Search

Effective Protein Quality Control (PQC), essential for cellular health, relies on spatial sequestration of misfolded proteins into defined inclusions. Here we elucidate the coordination of nuclear and cytoplasmic spatial PQC. While cytoplasmic misfolded proteins concentrate in a cytoplasmic, perinuclear Juxta Nuclear Quality control compartment (JUNQ), nuclear misfolded proteins sequester into a perinucleolar IntraNuclear Quality control compartment (INQ). Particle tracking reveals the INQ and JUNQ converge to face each other across the nuclear envelope at a site proximal to the Nuclear-Vacuolar Junction (NVJ) marked by perinuclear ESCRT-II/-III protein Chm7. Strikingly, this ESCRT-dependent convergence facilitates VPS4-dependent vacuolar clearance of misfolded cytoplasmic and nuclear proteins, the latter entailing extrusion of nuclear INQ into the vacuole. We propose perinuclear ESCRT coordinates spatial PQC at nuclear-vacuolar contacts to facilitate vacuolar clearance of nuclear and cytoplasmic misfolded proteins.

There is controversy surrounding the efficacy and safety of colonic stents as a bridge to surgery compared with immediate resection in patients presenting with an acute malignant large bowel obstruction. Retrospective longitudinal cohort study using the NYS SPARCS Database. Patients with acute malignant large bowel obstruction who either had stent followed by elective surgery within 3 weeks (bridge to surgery) or underwent immediate resection between October 2009 and June 2016 in the state of New York were included. The primary outcome was rate of stoma creation at index resection. Secondary outcomes were 90-day readmission, reoperation, procedural complications, and discharge disposition. A total of 3059 patients were included, n = 2917 (95.4%) underwent an immediate resection and n = 142 (4.6%) underwent bridge to surgery. We analyzed 139 patients in propensity score-matched groups. Patients in the bridge to surgery group were less likely than those in the immediate resection group to get a stoma at the time of surgery (OR 0.33, 95% CI 0.18–0.60). They were also less likely to be discharged to a rehabilitation facility or require a home health aide upon discharge (OR 0.36, 95% CI 0.22–0.61). There were no differences in rates of 90-day readmission, reoperation, or procedural complications between groups. Colonic stenting as a bridge to surgery leads to less stoma creation, a significant quality of life advantage, compared with immediate resection. Patients should be counseled regarding these potential benefits when the technology is available.

Background Attrition from general surgery residency is high with a national rate of 20%. We evaluated potential associations between financial considerations and attrition. Methods National prospective cohort study of categorical general surgery trainees. Results Of the 1048 interns who started training in 2007, 681 (65%) had complete survey and follow-up data. In logistic regression, those with higher starting attending salary expectations (>$300K) were more likely to leave training (OR 2.9, 95% CI 1.2–6.9). Women with a partner who earned more (>$50K/year) were more likely to leave training (OR 4.1, 95% CI 1.6–10.5). In a subgroup of interns undecided about their future practice setting (academic, community, private practice, industry), those with less debt (≤$100K) were more likely to leave training (OR 2.4, 95% CI 1.1–5.2). Conclusions Several financial matters were associated with attrition. Addressing these financial concerns may help decrease attrition in surgical training and improve surgical training.

