Inhibition of IKKα by BAY61-3606 Reveals IKKα-Dependent Histone H3 Phosphorylation in Human Cytomegalovirus Infected Cells

Protein kinase inhibitors can be used as tools to identify proteins and pathways required for virus replication. Using virus replication assays and western blotting we found that the widely used protein kinase inhibitor BAY61-3606 inhibits replication of human cytomegalovirus (HCMV) strain AD169 and the accumulation of HCMV immediate-early proteins in AD169 infected cells, but has no effect on replication of HCMV strain Merlin. Using in vitro kinase assays we found that BAY61-3606 is a potent inhibitor of the cellular kinase IKKα. Infection of cells treated with siRNA targeting IKKα indicated IKKα was required for efficient AD169 replication and immediate-early protein production. We hypothesized that IKKα was required for AD169 immediate-early protein production as part of the canonical NF-κB signaling pathway. However, although BAY61-3606 inhibited phosphorylation of the IKKα substrate IκBα, we found no canonical or non-canonical NF-κB signaling in AD169 infected cells. Rather, we observed that treatment of cells with BAY61-3606 or siRNA targeting IKKα decreased phosphorylation of histone H3 at serine 10 (H3S10p) in western blotting assays. Furthermore, we found treatment of cells with BAY61-3606, but not siRNA targeting IKKα, inhibited the accumulation of histone H3 acetylation (H3K9ac, H3K18ac and H3K27ac) and tri-methylation (H3K27me3 and H3K36me3) modifications. Therefore, the requirement for IKKα in HCMV replication was strain-dependent and during replication of an HCMV strain requiring IKKα, IKKα-dependent H3S10 phosphorylation was associated with efficient HCMV replication and immediate-early protein production. Plus, inhibition of HCMV replication by BAY61-3606 is associated with acetylation and tri-methylation modifications of histone H3 that do not involve IKKα

Deep splicing plasticity of the human adenovirus type 5 transcriptome as a driver of virus evolution

Viral genomes are characterised by having high gene density and complex transcription strategies. One of the most complex is adenovirus which has a double stranded DNA genome and is the archetypal viral system in which splicing was first discovered. Understanding the transcriptional landscape using conventional mRNA cloning or more recent Illumina-based deep sequencing methods offers insight but also has limitations, including the potential for reverse transcription or PCR amplification artefacts and bias. Here we used direct RNA long read length sequencing on an Oxford Nanopore MinION device to gain a quantitative system-wide overview of transcription and splicing as it dynamically changes during a human adenovirus type 5 infection. This global overview revealed an extensive and hitherto unappreciated complexity of alternative splicing and secondary initiating codon usage. Allied to this, analysis of viral polyadenylation patterns over time showed that most viral transcripts tended to shorter polyadenylation lengths as the infection progressed. Moreover, development and use of an ORF-centric bioinformatics pipeline for analysis of sequenced mRNA, provided both a quantitative and deeper qualitative understanding of the genetic potential of this virus. The data strikingly illustrated that across the viral genome adenovirus made multiple distinctly spliced transcripts that coded for the same ORF. Indeed, as many as 11,000 different splicing patterns were recorded across the viral genome over the three time points analysed. This constitutive low level use of alternative splicing patterns and secondary ORFs potentially enables the virus to maximise its coding potential over evolutionary timescales.</jats:p

A molnupiravir-associated mutational signature in global SARS-CoV-2 genomes

Acknowledgements: We thank all data contributors, that is, the authors and their originating laboratories responsible for obtaining the specimens, and their submitting laboratories for generating the genetic sequence and metadata and sharing via the GISAID initiative, on which this research is based. We also thank everyone who contributed to the generation of the genomes deposited in the INSDC databases, on which this research is also based. We thank A. Hinrichs and colleagues for access to an UShER mutation-annotated tree built with all available genomic data. We thank NHS England for providing the Blueteq data on treatment records. We thank the UKHSA COVID-19 Therapeutics Programme Team past and present, in particular J. Charlesworth, A. Lackenby, A. Demirjian, M. Chand and C. Brown. We thank J. Bloom, M. Lin, R. Neher, K. Harris and F. Débarre for useful discussions. T.S. was supported by the Wellcome Trust (no. 210918/Z/18/Z) and the Francis Crick Institute, which receives its core funding from Cancer Research UK (no. FC001043), the UK Medical Research Council (MRC) (no. FC001043) and the Wellcome Trust (no. FC001043). This research was funded in whole, or in part, by the Wellcome Trust (nos. 210918/Z/18/Z, FC001043). For the purpose of open access, the authors have applied a CC-BY public copyright licence to any author-accepted manuscript resulting from this article. I.D.-B. is supported by PhD funding from the National Institute for Health and Care Research (NIHR) Health Protection Research Unit in Emerging and Zoonotic Infections at the University of Liverpool in partnership with Public Health England (now UKHSA), in collaboration with the Liverpool School of Tropical Medicine and the University of Oxford (award no. 200907). The views expressed are those of the authors and not necessarily those of the Department of Health and Social Care or NIHR. Neither the funders nor the trial sponsor were involved in study design, data collection, analysis, interpretation or preparation of the manuscript. T.P.P. was funded by the G2P-UK National Virology Consortium, which is funded by the MRC (no. MR/W005611/1). C.R. was supported by a Fondation Botnar Research Award (programme grant no. 6063), the UK Cystic Fibrosis Trust (Innovation Hub Award 001) and funding from the Oxford Martin School.Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load1, 2. However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir

