In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.

Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.ImportanceTo fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available

In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus

双链RNA病毒是病毒中最大的类群，其中轮状病毒作为双链RNA病毒家族最知名的病毒之一，每年引起接近百万的新生儿死亡，而单链RNA病毒虽然没有双链RNA病毒那么多，但是却包含了许多很有名的病毒：HIV，埃博拉病毒，小核糖核苷酸病毒（包括甲肝病毒HAV、肠道病毒、脊髓灰质炎病毒、口蹄疫病毒、感冒病毒等），SARS病毒，丙肝病毒HCV等等。而且与双链RNA病毒不同的是，单链RNA病毒不会把它们的基因组包裹到预制的外壳蛋白中，而是利用基因组共同装配壳体，关于这个共同组装的过程，科学家们了解的很少。最新研究中，研究人员获取了单链RNA病毒：大肠杆菌噬菌体MS2的冷冻电镜结构（分辨率为3.6Å），并追踪了80%病毒基因组结构，从而发现了这种病毒的壳体共同组装过程中的分子机制，这将为解析核蛋白复合物与病毒起源之间的联系提供了重要的信息。【Abstract】Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid1–3 , single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome4–7 ; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host ‘sex pilus’(F-pilus)8 ; it was the first fully sequenced organism9 and is a model system for studies of translational gene regulation10,11,RNA–protein interactions12–14 , and RNA virus assembly15–17 .Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T=3 icosahedral lattice18,19 . The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection8 ,but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryoelectron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome,built atomic models for 16 RNA stem–loops, and identified three conserved motifs of RNA–coat protein interactions among 15 of these stem–loops with diverse sequences. The stem–loop at the 3′end of the genome interacts extensively with the maturation protein,which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome–capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA–capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.This project was supported in part by grants from the National Institutes of Health (GM071940, DE025567, DE023591, CA177322 and AI094386) and National Science Foundation (DMR-1548924). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines (supported by UCLA and by instrumentation grants from the NIH (1S10OD018111, 1U24GM116792) and NSF (DBI-1338135))

A high-quality human reference panel reveals the complexity and distribution of genomic structural variants

Structural variation (SV) represents a major source of differences between individual human genomes and has been linked to disease phenotypes. However, the majority of studies provide neither a global view of the full spectrum of these variants nor integrate them into reference panels of genetic variation. Here, we analyse whole genome sequencing data of 769 individuals from 250 Dutch families, and provide a haplotype-resolved map of 1.9 million genome variants across 9 different variant classes, including novel forms of complex indels, and retrotransposition-mediated insertions of mobile elements and processed RNAs. A large proportion are previously under reported variants sized between 21 and 100 bp. We detect 4 megabases of novel sequence, encoding 11 new transcripts. Finally, we show 191 known, trait-associated SNPs to be in strong linkage disequilibrium with SVs and demonstrate that our panel facilitates accurate imputation of SVs in unrelated individuals

