Human papillomavirus E2 regulates SRSF3 (SRp20) to promote capsid protein expression in infected differentiated keratinocytes

The human papillomavirus (HPV) life cycle is tightly linked to differentiation of the infected epithelial cell suggesting a sophisticated interplay between host cell metabolism and virus replication. Previously we demonstrated in differentiated keratinocytes in vitro and in vivo that HPV16 infection caused increased levels of the cellular SR splicing factors (SRSFs) SRSF1 (ASF/SF2), SRSF2 (SC35) and SRSF3 (SRp20). Moreover, the viral E2 transcription and replication factor that is expressed at high levels in differentiating keratinocytes could bind and control activity of the SRSF1 gene promoter. Here we reveal that E2 proteins of HPV16 and HPV31 control expression of SRSFs 1, 2 and 3 in a differentiation-dependent manner. E2 has the greatest trans-activation effect on expression of SRSF3. siRNA depletion experiments in two different models of the HPV16 life cycle (W12E and NIKS16) and one model of the HPV31 life cycle (CIN612-9E) revealed that only SRSF3 contributed significantly to regulation of late events in the virus life cycle. Increased levels of SRSF3 are required for L1 mRNA and capsid protein expression. Capsid protein expression was regulated specifically by SRSF3 and appeared independent of other SRSFs. Taken together these data suggest a significant role of the HPV E2 protein in regulating late events in the HPV life cycle through transcriptional regulation of SRSF3 expression. IMPORTANCE Human papillomavirus replication is accomplished in concert with differentiation of the infected epithelium. Virus capsid protein expression is confined to the upper epithelial layers so as to avoid immune detection. In this study we demonstrate that the viral E2 transcription factor activates the promoter of the cellular SRSF3 RNA processing factor. SRSF3 is required for expression of the E4̂L1 mRNA and so controls expression of the HPV L1 capsid protein. Thus we reveal a new dimension of virus-host interaction crucial for production of infectious virus. SRSF proteins are known drug targets. Therefore, this study provides an excellent basis for developing strategies to regulate capsid protein production in the infected epithelium and production of new virions

Metagenomic next-generation sequencing aids the diagnosis of viral infections in febrile returning travellers

Objectives Travel-associated infections are challenging to diagnose because of the broad spectrum of potential aetiologies. As a proof-of-principle study, we used MNGS to identify viral pathogens in clinical samples from returning travellers in a single center to explore its suitability as a diagnostic tool. Methods Plasma samples from 40 returning travellers presenting with a fever of ≥38°C were sequenced using MNGS on the Illumina MiSeq platform and compared with standard-of-care diagnostic assays. Results In total, 11/40 patients were diagnosed with a viral infection. Standard of care diagnostics revealed 5 viral infections using plasma samples; dengue virus 1 (n = 2), hepatitis E (n = 1), Ebola virus (n = 1) and hepatitis A (n = 1), all of which were detected by MNGS. Three additional patients with Chikungunya virus (n = 2) and mumps virus were diagnosed by MNGS only. Respiratory infections detected by nasal/throat swabs only were not detected by MNGS of plasma. One patient had infection with malaria and mumps virus during the same admission. Conclusions MNGS analysis of plasma samples improves the sensitivity of diagnosis of viral infections and has potential as an all-in-one diagnostic test. It can be used to identify infections that have not been considered by the treating physician, co-infections and new or emerging pathogens. Summary Next generation sequencing (NGS) has potential as an all-in-one diagnostic test. In this study we used NGS to diagnose returning travellers with acute febrile illness in the UK, highlighting cases where the diagnosis was missed using standard methods

Rituximab and obinutuzumab differentially hijack the B-cell receptor and NOTCH1 signaling pathways