Investigation of histone H3 modifications in BAY61-3606 and siRNA treated cells.

<p>(A, C and D) Western blotting of HFF cells treated with BAY61-3606. HFF cells were uninfected or infected with AD169 at an MOI of 1, then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO. Cell lysates were prepared for western blotting at the time points (hours post infection (h.p.i.)) indicated above the figure. Uninfected cells harvested at the time of infection are shown as 0 h.p.i.. (B) Western blotting of HFF cells treated with siRNA. HFF cells were treated with either Crtl or IKKα siRNA. After 72 hours incubation cells infected with 1x10⁵ p.f.u. of AD169 and then prepared for western blotting at 72 h.p.i.. The siRNA used is indicated above the figure. In each figure proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure.</p

Investigation of canonical and non-canonical NF-κB signaling.

<p>(A) Canonical NF-κB signaling in uninfected HFF cells treated with TNF-α. Lysates of HFF cells prepared for western blotting after pre-treatment with either 1μM BAY61-3606 or the equivalent volume of DMSO and then treated with 10 ng/ml TNF-α for 5 mins. The number of hours pre-treatment (h.pre.t.) with DMSO or BAY61-3606 is indicated above the Fig 4A. Where cells were simultaneously treated with TNF-α and either DMSO or BAY61-3606 is indicated as 0 h.p.t. (B) Canonical NF-κB signaling in uninfected and infected HFF cells. HFF cells were uninfected or infected with AD169 at an MOI of 1, then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO. Cell lysates were prepared for western blotting at the time points (hours post infection (h.p.i.)) indicated above the figure. Uninfected cells harvested at the time of infection are shown as 0 h.p.i.. (C) Analysis of IκBα degradation at early time points. HFF cells were uninfected (0 h.p.i.) (lane 1), uninfected and treated with 10 ng/ml TNF-α for 5 minutes (lane 2), or infected with AD169 at an MOI of 1, then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO (lanes 6–8 and 3–5, respectively). Cell lysates were prepared for western blotting at the time points (hours post infection (h.p.i.)) indicated above the figure. (D) Analysis of cycloheximide treatment on IκBα degradation. HFF cells were infected with AD169 at an MOI of 1 then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO for 72 hours. At 72 h.p.i. cell lysates were prepared for western blotting after treatment with 100 μg/ml cycloheximide for the time points (hours post treatment (h.po.t.)) indicated above the figure. (E) Non-canonical NF-κB signaling in uninfected and infected HFF cells. HFF cells were uninfected or infected with AD169 at an MOI of 1, then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO. Cell lysates were prepared for western blotting at the time points (hours post infection (h.p.i.)) indicated above the figure. Uninfected cells harvested at the time of infection are shown as 0 h.p.i.. An equivalent volume of lysate from the EBV infected B cell line HB7 was also analyzed. Where indicated, uninfected cells harvested at the time of infection are shown as 0 h.p.i.. In each figure proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure.</p

Investigation of roles for SYK and GCK in HCMV infected cells.

<p>(A and B) Analysis of siRNA treated cells infected with AD169. HFF were treated with the siRNAs indicated in each figure for 72 hours and infected with 1x10⁵ plaque forming units (p.f.u.) of HCMV. At 96 h.p.i cell lysates were prepared for western blotting (Fig 2A) and viral supernatants were harvested for virus titration (Fig 2B). In Fig 2B viral titre is expressed as plaque forming units/ml (p.f.u./ml) and the mean and standard deviation of 3 experiments is shown. (C) Analysis of HCMV infected cells treated with inhibitors of GCK. HFF cells were infected with AD169 at an MOI of 1 then treated with 1μM of the drug indicated in the figure or the equivalent volume of DMSO. Viral supernatants were harvested at the indicated time points and viral titre (p.f.u./ml) at each time point was determined. (D and E) Western blotting of lysate from uninfected or infected cell lines. Unless stated otherwise, all lysates are from HFF cells. Where indicated, HFF cells were either uninfected or infected with AD169 at an MOI of 1 then treated with either 1μM BAY61-3606, 1μM Maribavir or the equivalent volume of DMSO. Cell lysates were prepared for western blotting at 72 hours post infection. In each panel showing western blotting proteins recognized by the antibodies used in each experiment are indicated to the right of each figure and the positions of molecular weight markers (kDa) are indicated to the left of each figure.</p

Analysis of H3 phosphorylation by IKKα.