Dengue virus (DENV) cycles between mosquito and mammalian hosts. To examine how DENV populations adapt to these different host environments, we used serial passage in human and mosquito cell lines and estimated fitness effects for all single-nucleotide variants in these populations using ultra-deep sequencing. This allowed us to determine the contributions of beneficial and deleterious mutations to the collective fitness of the population. Our analysis revealed that the continuous influx of a large burden of deleterious mutations counterbalances the effect of rare, host-specific beneficial mutations to shape the path of adaptation. Beneficial mutations preferentially map to intrinsically disordered domains in the viral proteome and cluster to defined regions in the genome. These phenotypically redundant adaptive alleles may facilitate host-specific DENV adaptation. Importantly, the evolutionary constraints described in our simple system mirror trends observed across DENV and Zika strains, indicating it recapitulates key biophysical and biological constraints shaping long-term viral evolution., eLife digest Viruses are constantly evolving as a result of mutations in their genetic material and environmental pressures. Viruses switching between insects and mammals face unique evolutionary pressures because they must retain their ability to infect both types of organisms. Yet, the mutations in a virus that may be beneficial in an insect may be different from the ones that may be beneficial in a mammal. Mutations in one host may be even harmful in the other. To learn more about how such viruses thrive as they switch between hosts, Dolan, Taguwa et al. studied the dengue virus, which causes over 390 million infections and over 10,000 deaths each year around the globe. They compared the mutations that occurred as the virus multiplied in human and mosquito cells grown in a laboratory. In the experiments, they used a method called ultra-deep RNA sequencing to identify every change that occurred in the genetic material of the virus each time it multiplied. They determined whether the mutations were beneficial or harmful based on whether they became more common – suggesting they helped the virus survive – or whether they did not persist because they were likely harmful or even fatal to the virus. The experiments showed that many harmful mutations constantly occur in the virus, in both human and mosquito cells. Beneficial changes happen rarely, and those that do are usually only helpful in one type of cell. Fatal mutations tended to occur in parts of the genetic material that encodes regions in the viral proteins that must remain the same. These structural elements appear to be essential to the virus’s survival and unable to undergo change, which makes them good targets for antiviral drugs or vaccines. The techniques used in the study may be useful for investigating other viruses and for understanding the evolutionary constraints on viruses more generally. This may help scientists develop antiviral drugs or vaccines that will remain effective even as viruses continue to evolve and mutate.

SummaryNeural stem and progenitor cells (NSPCs) are critical for continued cellular replacement in the adult brain. Life-long maintenance of a functional NSPC pool necessitates stringent mechanisms to preserve a pristine proteome. We find that the NSPCs chaperone network robustly maintains misfolded protein solubility and stress resilience through high levels of the ATP-dependent chaperonin TRiC/CCT. Strikingly, NSPC differentiation rewires the cellular chaperone network, reducing TRiC/CCT levels and inducing those of the ATP-independent small heat shock proteins (sHSPs). This switches the proteostasis strategy in neural progeny cells to promote sequestration of misfolded proteins into protective inclusions. The chaperone network of NSPCs is more effective than that of differentiated cells, leading to improved management of proteotoxic stress and amyloidogenic proteins. However, NSPC proteostasis is impaired by brain aging. The less efficient chaperone network of differentiated neural progeny may contribute to their enhanced susceptibility to neurodegenerative diseases characterized by aberrant protein misfolding and aggregation.

RNA viruses are unique in their evolutionary capacity, exhibiting high mutation rates and frequent recombination. They rapidly adapt to environmental changes, such as shifts in immune pressure or pharmacological challenge. The evolution of RNA viruses has been brought into new focus with the recent developments of genetic and experimental tools to explore and manipulate the evolutionary dynamics of viral populations. These studies have uncovered new mechanisms that enable viruses to overcome evolutionary challenges in the environment and have emphasized the intimate relationship of viral populations with evolution. Here, we review some of the emerging viral and host mechanisms that underlie the evolution of RNA viruses. We also discuss new studies that demonstrate that the relationship between evolutionary dynamics and virus biology spans many spatial and temporal scales, affecting transmission dynamics within and between hosts as well as pathogenesis.

This study provides an updated description of diversity along the academic surgical pipeline to determine what progress has been made.Data was extracted from a variety of publically available data sources to determine proportions of minorities in medical school, general surgery training, and academic surgery leadership.In 2014-2015, Blacks represented 12.4% of the U.S. population, but only 5.7% graduating medical students, 6.2% general surgery trainees, 3.8% assistant professors, 2.5% associate professors and 2.0% full professors. From 2005-2015, representation among Black associate professors has gotten worse (-0.07%/year, p 0.01). Similarly, in 2014-2015, Hispanics represented 17.4% of the U.S. population but only 4.5% graduating medical students, 8.5% general surgery trainees, 5.0% assistant professors, 5.0% associate professors and 4.0% full professors. There has been modest improvement in Hispanic representation among general surgery trainees (0.2%/year, p 0.01), associate (0.12%/year, p 0.01) and full professors (0.13%/year, p 0.01).Despite efforts to promote diversity in surgery, Blacks and Hispanics remain underrepresented. A multi-level national focus is imperative to elucidate effective mechanisms to make academic surgery more reflective of the US population.