The anti-CD20 monoclonal antibodies rituximab and obinutuzumab differ in their mechanisms of action, with obinutuzumab evoking greater direct B-cell death. To characterize the signaling processes responsible for improved B-cell killing by obinutuzumab, we undertook a phosphoproteomics approach and demonstrate that rituximab and obinutuzumab differentially activate pathways downstream of the B-cell receptor. While both antibodies induce strong ERK and MYC activation sufficient to promote cell cycle arrest and B-cell death, obinutuzumab exceeds rituximab in supporting apoptosis induction by means of aberrant SYK phosphorylation. In contrast, rituximab elicits stronger anti-apoptotic signals by activating AKT, impairing pro-apoptotic BAD, and by releasing membrane-bound NOTCH1 to up-regulate pro-survival target genes. As a consequence, rituximab appears to reinforce BCL2-mediated apoptosis resistance. The unexpected complexity and differences by which rituximab and obinutuzumab interfere with signaling pathways essential for lymphoma pathogenesis and treatment provide important impetus to optimize and personalize the application of different anti-CD20 treatments

Stromal cell inhibition of anti-CD20 antibody mediated killing of B-cell malignancies

Introduction: The glycoengineered type II anti-CD20 monoclonal antibody obinutuzumab has been licensed for treatment in follicular non-Hodgkin lymphoma and B-CLL following clinical trials demonstrating superior outcomes to standard of care treatment. However, ultimately many patients still relapse, highlighting the need to understand the mechanisms behind treatment failure to improve patient care. Resistance to chemotherapy is often caused by the ability of malignant B-cells to migrate to the bone marrow and home into the stromal layer. Therefore, this study aimed to investigate whether stromal cells were also able to inhibit type II anti-CD20 antibody mechanisms of action, contributing to resistance to therapy.Methods: A stromal-tumor co-culture was established in vitro between Raji or Daudi B-cell tumor cells and M210B4 stromal cells in 24 well plates.Results: Contact with stromal cells was able to protect tumor cells from obinutuzumab mediated programmed cell death (PCD), antibody dependent cellular phagocytosis and antibody dependent cellular cytotoxicity. Furthermore, such protection required direct contact between stroma and tumor cells. Stromal cells appeared to interfere with obinutuzumab mediated B-cell homotypic adhesion through inhibiting and reversing actin remodelling, potentially as a result of stromal-tumor cell contact leading to downregulation of CD20 on the surface of tumor cells. Further evidence for the potential role of CD20 downregulation comes through the reduction in surface CD20 expression and inhibition of obinutuzumab mediated PCD when tumor cells are treated with Ibrutinib in the presence of stromal cells. The proteomic analysis of tumor cells after contact with stromal cells led to the identification of a number of altered pathways including those involved in cell adhesion and the actin cytoskeleton and remodeling.Discussion: This work demonstrates that contact between tumor cells and stromal cells leads to inhibition of Obinutuzumab effector functions and has important implications for future therapies to improve outcomes to anti-CD20 antibodies. A deeper understanding of how anti-CD20 antibodies interact with stromal cells could prove a useful tool to define better strategies to target the micro-environment and ultimately improve patient outcomes in B-cell malignancies

Human papillomavirus gene expression is controlled by host cell splicing factors

Human papillomaviruses (HPV) infect stratified epithelia and cause a variety of lesions ranging from benign warts to invasive tumours. The virus life cycle is tightly linked to differentiation of the keratinocyte it infects: papillomaviruses modulate host gene expression to ensure efficient virus replication. For example, the viral transcription factor E2 can directly upregulate, in an epithelial differentiation-dependent manner, cellular SR (serine/arginine-rich) splicing factors that control constitutive and alternative splicing. Changes in alternative splicing and the mechanisms controlling this for viral mRNAs have been a subject to intense exploration. However, to date experiments have only been carried out in model systems because the genetic systems suitable for studying alternative splicing of viral RNAs in the context of the virus life cycle are relatively recent and technically challenging. Now using these life cycle-supporting systems, our laboratory has identified SR proteins as important players in differentiation-dependent regulation of HPV gene expression. Better understanding of the role of cellular factors in regulating the virus life cycle is needed as it may help development of novel diagnostic approaches and antiviral therapies in the future

