Interactome Analysis Of The Human Respiratory Syncytial Virus Rna Polymerase Complex Identifies Protein Chaperones As Important Cofactors That Promote L-protein Stability And Rna Synthesis

The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L-and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L-or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.892917930Medical Research Council (MRC) [MR/K000276/1]MRC studentshipNIHRNIH [R01AI074903

Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach

Human respiratory syncytial virus (HRSV) is a major cause of serious respiratory tract infection. Treatment options include administration of ribavirin, a purine analog, although the mechanism of its anti-HRSV activity is unknown. We used transcriptome sequencing (RNA-seq) to investigate the genome mutation frequency and viral mRNA accumulation in HRSV-infected cells that were left untreated or treated with ribavirin. In the absence of ribavirin, HRSV-specific transcripts accounted for up to one-third of total RNA reads from the infected-cell RNA population. Ribavirin treatment resulted in a>90% reduction in abundance of viral mRNA reads, while at the same time no such reduction was detected for the abundance of cellular transcripts. The presented data reveal that ribavirin significantly increases the frequency of HRSV-specific RNA mutations, suggesting a direct influence on the fidelity of the HRSV polymerase. The presented data show that transitions and transversions occur during HRSV replication and that these changes occur in hot spots along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, our data indicate that in the continuous cell types used and at the time points analyzed, the abundances of some HRSV mRNAs do not reflect the order in which the mRNAs are transcribed

In the dedicated pursuit of dedicated capital: restoring an indigenous investment ethic to British capitalism

Tony Blair’s landslide electoral victory on May 1 (New Labour Day?) presents the party in power with a rare, perhaps even unprecedented, opportunity to revitalise and modernise Britain’s ailing and antiquated manufacturing economy.* If it is to do so, it must remain true to its long-standing (indeed, historic) commitment to restore an indigenous investment ethic to British capitalism. In this paper we argue that this in turn requires that the party reject the very neo-liberal orthodoxies which it offered to the electorate as evidence of its competence, moderation and ‘modernisation’, which is has internalised, and which it apparently now views as circumscribing the parameters of the politically and economically possible

Developing a computerised search to help UK General Practices identify more patients for palliative care planning:a feasibility study

We would like to thank all practices, patients and their carers who helped us successfully conduct this project. We are grateful for advice from Libby Morris, the eHealth Clinical Lead for NHS Scotland and a GP in Lothian. The project was funded by Marie Curie Cancer Care (ref A13575).Peer reviewedPublisher PD

Werewolf, there wolf : Variants in hairless associated with hypotrichia and roaning in the lykoi cat breed

Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland.A variety of cat breeds have been developed via novelty selection on aesthetic, dermatological traits, such as coat colors and fur types. A recently developed breed, the lykoi (a.k.a. werewolf cat), was bred from cats with a sparse hair coat with roaning, implying full color and all white hairs. The lykoi phenotype is a form of hypotrichia, presenting as a significant reduction in the average numbers of follicles per hair follicle group as compared to domestic shorthair cats, a mild to severe perifollicular to mural lymphocytic infiltration in 77% of observed hair follicle groups, and the follicles are often miniaturized, dilated, and dysplastic. Whole genome sequencing was conducted on a single lykoi cat that was a cross between two independently ascertained lineages. Comparison to the 99 Lives dataset of 194 non‐lykoi cats suggested two variants in the cat homolog for Hairless (HR) (HR lysine demethylase and nuclear receptor corepressor) as candidate causal gene variants. The lykoi cat was a compound heterozygote for two loss of function variants in HR, an exon 3 c.1255_1256dupGT (chrB1:36040783), which should produce a stop codon at amino acid 420 (p.Gln420Serfs*100) and, an exon 18 c.3389insGACA (chrB1:36051555), which should produce a stop codon at amino acid position 1130 (p.Ser1130Argfs*29). Ascertainment of 14 additional cats from founder lineages from Canada, France and different areas of the USA identified four additional loss of function HR variants likely causing the highly similar phenotypic hair coat across the diverse cats. The novel variants in HR for cat hypotrichia can now be established between minor differences in the phenotypic presentations.Peer reviewe

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Abstracts from the NIHR INVOLVE Conference 2017

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