Dolan, P, Witherbee, KE, Peterson, KM, and Kerksick, CM. Effect of carbohydrate, caffeine, and carbohydrate + caffeine mouth rinsing on intermittent running performance in collegiate male lacrosse athletes. J Strength Cond Res 31(9): 2473-2479, 2017-Recently, an interest has developed in the potential to rinse the oral cavity with key nutrients to impact various types of exercise and presumably sporting performance. Although multiple studies examining carbohydrate mouth rinsing have been completed, conflicting evidence surrounding caffeine mouth rinsing persists, and no research has explored its ability to impact high-intensity, intermittent running performance. This study investigated the independent and synergistic ability of carbohydrate and caffeine mouth rinsing to improve intermittent running performance. The Yo-Yo Intermittent Recovery Test-Level 1 (Yo-Yo Level 1) was completed in 10 collegiate (National Collegiate Athletic Association [NCAA] Division II) male lacrosse players after a 10-second mouth rinse with a solution of either carbohydrate (CHO), caffeine (CAF), carbohydrate + caffeine (CHO + CAF), placebo (H2O), or a no rinse control (CON). No significant improvements in Yo-Yo IRT-1 performance were found (p > 0.05). Perceptual indications of effort (i.e., rating of their perceived exertion [RPE]) were significantly lower (p ≤ 0.05) in CHO and CHO + CAF when compared with CON after speed level 11. Interestingly, RPE levels were nonsignificantly lower in all but one level of the Yo-Yo Level 1 for CHO in comparison with other groups. Carbohydrate and caffeine mouth rinsing seems to exert no impact on running performance before maximal intermittent running in a group of male collegiate lacrosse players.

There is controversy surrounding the efficacy and safety of colonic stents as a bridge to surgery compared with immediate resection in patients presenting with an acute malignant large bowel obstruction.Retrospective longitudinal cohort study using the NYS SPARCS Database. Patients with acute malignant large bowel obstruction who either had stent followed by elective surgery within 3 weeks (bridge to surgery) or underwent immediate resection between October 2009 and June 2016 in the state of New York were included. The primary outcome was rate of stoma creation at index resection. Secondary outcomes were 90-day readmission, reoperation, procedural complications, and discharge disposition.A total of 3059 patients were included, n = 2917 (95.4%) underwent an immediate resection and n = 142 (4.6%) underwent bridge to surgery. We analyzed 139 patients in propensity score-matched groups. Patients in the bridge to surgery group were less likely than those in the immediate resection group to get a stoma at the time of surgery (OR 0.33, 95% CI 0.18-0.60). They were also less likely to be discharged to a rehabilitation facility or require a home health aide upon discharge (OR 0.36, 95% CI 0.22-0.61). There were no differences in rates of 90-day readmission, reoperation, or procedural complications between groups.Colonic stenting as a bridge to surgery leads to less stoma creation, a significant quality of life advantage, compared with immediate resection. Patients should be counseled regarding these potential benefits when the technology is available.

Summary The live-attenuated oral poliovirus vaccine (OPV or Sabin vaccine) replicates in gut-associated tissues, eliciting mucosa and systemic immunity. OPV protects from disease and limits poliovirus spread. Accordingly, vaccination with OPV is the primary strategy used to end the circulation of all polioviruses. However, the ability of OPV to regain replication fitness and establish new epidemics represents a significant risk of polio re-emergence should immunization cease. Here, we report the development of a poliovirus type 2 vaccine strain (nOPV2) that is genetically more stable and less likely to regain virulence than the original Sabin2 strain. We introduced modifications within at the 5′ untranslated region of the Sabin2 genome to stabilize attenuation determinants, 2C coding region to prevent recombination, and 3D polymerase to limit viral adaptability. Prior work established that nOPV2 is immunogenic in preclinical and clinical studies, and thus may enable complete poliovirus eradication., Graphical Abstract, Highlights • The Sabin vaccine protects from poliomyelitis but can regain neurovirulence • Specific modifications into the Sabin2 genome increase its genetic stability • Slowing down Sabin2 virus evolution prevents reversion and leads to a safer vaccine • The new strain (nOPV2) is safe and immunogenic in preclinical and clinical studies, The live-attenuated oral poliovirus vaccine (OPV; Sabin vaccine) can revert to neurovirulent variants, causing safety concerns. Yeh et al. engineer a poliovirus vaccine strain (nOPV2) that preserves the antigenic and immunogenic characteristics of Sabin2 while stabilizing determinants of attenuation and reducing evolvability. nOPV2 is safe and immunogenic in preclinical studies.

