Type I interferon (IFN-α/β) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN-α/β in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis following inoculation of the live-attenuated measles, mumps and rubella (MMR) vaccine. By targeted resequencing we identified a homozygous mutation in the high-affinity * Correspondence to: christopher.duncan@ncl.ac.uk or sophie.hambleton@ncl.ac.uk.

Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.

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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. IMPORTANCEHuman 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. H uman respiratory syncytial virus (HRSV) is the leading cause of severe respiratory tract infections in newborn children worldwide (1). It infects almost all infants within the first 2 years of life and is the main cause of infant bronchiolitis. Currently, only ribav...

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.