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

Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNγ-like response

MN and CP were supported by the Wellcome Trust (www.wellcome.ac.uk) Institutional Strategic Support Fund and CP was supported by the Deutsche Forschungsgemeinschaft (PA 815/2-1; www.dfg.de).The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication.Publisher PDFPeer reviewe

The contribution of hospital-acquired infections to the COVID-19 epidemic in England in the first half of 2020

Background: SARS-CoV-2 is known to transmit in hospital settings, but the contribution of infections acquired in hospitals to the epidemic at a national scale is unknown. Methods: We used comprehensive national English datasets to determine the number of COVID-19 patients with identified hospital-acquired infections (with symptom onset > 7 days after admission and before discharge) in acute English hospitals up to August 2020. As patients may leave the hospital prior to detection of infection or have rapid symptom onset, we combined measures of the length of stay and the incubation period distribution to estimate how many hospital-acquired infections may have been missed. We used simulations to estimate the total number (identified and unidentified) of symptomatic hospital-acquired infections, as well as infections due to onward community transmission from missed hospital-acquired infections, to 31st July 2020. Results: In our dataset of hospitalised COVID-19 patients in acute English hospitals with a recorded symptom onset date (n = 65,028), 7% were classified as hospital-acquired. We estimated that only 30% (range across weeks and 200 simulations: 20–41%) of symptomatic hospital-acquired infections would be identified, with up to 15% (mean, 95% range over 200 simulations: 14.1–15.8%) of cases currently classified as community-acquired COVID-19 potentially linked to hospital transmission. We estimated that 26,600 (25,900 to 27,700) individuals acquired a symptomatic SARS-CoV-2 infection in an acute Trust in England before 31st July 2020, resulting in 15,900 (15,200–16,400) or 20.1% (19.2–20.7%) of all identified hospitalised COVID-19 cases. Conclusions: Transmission of SARS-CoV-2 to hospitalised patients likely caused approximately a fifth of identified cases of hospitalised COVID-19 in the “first wave” in England, but less than 1% of all infections in England. Using time to symptom onset from admission for inpatients as a detection method likely misses a substantial proportion (> 60%) of hospital-acquired infections

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

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Residues within IE1 region 410–445 are required for targeting of STAT3 and down-regulation of STAT3-responsive genes.

<p>(A) TetR cells without (w/o) or with inducible expression of the indicated HA-IE1 proteins were treated with dox for 48 h. During the final 24 h of dox treatment, cells were kept in medium with 0.5% FBS. Subcellular localization of endogenous STAT3α in IE1 expressing cells was analyzed by indirect immunofluorescence microscopy. Samples were simultaneously reacted with a rabbit monoclonal antibody to STAT3α and a mouse monoclonal antibody to HA-tagged IE1, followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate and a mouse-specific Alexa Fluor 488 conjugate. Host cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Additionally, merge images of STAT3α, IE1 and DAPI signals are presented. (B) The percentage of cells with i) predominantly nuclear STAT3α staining (N>C), ii) equally strong nuclear and cytoplasmic STAT3α staining (N = C) and iii) predominantly cytoplasmic STAT3α staining (C>N) was determined for 100 randomly selected cells per sample described in (A). (C) TetR cells without or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent (w/o) or IL6 plus IL6R (IL6/Rα) for 30 min. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting for histone H2B, STAT2, STAT3α and IE1. (D) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h. Whole cell extracts were prepared and used for immunoprecipitations (IPs) with anti-HA-agarose. Samples of lysates and immunoprecipitates were analyzed by immunoblotting for IE1 and STAT3α. (E) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with IL6 plus IL6R for 30 min. Samples were subjected to ChIP with rabbit polyclonal antibodies to STAT3 or normal rabbit IgG and primers specific for sequences in the SOCS3 promoter or coding region. The percentage of output versus input DNA is presented as the difference between STAT3 and normal IgG ChIPs. Means and standard deviations of two biological and two technical replicates are shown. (F) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT3 target genes CXCL12 and SOCS3. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).</p

Ingenuity analysis¹ of human genes repressed by IE1 and activated by STAT3, IL6 or OSM.

<p>Ingenuity analysis<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005748#t001fn001" target="_blank">¹</a> of human genes repressed by IE1 and activated by STAT3, IL6 or OSM.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Human genes repressed by IE1 are IL6- and OSM-responsive pSTAT3 target genes.

<p>(A) TetR (w/o) and TetR-IE1 (IE1) cells were treated with dox for 72 h. Relative mRNA levels were determined by RT-qPCR with primers specific for the C4A, CHL1, CXCL12, IFI16, RASL11A or SOCS3 genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown in comparison to TetR cells (set to 1). (B) TetR cells were treated with solvent or IL6 plus IL6R (IL6/Rα) for 6 h or 24 h. Relative mRNA levels in comparison to solvent-treated cells (set to 1) were determined by RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown. (C) TetR cells were treated with solvent or OSM for 24 h. Relative mRNA levels in comparison to solvent-treated cells (set to 1) were determined by RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown. (D) TetR cells were transfected with the indicated siRNA duplexes. Two and five days post transfection (p.t.), whole cell protein extracts were prepared and subjected to immunoblotting for STAT3α and GAPDH (left panel). Five days post transfection, relative mRNA levels were determined by RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to control siRNA-transfected cells (set to 1) (right panel). (E) Whole cell protein extracts from TetR cells without (w/o) or with stable expression of the indicated STAT3α-Myc proteins were subjected to immunoblotting for STAT3 (Myc tag), TUBA, pSTAT3 (Y705) and pSTAT3 (S727) (left panel). Total RNA samples from TetR cells overexpressing either wild-type STAT3α-Myc or STAT3α_Y705F-Myc were subjected to RT-qPCR with primers specific for the indicated genes. Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown in comparison to cells expressing wild-type STAT3α-Myc (set to 1) (right panel).</p