Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP–mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Aquareovirus and orthoreovirus are two genera of the Reoviridae family of segmented double-stranded RNA viruses that form multinucleated syncytia after infection, which can increase viral spread and pathogenicity (1–4). To drive cell-cell fusion, both aquareovirus and orthoreovirus express a nonstructural, fusion-associated small transmembrane (FAST) protein on the plasma membrane of infected cells. The FAST protein is not required for viral entry, and expression of FAST protein alone is sufficient to cause cells to fuse with naïve neighboring cells, forming large multinucleated syncytium (1, 2, 5–12), confirming they are bona fide cell-cell fusogens. Although they have similar function and topology in the membrane, FAST proteins from aquareovirus and orthoreovirus share minimal sequence identity (13). Based on phylogenetic analysis, they are hypothesized to have evolved from a common, likely nonfusogenic, ancestor 510 million years ago (4, 13, 14). Separate gain-of-function events are believed to have produced fusogenic proteins in both aquareovirus and orthoreovirus, with further divergence or acquisition events resulting in the diversity of FAST proteins found in reoviruses today (13).Aquareovirus and orthoreovirus FAST proteins are single-pass membrane proteins of fewer than 200 residues comprised of a mostly disordered cytoplasmic tail, a transmembrane domain, and a small ectodomain of fewer than 40 residues (1, 2). The membrane-disruptive ectodomains of FAST proteins typically have solvent-exposed hydrophobic residues and/or myristoylation motifs that are necessary for cell-cell fusion (5, 15–17). In contrast to other cell-cell fusogens that fuse membranes by pulling them together using conformational changes in their ∼10 nm-tall ectodomains, the ectodomains of FAST proteins have minimal predicted secondary structure, are unlikely to undergo conformational changes to drive membrane fusion (1, 2), and extend only ∼1 nm above the bilayer (5, 18). How such short fusogens can overcome the ∼2 nm repulsive hydration barrier and larger barrier presented by cell surface proteins to reach and fuse with an opposing membrane (5, 18) has been a long-standing question for FAST proteins and other short cell-cell fusogens, such as myomixer and myomaker that are involved in myoblast fusion (19–22).Recently, we found that the FAST protein from reptilian orthoreovirus, p14, hijacks the host cell actin cytoskeleton to drive cell-cell fusion by forming localized branched actin networks (23). This is accomplished through a c-src phosphorylated tyrosine motif, YVNI, in p14’s disordered cytoplasmic tail that binds to a host adaptor protein, Grb2, which then binds to N-WASP and nucleates branched actin assembly. We hypothesize that this directly couples local actin-generated forces to push p14’s short, fusogenic ectodomain into the opposing cell’s plasma membrane (23). While all FAST family proteins have similarly short ectodomains, it is unclear if this is a general strategy used by other FAST proteins to drive cell-cell fusion.Here, we report that a FAST protein from the divergent aquareovirus, p22, also hijacks the host actin cytoskeleton but does so using a molecular strategy distinct from that of the orthoreovirus FAST protein p14. Instead of binding to Grb2, we find that p22 binds to Intersectin-1 through an SH3 binding motif in its cytoplasmic tail, which binds Cdc42 to activate N-WASP–mediated branched actin assembly. We show that despite minimal sequence identity, the p22 cytoplasmic tail can be functionally swapped with that of p14, suggesting that while the cytoplasmic tails of the two FAST proteins evolved independently, they serve a similar function. By directly coupling the ectodomain to a different actin nucleator, we suggest that actin’s functional role is applying mechanical pressure to a fusogenic ectodomain at the plasma membrane. This biophysical role may be shared across other members of the FAST protein family and could be more generally employed by other cell-cell fusogens.     

New-Onset Diabetes and Preexisting Diabetes Are Associated With Comparable Reduction in Long-Term Survival After Liver Transplant: A Machine Learning Approach

Objective

To identify key predictors and survival outcomes of new-onset diabetes after transplant (NODAT) in liver transplant (LT) recipients by using the Scientific Registry of Transplant Recipients.

