Various species of the intestinal microbiota have been associated with the development of colorectal cancer
, but it has not been demonstrated that bacteria have a direct role in the occurrence of ...oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin
. This compound is believed to alkylate DNA on adenine residues
and induces double-strand breaks in cultured cells
. Here we expose human intestinal organoids to genotoxic pks
E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.
SARS-CoV-2, a positive-sense RNA virus in the family Coronaviridae, has caused a worldwide pandemic of coronavirus disease 2019 or COVID-19. Coronaviruses generate a tiered series of subgenomic RNAs ...(sgRNAs) through a process involving homology between transcriptional regulatory sequences (TRS) located after the leader sequence in the 5' UTR (the TRS-L) and TRS located near the start of ORFs encoding structural and accessory proteins (TRS-B) near the 3' end of the genome. In addition to the canonical sgRNAs generated by SARS-CoV-2, non-canonical sgRNAs (nc-sgRNAs) have been reported. However, the consistency of these nc-sgRNAs across viral isolates and infection conditions is unknown. The comprehensive definition of SARS-CoV-2 RNA products is a key step in understanding SARS-CoV-2 pathogenesis.
Here, we report an integrative analysis of eight independent SARS-CoV-2 transcriptomes generated using three sequencing strategies, five host systems, and seven viral isolates. Read-mapping to the SARS-CoV-2 genome was used to determine the 5' and 3' coordinates of all junctions in viral RNAs identified in these samples.
Using junctional abundances, we show nc-sgRNAs make up as much as 33% of total sgRNAs in cell culture models of infection, are largely consistent in abundance across independent transcriptomes, and increase in abundance over time during infection. By assessing the homology between sequences flanking the 5' and 3' junction points, we show that nc-sgRNAs are not associated with TRS-like homology. By incorporating read coverage information, we find strong evidence for subgenomic RNAs that contain only 5' regions of ORF1a. Finally, we show that non-canonical junctions change the landscape of viral open reading frames.
We identify canonical and non-canonical junctions in SARS-CoV-2 sgRNAs and show that these RNA products are consistently generated by many independent viral isolates and sequencing approaches. These analyses highlight the diverse transcriptional activity of SARS-CoV-2 and offer important insights into SARS-CoV-2 biology.
Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus-host interactions. CRISPR systems enable focused or systematic, ...genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR-Cas9 for the loss-of-function analysis of host dependency factors. We focus on the use of CRISPR-pooled screens for the systematic identification of host dependency factors, particularly in Epstein-Barr virus-transformed B cells. We also discuss the use of CRISPR interference (CRISPRi) and gain-of-function CRISPR activation (CRISPRa) approaches to probe virus-host interactions. Finally, we comment on the future directions enabled by combinatorial CRISPR screens.
Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B cell lymphomas. How EBV remodels metabolic pathways to support rapid B cell outgrowth remains largely unknown. To gain ...insights, primary human B cells were profiled by tandem-mass-tag-based proteomics at rest and at nine time points after infection; >8,000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production, and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism.
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•Global analysis of EBV proteome and metabolism remodeling in B cell transformation•EBV induces mitochondrial 1C metabolic enzyme expression and flux•EBNA2 is a viral master regulator of B cell metabolic reprogramming•1C drives nucleotide, mitochondrial NADPH, and glutathione production
Global unbiased proteomic analysis reveals key metabolic pathways induced by Epstein-Barr virus critical for B cell growth transformation. In this issue of Cell Metabolism, Wang et al. utilized multiplexed proteomics to identify key virus-induced metabolic pathways important for outgrowth of newly infected primary human B cells. The authors describe virus-activated mitochondrial one-carbon metabolism as being crucial for nucleotide and glutathione syntheses, as well as generation of intramitochondrial NADPH.
Polyomaviruses (PyV) are ubiquitous pathogens that can cause devastating human diseases. Due to the small size of their genomes, PyV utilize complex patterns of RNA splicing to maximize their coding ...capacity. Despite the importance of PyV to human disease, their transcriptome architecture is poorly characterized. Here, we compare short- and long-read RNA sequencing data from eight human and non-human PyV. We provide a detailed transcriptome atlas for BK polyomavirus (BKPyV), an important human pathogen, and the prototype PyV, simian virus 40 (SV40). We identify pervasive wraparound transcription in PyV, wherein transcription runs through the polyA site and circles the genome multiple times. Comparative analyses identify novel, conserved transcripts that increase PyV coding capacity. One of these conserved transcripts encodes superT, a T antigen containing two RB-binding LxCxE motifs. We find that superT-encoding transcripts are abundant in PyV-associated human cancers. Together, we show that comparative transcriptomic approaches can greatly expand known transcript and coding capacity in one of the simplest and most well-studied viral families.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced ...by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication.
Measles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus.