Background Weight loss surgery is safe and effective, but fluid and electrolyte balance remains problematic postoperatively. We developed a mobile app to help patients recover after weight loss surgery. Material and methods Single-center prospective, mixed-methods, participatory research design study of eligible English-speaking adults undergoing weight loss surgery was used. Patients used the app for 30 d after surgery. We developed and refined the app from July 2017 to October 2018. The principal component of the app is a daily survey designed to assess patients' recovery. The app was revised based on patient feedback, and we compared app utilization between initial and updated versions of the app. Primary outcome was successful patient engagement, which we defined as 80% of patients completing at least 70% of the surveys. Results Ten patients completed the trial period, four with the initial version of the app, and six with the updated version. All patients expressed satisfaction with the app and most frequently reported that push notifications were helpful. We found that one (25%) patient completed at least 70% of the surveys in the initial version of the app. In the updated version, five (83.3%) of patients completed at least 70% of the surveys, passing our criteria for successful engagement. Conclusions Participatory research design in app development requires continuous evaluation and refinement to patient and clinician needs. This effort is essential as we observed significant improvement in app utilization. Our next step is to pilot the app in a larger set of patients to assess utility and feasibility.

Bariatric surgery is effective at achieving sustained weight loss and improving the control and resolution of obesity-related co-morbidities. Most studies that have demonstrated co-morbidity resolution in patients undergoing laparoscopic sleeve gastrectomy (LSG) only follow patients for the short term (less than 1 year) or follow a relatively small cohort (

Importance American Board of Surgery board certification requires passing both a written qualifying examination and an oral certifying examination. No studies have been conducted assessing the effect of sociodemographic variables on board passage rates. Objective To evaluate if trainee sociodemographic factors are associated with board passage rates. Design, Setting, and Participants This national and multi-institutional prospective observational cohort study of 1048 categorical general surgery trainees starting in 2007-2008 were surveyed. Data collection began in June 2007, follow-up was completed on December 31, 2016, and analysis began September 2018. Main Outcomes and Measures Survey responses were linked to American Board of Surgery board passage data. Results Of 662 examinees who had complete survey and follow-up data, 443 (65%) were men and 459 (69%) were white, with an overall board passage rate of 87% (n = 578). In a multinomial regression model, trainees of Hispanic ethnicity were more likely to not attempt the examinations (vs passed both) than non-Hispanic trainees (odds ratio [OR], 4.7; 95% CI, 1.5-14). Compared with examinees who were married with children during internship, examinees who were married without children (OR, 0.3; 95% CI, 0.1-0.8) or were single (OR, 0.4; 95% CI, 0.2-0.9) were less likely to fail the examinations. Logistic regression showed white examinees compared with nonwhite examinees (black individuals, Asian individuals, and individuals of other races) (OR, 1.8; 95% CI, 1.03-3.0) and examinees who performed better on their first American Board of Surgery In-Training Examination (OR, 1.03; 95% CI, 1.02-1.05) were more likely to pass the qualifying examination on the first try. White examinees compared with nonwhite examinees (OR, 1.8; 95% CI, 1.1-2.8), non-Hispanic compared with Hispanic examinees (OR, 2.4; 95% CI, 1.2-4.7), and single women compared with women who were married with children during internship (OR, 10.3; 95% CI, 2.1-51) were more likely to pass the certifying examination on the first try. Conclusions and Relevance Resident race, ethnicity, sex, and family status at internship were observed to be associated with board passage rates. There are multiple possible explanations for these worrisome observations that need to be explored. Tracking demographics of trainees to help understand passage rates based on demographics will be important. The American Board of Surgery already has begun addressing the potential for unconscious bias among board examiners by increasing diversity and adding implicit bias training.