A randomized controlled trial of artemotil (beta-arteether) in Zambian children with cerebral malaria

The efficacy and safety of intramuscular artemotil (ARTECEF) was compared to intravenous quinine in African children with cerebral malaria. This prospective block randomized open-label study was conducted at two centers in Zambia. Subjects were children aged 0 to 10 years of age with cerebral malaria and a Blantyre Coma Score of 2 or less. Ninety two children were studied; 48 received artemotil and 44 quinine. No significant differences in survival, coma resolution time, neurologic sequelae, parasite clearance time, and fever resolution time were seen between the two regimens. Rates for negative malaria smears one month after therapy were similar in both groups. Artemotil was a well-tolerated drug in the 48 patients in this study. It appears to be at least therapeutically equivalent to quinine for the treatment of pediatric cerebral malaria. It has the advantage of being able to be given intramuscularly once daily for only five days.     

Body fat measurement in Indian men: comparison of three methods based on a two-compartment model

Obesity is a major risk factor for diabetes and related disorders. The current classification of obesity is based on body mass index (BMI, kg/m(2)), which is a surrogate for the total body fat. Since the relationship between BMI and body fat varies in different populations, an independent validation of the BMI-body fat relationship in the population of interest is desirable. OBJECTIVES: (1) To study the validity of field methods of measuring body fat (multiple skinfolds and bioimpedance) against a criterion method (deuterium dilution) and (2) To compare the prevalence of obesity (WHO 2000 criteria for BMI) with adiposity (body fat >25%) in middle-aged Indian men in rural and urban Pune. DESIGN: Community-based multistage stratified random sampling of middle-aged men from rural and urban Pune for study of body composition and cardiovascular risk. A third of these men, selected to represent wide BMI distribution, were studied for body fat measurements by specific methods. SUBJECTS: A total of 141 healthy men, approximately similar number from rural, urban slums and middle class from Pune. They were 39.3 (+/-6.2) y old and had a BMI of 21.9 (+/-3.7) kg/m(2). MEASUREMENTS: Anthropometry (height, weight and multiple skinfold thicknesses) by trained observers using standardised technique to calculate body fat by Durnin and Womersley's equation. Total body water and body fat by bioelectrical impedance analysis (BIA) and deuterium oxide dilution (D(2)O). RESULTS: Mean total body fat was 14.3 kg (23.0%) by anthropometry, 16.5 kg (26.0%) by BIA and 15.3 kg (24.6%) by D(2)O method. Although there was a good correlation between fat estimation by three methods (r= approximately 0.9, P<0.001 all), compared to D(2)O method anthropometry underestimated body fat by 1.0 kg and BIA overestimated fat by 1.2 kg (P<0.001 both). Using the standard cut-point of 25% body fat for 'adiposity' 29.5% rural, 46.0% slum and 75.0% middle class men were adipose. These proportions were considerably higher than the number of men who were 'preobese' (BMI> or =25-29.9 kg/m(2), 9.0% rural, 22.0% urban slums and 27.0% urban middle class) and 'obese' (BMI >30 kg/m(2), 4.0% urban slums, none in rural and urban middle class). CONCLUSION: We recommend that future studies assessing risk for chronic diseases in Indians should measure adiposity by anthropometry (multiple skinfolds) or BIA (calibrated for Indians) rather than relying only on BMI cut-points.     

Research on tuberculosis in tribal areas in India: A systematic review

Background

Tuberculosis (TB) remains a major public health problem in resource-poor countries including India. Scientific knowledge is used to guide policy and practice. There is however, a limited, systematically collected data required for guiding the scale-up of interventions particularly amongst vulnerable populations including tribal groups in the country. In view of this, a systematic review of the TB research studies carried out in tribal areas of different parts of the country was undertaken.

Mammalian target of rapamycin inhibition after solid organ transplantation: can it,and does it,reduce cancer risk?