Quantitative high-throughput genomics in RNA viruses

The high mutation rate and rapid genome replication of RNA viruses drive their adaptation to diverse selection pressures. The emergence of drug resistant or immune escape viral strains is always a major concern to public health. A comprehensive understanding of the mutation tolerability of viral genome is thus crucial to understand the evolution potential of viruses and guild the accurate risk assessments.Traditional genetics has proven to be a powerful tool for virology studies. Including forward genetics – determine the genetic basis responsible for a phenotype, and reverse genetics – determine the phenotype of a genetic change, it reveals the functional role of many important mutations. However, traditional genetics is usually restricted by limited and biased sampling, and is time and money consuming. To overcome these limitations, we have developed a qantatative high-throughput genomic system that enables us to quantify the phenotype of thousands to millions of mutations as a massive parallel process. Using random mutagenesis or satuated mutagenesis, we can generate a diverse pool of viral library containing desired mutations. The library can be used to assess the function of every amino acid/nucleotide in a variety of protein functional assays as well as viral growth assay, with the frequency of each mutant changed according to their competitive strength. We were able to quantify the relative frequency change of each variant pre and post selection by high-throughput sequencing, which represented their “relative fitness” under the particular selection condition. Since the first inception of the system, we have optimized and successfully applied it to human immunodeficiency virus (HIV), Hepatitis C Virus (HCV) and influenza A virus. We also explored the applications of the system to a variety of biological questions, with a specicial focus in the following 4 areas:Firstly, a direct application of the system is to better understand the distribution of fitness effect (DFE), which is fundamental to a variety of evolution theories. We systematically quantified the DFE of single amino acid substitutions (86 amino acids total) in the drug-targeted region of NS5A protein of Hepatitis C Virus (HCV). We found that the majority of non-synonymous substitutions incur large fitness costs, suggesting that NS5A protein is highly optimized in natural conditions. Furthermore, we characterized the evolutionary potential of HCV by subjecting the mutant viruses to varying concentrations of an NS5A inhibitor Daclatasvir. As the selection pressure increases, the DFE of beneficial mutations shifts from an exponential distribution to a heavy-tailed distribution with a disproportionate number of exceptionally fit mutants. The number of available beneficial mutations and the selection coefficient both increase at higher levels of antiviral drug concentration, as predicted by a pharmacodynamics model describing viral fitness as a function of drug concentration. Our large-scale fitness data of mutant viruses also provide insights into the biophysical basis of evolutionary constraints and the role of the genetic code in protein evolution.Secondly, we explored the usage of fitness profiling to identify and annotate protein functional residues. Using influenza A virus PB1 protein as an example, we developed an approach to achieve this task: Firstly, the effect of PB1 point mutations on viral replication was examined by saturation mutagenesis and high-throughput sequencing. Secondly, functional PB1 residues that are essential for viral growth but do not affect protein stability were identified by protein stability prediction. Lastly, homologous structural alignment was utilized to further annotate specific biological functions (canonical versus non-canonical functions) for each functional residue. We achieved high sensitivity in identifying and annotating the canonical polymerase functional residues. Moreover, we identified non-canonical functional residues, which are exemplified by a cluster of residues located in the loop region of PB1 β ribbon. These previously uncharacterized residues were shown to be important for PB1 protein nuclear import by interacting with Ran-binding protein 5 (RanBP5).Thirdly, the system was shown to be valuable for the identification of drug resistant mutations and the design of personalized therapy. Using influenza NA protein as an example, we characterized the fitness effects of single nucleotide mutations of neuraminidase (NA) and systematically identified resistant mutations for three neuraminidase inhibitors (NAIs): zanamivir, oseltamivir and AV5080. We observed that both the numbers and the effects of resistant mutations of AV5080 are smaller than those of zanamivir and oseltamivir, but so are their fitness costs. We used population genetic models to estimate the rate of increase in fitness under drug selection as a function of drug dosage. AV5080 showed a higher rate of increase in fitness at low drug concentrations due to the low fitness cost of resistant mutations, but also exhibited a steep drop with high drug concentrations because of lower strength of resistance. Our approach also enabled the systematic analyses of cross-resistance against different drugs, which showed to be uncommon between AV5080 and zanamivir. Lastly and importantly, the system can be utilized to explore new functions of viral proteins. To this end, we systematically identified type I interferon sensitive mutations across the entire influenza A viral genome. We have identified novel IFN-sensitive mutations on PB2, PA, PB1 and M1, in addition to NS1, which provides a foundation to determine multiple anti-IFN mechanisms encoded in different viral segments. Moreover, this quantitative functional information of every amino acid in the genome enabled us to rationally design vaccine to increase the safety and immunogenicity. By selecting and combining 8 mutations into one viral genome, we successfully generated a deficient in anti-interferon (DAI) influenza strain as a live attenuated vaccine candidate. DAI is replication-competent in IFN-deficient host, but able to induce transient IFN response and highly attenuated in IFN competent host. Impressively, DAI is capable of inducing a robust humoral response and a strong T cell response, which collectively leads to broad protection. The superior property of DAI strain demonstrated the capacity of our approach to construct a safe, effective and broadly protecting live attenuated influenza vaccine. Thus we proposed a novel and generally applicable approach for vaccine design: systematically identifying and eliminating immune evasion functions on the virus genome, while maintaining the replication fitness in vitro for vaccine production. In summary, we have developed the quantitative high-throughput genomic system, and applied it to a variety of biological questions. It is proven to be a powerful system to investigate fundamental evolution problems, identify functional residues and new functions of target proteins, and facilitate drug development. With the maturation of DNA systhesis technology and ever increasing sequencing power, we foresee the further improvement and more broad applications of this system to address foundamental mechanistic questions and practical applications

The Effect Mechanism of Tie Strength of Supply Networks on Risk Sharing: Based on the Empirical Data of China’s Automobile Manufacturing Industry

Based on the research perspective of the cooperation risk and opportunistic risk between supply network enterprises, this article investigates the mechanism of how tie strength between manufacturers and suppliers influences risk sharing among enterprises from two dimensions of tie strength: structural strength and relational strength. In particular, we introduce how asymmetry of dependence moderates the relationship between tie strength and risk sharing. We surveyed China’s domestic auto OEMs and their first-tier suppliers in China through 260 questionnaires and used a hierarchical regression model as a research method to carry out the empirical analysis and test. We found an inverted U-shaped relationship between tie strength and risk sharing among enterprises, and asymmetry of dependence has a significant negative adjustment function on relational strength of the tie and risk-sharing relationship, while there is no significant adjustment function on the structural strength of it. Our findings suggest that keeping moderate tie strength among enterprises is conducive to achieving risk sharing. Moreover, trust and reciprocity is inhibitory regarding the adjustment effect of asymmetry of the dependence influencing relational strength and risk-sharing relationship. However, the structural strength and risk-sharing relationship are not interfered with by the adjustment function of asymmetry of dependence; that is, structural strength plays a decisive role in risk sharing

Open knowledge disclosure and firm value:a signalling theory perspective

A growing number of firms are openly disclosing knowledge through academic journals and conferences; however, the impact of this practice on their market value needs further research. From a signalling theory perspective, we investigate the relationship between open knowledge disclosure and firm value and identify potential contingency factors. We propose that open knowledge disclosure conveys a firm’s technical capability and commitment to open science, consequently contributing to its market value. Drawing upon data from listed companies within China’s information and communication technology sector, we confirm that open knowledge disclosure enhances firm value. Furthermore, this enhancement is more pronounced for small firms, young firms, private firms, firms with few patents, firms drafting few technical standards, or firms operating in an immature technology market. Our findings suggest that firms, especially those facing high information asymmetry or lacking alternative signals, can increase their market value by sending positive signals through open knowledge disclosure.</p

Emergence of Integrase Resistance Mutations During Initial Therapy Containing Dolutegravir.

Dolutegravir (DTG) is a preferred drug for initial treatment of human immunodeficiency virus type 1 infection. We present next-generation sequencing analysis of integrase genotypes during a period of virologic failure in a treatment-naive man who initiated tenofovir disoproxil fumarate/emtricitabine plus DTG