Pcl-PRC2 is needed to generate high levels of H3-K27 trimethylation at Polycomb target genes

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-, di- and trimethylates H3-K27 in nucleosomes in vitro. Analysis of Drosophila mutants that lack Pcl unexpectedly reveals that Pcl-PRC2 is required to generate high levels of H3-K27 trimethylation at Polycomb target genes but is dispensable for the genome-wide H3-K27 mono- and dimethylation that is generated by PRC2. In Pcl mutants, Polycomb target genes become derepressed even though H3-K27 trimethylation at these genes is only reduced and not abolished, and even though targeting of the Polycomb protein complexes PhoRC and PRC1 to Polycomb response elements is not affected. Pcl-PRC2 is thus the HMTase that generates the high levels of H3-K27 trimethylation in Polycomb target genes that are needed to maintain a Polycomb-repressed chromatin state

Multi-platform profiling characterizes molecular subgroups and resistance networks in chronic lymphocytic leukemia

Knowledge of the genomic landscape of chronic lymphocytic leukemia (CLL) grows increasingly detailed, providing challenges in contextualizing the accumulated information. To define the underlying networks, we here perform a multi-platform molecular characterization. We identify major subgroups characterized by genomic instability (GI) or activation of epithelial-mesenchymal-transition (EMT)-like programs, which subdivide into non-inflammatory and inflammatory subtypes. GI CLL exhibit disruption of genome integrity, DNA-damage response and are associated with mutagenesis mediated through activation-induced cytidine deaminase or defective mismatch repair. TP53 wild-type and mutated/deleted cases constitute a transcriptionally uniform entity in GI CLL and show similarly poor progression-free survival at relapse. EMT-like CLL exhibit high genomic stability, reduced benefit from the addition of rituximab and EMT-like differentiation is inhibited by induction of DNA damage. This work extends the perspective on CLL biology and risk categories in TP53 wild-type CLL. Furthermore, molecular targets identified within each subgroup provide opportunities for new treatment approaches

The novel Bcl-2 inhibitor ABT-737 is more effective in hypoxia and is able to reverse hypoxia-induced drug resistance in neuroblastoma cells

Neuroblastoma is a common solid tumour of childhood and advanced disease carries a poor prognosis despite intensive multi-modality therapy. Hypoxia is a common feature of solid tumours due to poorly organised tumour-induced neovasculature. Hypoxia is associated with advanced stage and poor outcome in a range of tumour types, and leads to resistance to clinically relevant cytotoxic agents in neuroblastoma and other paediatric tumours in vitro. Resistance to apoptosis is a common feature of tumour cells, and leads to pleiotropic drug resistance, mediated by Bcl-2 family proteins. ABT-737 is a novel small molecule inhibitor of Bcl-2 and Bcl-x(L) that is able to induce apoptosis in a range of tumour types. Neuroblastoma cell lines are relatively resistant to ABT-737-induced apoptosis in normoxia, but in contrast to the situation with conventional cytotoxic agents, are more sensitive in hypoxia. This sensitisation is due to an increase in ABT-737-induced apoptosis and is variably dependent upon the presence of functional HIF-1α. In contrast to the situation in colon carcinoma and non small cell lung cancer cells hypoxia does not result in down-regulation of the known ABT-737 resistance factor, Mcl-1, nor any other Bcl-2 family proteins. ABT-737 sensitises neuroblastoma cells to clinically relevant cytotoxic agents under normal levels of oxygen, and importantly this sensitisation is maintained under hypoxia, when neuroblastoma cells are resistant to these agents. Thus rational combinations of ABT-737 and conventional cytotoxics offer a novel approach to overcoming hypoxia-induced drug resistance in neuroblastoma