Merkel cell carcinoma (MCC) is a highly aggressive neuroendocrine carcinoma of the skin caused by either the integration of Merkel cell polyomavirus (MCPyV) and expression of viral T antigens or by ...ultraviolet-induced damage to the tumor genome from excessive sunlight exposure. An increasing number of deep sequencing studies of MCC have identified significant differences between the number and types of point mutations, copy number alterations, and structural variants between virus-positive and virus-negative tumors. However, it has been challenging to reliably distinguish between virus positive and UV damaged MCC.
In this study, we assembled a cohort of 71 MCC patients and performed deep sequencing with OncoPanel, a clinically implemented, next-generation sequencing assay targeting over 400 cancer-associated genes. To improve the accuracy and sensitivity for virus detection compared to traditional PCR and IHC methods, we developed a hybrid capture baitset against the entire MCPyV genome and software to detect integration sites and structure.
Sequencing from this approach revealed distinct integration junctions in the tumor genome and generated assemblies that strongly support a model of microhomology-initiated hybrid, virus-host, circular DNA intermediate that promotes focal amplification of host and viral DNA. Using the clear delineation between virus-positive and virus-negative tumors from this method, we identified recurrent somatic alterations common across MCC and alterations specific to each class of tumor, associated with differences in overall survival. Finally, comparing the molecular and clinical data from these patients revealed a surprising association of immunosuppression with virus-negative MCC and significantly shortened overall survival.
These results demonstrate the value of high-confidence virus detection for identifying molecular mechanisms of UV and viral oncogenesis in MCC. Furthermore, integrating these data with clinical data revealed features that could impact patient outcome and improve our understanding of MCC risk factors.
Cancers avoid immune surveillance through an array of mechanisms, including perturbation of HLA class I antigen presentation. Merkel cell carcinoma (MCC) is an aggressive, HLA-I-low, neuroendocrine ...carcinoma of the skin often caused by the Merkel cell polyomavirus (MCPyV). Through the characterization of 11 newly generated MCC patient-derived cell lines, we identified transcriptional suppression of several class I antigen presentation genes. To systematically identify regulators of HLA-I loss in MCC, we performed parallel, genome-scale, gain- and loss-of-function screens in a patient-derived MCPyV-positive cell line and identified MYCL and the non-canonical Polycomb repressive complex 1.1 (PRC1.1) as HLA-I repressors. We observed physical interaction of MYCL with the MCPyV small T viral antigen, supporting a mechanism of virally mediated HLA-I suppression. We further identify the PRC1.1 component USP7 as a pharmacologic target to restore HLA-I expression in MCC.
Objectives
Kikuchi-Fujimoto disease (KFD) is a self-limited lymphadenitis of unclear etiology. We aimed to further characterize this disease in pediatric patients, including evaluation of the CD123 ...immunohistochemical (IHC) staining and investigation of potential immunologic and infectious causes.
Methods
Seventeen KFD cases and 12 controls were retrospectively identified, and the histologic and clinical features were evaluated. CD123 IHC staining was quantified by digital image analysis. Next generation sequencing was employed for comparative microbial analysis via RNAseq (5 KFD cases) and to evaluate the immune repertoire (9 KFD cases).
Results
In cases of lymphadenitis with necrosis, >0.85% CD123+ cells by IHC was found to be six times more likely in cases with a final diagnosis of KFD (sensitivity 75%, specificity 87.5%). RNAseq based comparative microbial analysis did not detect novel or known pathogen sequences in KFD. A shared complementarity determining region 3 (CDR3) sequence and use of the same T-cell receptor beta variable region family was identified in KFD LNs but not controls, and was not identified in available databases.
Conclusions:
Digital quantification of CD123 IHC can distinguish KFD from other necrotizing lymphadenitides. The presence of a unique shared CDR3 sequence suggests that a shared antigen underlies KFD pathogenesis.
Systemic anaplastic large cell lymphoma (ALCL) is a rare CD30-expressing T-cell non-Hodgkin lymphoma. Risk of systemic ALCL is highly increased among immunosuppressed individuals. Because risk of ...cancers associated with viruses is increased with immunosuppression, we conducted a metagenomic analysis of systemic ALCL to determine whether a known or novel pathogen is associated with this malignancy. Total RNA was extracted and sequenced from formalin-fixed paraffin-embedded tumor specimens from 19 systemic ALCL cases (including one case from an immunosuppressed individual with human immunodeficiency virus infection), 3 Epstein-Barr virus positive diffuse large B-cell lymphomas (DLBCLs) occurring in solid organ transplant recipients (positive controls), and 3 breast cancers (negative controls). We used a pipeline based on the Genome Analysis Toolkit (GATK)-PathSeq algorithm to subtract out human RNA reads and map the remaining RNA reads to microbes. No microbial association with ALCL was identified, but we found Epstein-Barr virus in the DLBCL positive controls and determined the breast cancers to be negative. In conclusion, we did not find a pathogen associated with systemic ALCL, but because we analyzed only one ALCL tumor from an immunosuppressed person, we cannot exclude the possibility that a pathogen is associated with some cases that arise in the setting of immunosuppression.
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