The focus of this chapter will be on the available surgical techniques in managing challenging abdominal wall hernias. Although these hernias have different anatomic locations and etiologies, they are similar in that they are rare entities, and therefore there is little data to help guide optimal management and surgical approach. As with other hernia repairs, the operating surgeon can approach these hernias with open or minimally invasive surgery (MIS) techniques. Within MIS, there are total extraperitoneal (TEP), transabdominal preperitoneal (TAPP), and intraperitoneal onlay mesh (IPOM) options. Furthermore, the proliferation of single-incision laparoscopic surgery (SILS) and robotics offers other tools for the surgeon. Regardless of the approach, the essential tenets of a durable hernia repair still apply: reducing the hernia contents and sac and then performing a tension-free repair of the hernia defect with adequate overlap of a mesh. These approaches will be discussed in detail, along with associated risks and benefits based on the available data. Hernia-type specific challenges to repair will also be discussed.

The MESA erythrocyte cytoskeleton binding (MEC) motif is a 13-amino acid sequence found in 14 exported Plasmodium falciparum proteins. First identified in the P. falciparum Mature-parasite-infected Erythrocyte Surface Antigen (MESA), the MEC motif is sufficient to target proteins to the infected red blood cell cytoskeleton. To identify host cell targets, purified MESA MEC motif was incubated with a soluble extract from uninfected erythrocytes, precipitated and subjected to mass spectrometry. The most abundant co-purifying protein was erythrocyte ankyrin (ANK1). A direct interaction between the MEC motif and ANK1 was independently verified using co-purification experiments, the split-luciferase assay, and the yeast two-hybrid assay. A systematic mutational analysis of the core MEC motif demonstrated a critical role for the conserved aspartic acid residue at the C-terminus of the MEC motif for binding to both erythrocyte inside-out vesicles and to ANK1. Using a panel of ANK1 constructs, the MEC motif binding site was localized to the ZU5(C) domain, which has no known function. The MEC motif had no impact on erythrocyte deformability when introduced into uninfected erythrocyte ghosts, suggesting the MEC motif’s primary function is to target exported proteins to the cytoskeleton. Finally, we show that PF3D7_0402100 (PFD0095c) binds to ANK1 and band 4.1, likely through its MEC and PHIST motifs, respectively. In conclusion, we have provided multiple lines of evidence that the MEC motif binds to erythrocyte ANK1.

Mosquitoes spread pathogenic arboviruses while themselves tolerate infection. We here characterize an immunity pathway providing long-term antiviral protection and define how this pathway discriminates between self and non-self. Mosquitoes use viral RNAs to create viral derived cDNAs (vDNAs) central to the antiviral response. vDNA molecules are acquired through a process of reverse-transcription and recombination directed by endogenous retrotransposons. These vDNAs are thought to integrate in the host genome as endogenous viral elements (EVEs). Sequencing of pre-integrated vDNA revealed that the acquisition process exquisitely distinguishes viral from host RNA, providing one layer of self-nonself discrimination. Importantly, we show EVE-derived piRNAs have antiviral activity and are loaded onto Piwi4 to inhibit virus replication. In a second layer of self-non-self discrimination, Piwi4 preferentially loads EVE-derived piRNAs, discriminating against transposon-targeting piRNAs. Our findings define a fundamental virus-specific immunity pathway in mosquitoes that uses EVEs as a potent and specific antiviral transgenerational mechanism.

The deterministic force of natural selection and stochastic influence of drift shape RNA virus evolution. New deep-sequencing and microfluidics technologies allow us to quantify the effect of mutations and trace the evolution of viral populations with single-genome and single-nucleotide resolution. Such experiments can reveal the topography of the genotype-fitness landscapes that shape the path of viral evolution. By combining historical analyses, like phylogenetic approaches, with high-throughput and high-resolution evolutionary experiments, we can observe parallel patterns of evolution that drive important phenotypic transitions. These developments provide a framework for quantifying and anticipating potential evolutionary events. Here, we examine emerging technologies that can map the selective landscapes of viruses, focusing on their application to pathogenic viruses. We identify areas where these technologies can bolster our ability to study the evolution of viruses and to anticipate and possibly intervene in evolutionary events and prevent viral disease.

Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein–protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N-terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two-hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site-directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.

RNA viruses, such as poliovirus, have a great evolutionary capacity, allowing them to quickly adapt and overcome challenges encountered during infection. Here we show that poliovirus infection in immune-competent mice requires adaptation to tissue-specific innate immune microenvironments. The ability of the virus to establish robust infection and virulence correlates with its evolutionary capacity. We further identify a region in the multi-functional poliovirus protein 2B as a hotspot for the accumulation of minor alleles that facilitate a more effective suppression of the interferon response. We propose that population genetic dynamics enables poliovirus spread between tissues through optimization of the genetic composition of low frequency variants, which together cooperate to circumvent tissue-specific challenges. Thus, intrahost virus evolution determines pathogenesis, allowing a dynamic regulation of viral functions required to overcome barriers to infection., RNA viruses, such as polioviruses, have a great evolutionary capacity and can adapt quickly during infection. Here, the authors show that poliovirus infection in mice requires adaptation to innate immune microenvironments encountered in different tissues.

The Aedes aegypti mosquito transmits arboviruses, including dengue, chikungunya, and Zika virus. Understanding the mechanisms underlying mosquito immunity could provide new tools to control arbovirus spread. Insects exploit two different RNAi pathways to combat viral and transposon infection: short interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) [1, 2]. Endogenous viral elements (EVEs) are sequences from non-retroviral viruses that are inserted into the mosquito genome and can act as templates for the production of piRNAs [3, 4]. EVEs therefore represent a record of past infections and a reservoir of potential immune memory [5]. The large-scale organization of EVEs has been difficult to resolve with short-read sequencing because they tend to integrate into repetitive regions of the genome. To define the diversity, organization, and function of EVEs, we took advantage of the contiguity associated with long-read sequencing to generate a high-quality assembly of the Ae. aegypti-derived Aag2 cell line genome, an important and widely used model system. We show EVEs are acquired through recombination with specific classes of long terminal repeat (LTR) retrotransposons and organize into large loci (>50 kbp) characterized by high LTR density. These EVE-containing loci have increased density of piRNAs compared to similar regions without EVEs. Furthermore, we detected EVE-derived piRNAs consistent with a targeted processing of persistently infecting virus genomes. We propose that comparisons of EVEs across mosquito populations may explain differences in vector competence, and further study of the structure and function of these elements in the genome of mosquitoes may lead to epidemiological interventions.

Many viral proteins or their biologically important regions are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions do not possess unique structures and possess functions that complement the functional repertoire of "normal" ordered proteins and domains, with many protein functional classes being heavily dependent on the intrinsic disorder. Viruses commonly use these highly flexible regions to invade the host organisms and to hijack various host systems. These disordered regions also help viruses in adapting to their hostile habitats and to manage their economic usage of genetic material. In this article, we focus on the structural peculiarities of proteins from human hepatitis C virus (HCV) and use a wide spectrum of bioinformatics techniques to evaluate the abundance of intrinsic disorder in the completed proteomes of several human HCV genotypes, to analyze the peculiarities of disorder distribution within the individual HCV proteins, and to establish potential roles of the structural disorder in functions of ten HCV proteins. We show that the intrinsic disorder or increased flexibility is not only abundant in these proteins, but is also absolutely necessary for their functions, playing a crucial role in the proteolytic processing of the HCV polyprotein, the maturation of the individual HCV proteins, and being related to the posttranslational modifications of these proteins and their interactions with DNA, RNA, and various host proteins.

This national cross-sectional survey of the US general population assesses the association of negative attitudes toward weight loss surgery and its subsequent use in obese patients.