<p>(A) Analysis of H3S10p in HCMV infected HFF cells treated with BAY61-3606. HFF cells were uninfected or infected with AD169 at an MOI of 1, then treated with either 1μM BAY61-3606 or the equivalent volume of DMSO. Cell lysates were prepared for western blotting at the time points (hours post infection (h.p.i.)) indicated above the figure. Uninfected cells harvested at the time of infection are shown as 0 h.p.i.. Short and a long exposures of the western blot to film to detect H3S10p are shown. (B) Analysis of H3S10p in infected cells treated with siRNA. HFF cells were treated with either Crtl or IKKα siRNA. After 72 hours incubation with siRNA cells were prepared for western blotting (0 h.p.i.) or infected with 1x10⁵ p.f.u. of AD169 and then prepared for western blotting at the time points (h.p.i.) indicated above the figure. The siRNA used are also indicated above the figure. In each figure proteins recognized by the antibodies used in each experiment are indicated to the right of each figure. The positions of molecular weight markers (kDa) are indicated to the left of each figure. In Fig 5B band intensities are expressed in arbitrary units below certain panels.</p

Identification of IKKα as a target of BAY61-3606.

<p>(A) Inhibition of AD169 replication by BAY compounds. HFF cells were infected with AD169 at an MOI of 1 then treated with 1μM of the drug indicated in the figure or the equivalent volume of DMSO. Viral supernatants were harvested at 96 h.p.i. and viral titre (p.f.u./ml) was determined. (B and C) In vitro kinase assays in the presence of BAY61-3606. The ability of 10 μM BAY61-3606 (Fig 3B) or a range of BAY61-3606 concentrations (Fig 3C) to inhibit the kinase activity of each of the indicated kinase proteins was assayed. Each data point in each figure represents the percentage kinase activity in the presence of drug compared to DMSO treated controls. Each data point shows the mean value of two experiments. (D-G) Analysis of HFF treated with siRNA. HFF cells were treated with either Crtl or IKKα siRNA. After 72 hours incubation with siRNA cell lysates were prepared for western blotting (Fig 3D) or infected with 1x10⁵ p.f.u. of HCMV to analyze virus replication by virus titration (Fig 3E). The data in Fig 3E is represented as p.f.u./ml at 96 h.p.i. and shows the mean and standard deviation of 3 experiments. Also, cell lysates from siRNA treated cells infected AD169 were prepared for western blotting 24 h.p.i. (Fig 3F). In Fig 3G, samples from lanes 1 and 5 of Fig 3E were diluted in a 2-fold series (lanes 2–4 and 6–8, respectively). The siRNA used in each case is indicated in each panel. In Fig 3D, 3F and 3G proteins recognized by the antibodies used in each experiment are indicated to the right of each figure and the positions of molecular weight markers (kDa) are indicated to the left of each figure. Band intensities are expressed in arbitrary units above or below each panel.</p

Amplicon and metagenomic analysis of MERS-CoV and the microbiome in patients with severe Middle East respiratory syndrome (MERS)

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a zoonotic infection that emerged in the Middle East in 2012. Symptoms range from mild to severe and include both respiratory and gastrointestinal illnesses. The virus is mainly present in camel populations with occasional spill overs into humans. The severity of infection in humans is influenced by numerous factors and similar to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underlying health complications can play a major role. Currently, MERS-CoV and SARS-CoV-2 are co-incident in the Middle East and a rapid way is required of sequencing MERS-CoV to derive genotype information for molecular epidemiology. Additionally, complicating factors in MERS-CoV infections are co-infections that require clinical management. The ability to rapidly characterise these infections would be advantageous. To rapidly sequence MERS-CoV, we developed an amplicon-based approach coupled to Oxford Nanopore long read length sequencing. The advantage of this approach is that insertions and deletions can be identified – which are the major drivers of genotype change in coronaviruses. This and a metagenomic approach were evaluated on clinical samples from patients with MERS. The data illustrated that whole genome or near whole genome information on MERS-CoV could be rapidly obtained. This approach provided data on both consensus genomes and the presence of minor variants including deletion mutants. Whereas, the metagenomic analysis provided information of the background microbiome