The mammalian target of rapamycin (mTOR) inhibitors sirolimus and everolimus has been increasingly used as immunosuppressants for recipients of solid organ transplants. Over the years, potential advantages unique to this class of immunosuppressants have been recognized, including chemoprevention by virtue of their antiproliferative effects. Prevention of malignancy after transplant through mTOR inhibitor‐based immunosuppression may have a specific practical application in transplant recipients with preexisting malignancy including hepatocellular carcinoma or cholangiocarcinoma. This review will reveal how the biochemistry of the mTOR pathway, as it pertains to chemoprevention, can support a clinical role for mTOR inhibitors in the prevention of malignancies, recurrent or de novo, after solid organ transplantation in selected patients.     

Efficacy of Human Chorion Membrane Allograft for Recession Coverage: A Case Series

Background : Membranes of human placentas have been used in the field of medicine for skin grafts, treatment of burns, and ulcerated skin conditions with great success. The use of placenta allografts in dentistry is a more recent development, with the first commercial product being made available in 2008. The unique inherent biologic properties in placenta allografts enhance wound healing and may propagate regeneration. Methods: Ten healthy adult patients presenting with 21 Miller Class I gingival recession (GR) defects (isolated or adjacent multiple) were surgically treated with a modified coronally advanced flap and chorion membrane for root coverage. Clinical parameters measured at baseline, 3 months, and 6 months were probing depth, clinical attachment level, GR height, width of keratinized gingiva, and assessment of gingival biotype. Statistical analysis was performed to compare the treatment outcomes at the follow‐up intervals. Results: The results showed statistically significant (P <0.001) improvements in all clinical parameters at the 3‐ and 6‐month follow‐ups. The mean percentage of root coverage at the end of 6 months was 89.92% ± 15.59%, and 14 of 21 treated GR defects showed 100% root coverage. The gingival biotype also showed a thick biotype in nine sites that had an initial thin biotype. Conclusions: Fetal membranes possess distinctive properties that can be harnessed to promote periodontal healing. The chorion membrane covered by a modified coronally advanced flap is a new approach that has shown promising results in terms of root coverage, increased width of keratinized tissue, and thickness of the gingival biotype.     

Coexistence of phosphotyrosine-dependent and -independent interactions between Cbl and Bcr-Abl

Cbl is one of the major tyrosine-phosphorylated proteins in Bcr-Abl-expressing cells. A direct association between the SH2 domain of Bcr-Abl and tyrosine-phosphorylated Cbl has been demonstrated. The purpose of this study was to determine if and how unphosphorylated Cbl and Bcr-Abl may associate.Interactions between Cbl and Bcr-Abl were investigated in yeast two- and three-hybrid systems, gel overlay assays, and immunoprecipitates from mammalian cells expressing wild-type and the Y177F mutant of Bcr-Abl.No direct interaction between Bcr-Abl and unphosphorylated Cbl was observed. Bcr-Abl did, however, associate with Grb2, an adaptor protein that binds tyrosine 177 of Bcr-Abl. Additionally, Grb2 interacted with Cbl. In a yeast three-hybrid assay, Grb2 mediated an interaction between Cbl and Bcr-Abl that was dependent on a functional Grb2 binding site. This interaction was confirmed in vitro using purified proteins. In cells expressing Bcr-Abl with a mutation in the Grb2 binding site, binding of Cbl to Bcr-Abl was significantly reduced, but Cbl tyrosine phosphorylation was maintained. Imatinib treatment of these cells further reduced but did not abrogate Cbl binding, reflecting residual kinase activity.Multiple phosphotyrosine-dependent and -independent interactions stabilize the interaction between Cbl and Abl. Grb2 or another, yet unidentified, protein may mediate an initial interaction between Cbl and Bcr-Abl that is independent of Cbl tyrosine phosphorylation. Following this initial interaction, Cbl can then become tyrosine phosphorylated and interact with the SH2 domain of Bcr-Abl, further stabilizing the complex.