APOBEC3G and APOBEC3F are cytidine deaminase with duplicative cytidine deaminase motifs that restrict HIV-1 replication by catalyzing C-to-U transitions on nascent viral cDNA. Despite 60% protein sequence similarity, APOBEC3F and APOBEC3G have a different target consensus sequence for editing, and importantly, APOBEC3G has 10-fold higher anti-HIV activity than APOBEC3F. Thus, APOBEC3F and APOBEC3G may have distinctive characteristics that account for their functional differences. Here, we have biochemically characterized human APOBEC3F and APOBEC3G protein complexes as a function of the HIV-1 life cycle. APOBEC3G was previously shown to form RNase-sensitive, enzymatically inactive, high molecular mass complexes in immortalized cells, which are converted into enzymatically active, low molecular mass complexes by RNase digestion. We found that APOBEC3F also formed high molecular mass complexes in these cells, but these complexes were resistant to RNase treatment. Further, the N-terminal half determined RNase sensitivity and was necessary for the high molecular mass complex assembly of APOBEC3G but not APOBEC3F. Unlike APOBEC3F, APOBEC3G strongly interacted with cellular proteins via disulfide bonds. Inside virions, both APOBEC3F and APOBEC3G were found in viral cores, but APOBEC3G was associated with low molecular mass, whereas APOBEC3F was still retained in high molecular mass complexes. After cell entry, both APOBEC3F and APOBEC3G were localized in low molecular mass complexes associated with viral reverse transcriptional machinery. These results demonstrate that APOBEC3F and APOBEC3G complexes undergo dynamic conversion during HIV-1 infection and also reveal biochemical differences that likely determine their different anti-HIV-1 activity.

Viral proteins bind to numerous cellular and viral proteins throughout the infection cycle. However, the mechanisms by which viral proteins interact with such large numbers of factors remain unknown. Cellular proteins that interact with multiple, distinct partners often do so through short sequences known as molecular recognition features (MoRFs) embedded within intrinsically disordered regions (IDRs). In this study, we report the first evidence that MoRFs in viral proteins play a similar role in targeting the host cell. Using a combination of evolutionary modeling, protein–protein interaction analyses and forward genetic screening, we systematically investigated two computationally predicted MoRFs within the N-terminal IDR of the hepatitis C virus (HCV) Core protein. Sequence analysis of the MoRFs showed their conservation across all HCV genotypes and the canine and equine Hepaciviruses. Phylogenetic modeling indicated that the Core MoRFs are under stronger purifying selection than the surrounding sequence, suggesting that these modules have a biological function. Using the yeast two-hybrid assay, we identified three cellular binding partners for each HCV Core MoRF, including two previously characterized cellular targets of HCV Core (DDX3X and NPM1). Random and site-directed mutagenesis demonstrated that the predicted MoRF regions were required for binding to the cellular proteins, but that different residues within each MoRF were critical for binding to different partners. This study demonstrated that viruses may use intrinsic disorder to target multiple cellular proteins with the same amino acid sequence and provides a framework for characterizing the binding partners of other disordered regions in viral and cellular proteomes.

Hepatitis C virus (HCV) alters the global behavior of the host cell to create an environment conducive to its own replication, but much remains unknown about how HCV proteins elicit these changes. Thus, a better understanding of the interface between the virus and host cell is required. Here we report the results of a large-scale yeast two-hybrid screen to identify protein–protein interactions between HCV genotype 2a (strain JFH1) and cellular factors. Our study identified 112 unique interactions between 7 HCV and 94 human proteins, over 40% of which have been linked to HCV infection by other studies. These interactions develop a more complete picture of HCV infection, providing insight into HCV manipulation of pathways, such as lipid and cholesterol metabolism, that were previously linked to HCV infection and implicating novel targets within microtubule-organizing centers, the complement system and cell cycle regulatory machinery. In an effort to understand the relationship between HCV and related viruses, we compared the HCV 2a interactome to those of other HCV genotypes and to the related dengue virus. Greater overlap was observed between HCV and dengue virus targets than between HCV genotypes, demonstrating the value of parallel screening approaches when comparing virus–host cell interactomes. Using siRNAs to inhibit expression of cellular proteins, we found that five of the ten shared targets tested (CUL7, PCM1, RILPL2, RNASET2, and TCF7L2) were required for replication of both HCV and dengue virus. These shared interactions provide insight into common features of the viral life cycles of the family Flaviviridae.