and Chikungunya virus. Alphaviruses are transmitted to humans primarily by mosquito vectors and include species that are classified as emerging pathogens. Alphaviruses assemble highly organized, spherical particles that bud from the plasma membrane. In this review, we discuss what is known about the alphavirus exit pathway during a cellular infection. We describe the viral protein interactions that are critical for virus assembly/budding and the host factors that are involved, and we highlight the recent discovery of cell-to-cell transmission of alphavirus particles via intercellular extensions. Lastly, we discuss outstanding questions in the alphavirus exit pathway that may provide important avenues for future research.

Effect of obesity on survival in epithelial ovarian cancer.

BACKGROUND: Epidemiologic studies suggest that obese women are more likely to die of ovarian cancer than those of ideal body weight, but it is not known whether increased incidence, comorbidities common to obese women, or altered tumor biology is responsible for this difference. The current study attempted to determine the influence of excess body weight on ovarian cancer survival, disease progression, and clinicopathologic factors. METHODS: The records of patients undergoing surgery for epithelial ovarian cancer at Cedars Sinai Medical Center between January 1, 1996 and June 30, 2003 were reviewed for height, weight, age, comorbidities, and treatment-specific details. Statistical analyses included the Fisher exact test, Kaplan-Meier survival, and Cox regression analyses. RESULTS: In all, 216 patients were identified. Eight percent were underweight (body mass index [BMI] < 18.5), 50% were ideal body weight (18.5 /= 30). Age, comorbidities including coronary artery disease and venous thromboembolism, and rates of optimal surgical cytoreduction were similar among BMI strata. Diabetes and hypertension were more common in obese women. Ten (29%) of the obese patients had International Federation of Gynecology and Obstetrics (FIGO) Stage I disease, compared with 19 (10%) of the patients with BMI < 30 (P = .01). In a subcohort of 149 patients with Stage III or IV disease, a significant trend was identified favoring increased BMI as an independent negative factor for disease-free (P = .02) and overall (P = .02) survival. CONCLUSIONS: Obese patients were more likely to have disease limited to the ovaries. For patients with advanced stage disease, obesity was independently associated with both shorter time to recurrence and shorter overall survival. These findings suggest an effect of excess body weight on tumor biology, and studies are under way to elucidate the molecular and hormonal mechanisms underlying these clinical observations.