Publications by authors named "Paul Cantalupo"

25 Publications

  • Page 1 of 1

Global Genome Demethylation Causes Transcription-Associated DNA Double Strand Breaks in HPV-Associated Head and Neck Cancer Cells.

Cancers (Basel) 2020 Dec 23;13(1). Epub 2020 Dec 23.

Department of Otolaryngology/Head and Neck Surgery, The University of North Carolina, Chapel Hill, NC 27599-7070, USA.

High levels of DNA methylation at CpG loci are associated with transcriptional repression of tumor suppressor genes and dysregulation of DNA repair genes. Human papilloma virus (HPV)-associated head and neck squamous cell carcinomas (HNSCC) have high levels of DNA methylation and methylation has been associated with dampening of an innate immune response in virally infected cells. We have been exploring demethylation as a potential treatment in HPV+ HNSCC and recently reported results of a window clinical trial showing that HNSCCs are particularly sensitive to demethylating agent 5-azacytidine (5-aza). Mechanistically, sensitivity is partially due to downregulation of HPV genes expression and restoration of tumor suppressors p53 and Rb. Here, for the first time, we show that 5-azaC treatment of HPV+ HNSCC induces replication and transcription-associated DNA double strand breaks (DSBs) that occur preferentially at demethylated genomic DNA. Blocking replication or transcription prevented formation of DNA DSBs and reduced sensitivity of HPV-positive head and neck cancer cells to 5-azaC, demonstrating that both replication and active transcription are required for formation of DSBs associated with 5-azaC.
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http://dx.doi.org/10.3390/cancers13010021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7793113PMC
December 2020

Single-Cell Transcriptomics Reveals a Heterogeneous Cellular Response to BK Virus Infection.

J Virol 2021 Feb 24;95(6). Epub 2021 Feb 24.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

BK virus (BKV) is a human polyomavirus that is generally harmless but can cause devastating disease in immunosuppressed individuals. BKV infection of renal cells is a common problem for kidney transplant patients undergoing immunosuppressive therapy. In cultured primary human renal proximal tubule epithelial (RPTE) cells, BKV undergoes a productive infection. The BKV-encoded large T antigen (LT) induces cell cycle entry, resulting in the upregulation of numerous genes associated with cell proliferation. Consistently, microarray and transcriptome sequencing (RNA-seq) experiments performed on bulk infected cell populations identified several proliferation-related pathways that are upregulated by BKV. These studies revealed few genes that are downregulated. In this study, we analyzed viral and cellular transcripts in single mock- or BKV-infected cells. We found that the levels of viral mRNAs vary widely among infected cells, resulting in different levels of LT and viral capsid protein expression. Cells expressing the highest levels of viral transcripts account for approximately 20% of the culture and have a gene expression pattern that is distinct from that of cells expressing lower levels of viral mRNAs. Surprisingly, cells expressing low levels of viral mRNA do not progress with time to high expression, suggesting that the two cellular responses are determined prior to or shortly following infection. Finally, comparison of cellular gene expression patterns of cells expressing high levels of viral mRNA with those of mock-infected cells or cells expressing low levels of viral mRNA revealed previously unidentified pathways that are downregulated by BKV. Among these are pathways associated with drug metabolism and detoxification, tumor necrosis factor (TNF) signaling, energy metabolism, and translation. The outcome of viral infection is determined by the ability of the virus to redirect cellular systems toward progeny production countered by the ability of the cell to block these viral actions. Thus, an infected culture consists of thousands of cells, each fighting its own individual battle. Bulk measurements, such as PCR or RNA-seq, measure the average of these individual responses to infection. Single-cell transcriptomics provides a window to the one-on-one battle between BKV and each cell. Our studies reveal that only a minority of infected cells are overwhelmed by the virus and produce large amounts of BKV mRNAs and proteins, while the infection appears to be restricted in the remaining cells. Correlation of viral transcript levels with cellular gene expression patterns reveals pathways manipulated by BKV that may play a role in limiting infection.
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http://dx.doi.org/10.1128/JVI.02237-20DOI Listing
February 2021

Analysis of viruses present in urine from patients with interstitial cystitis.

Virus Genes 2020 Aug 23;56(4):430-438. Epub 2020 May 23.

Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI, USA.

The question of whether some cases of interstitial cystitis may have an infectious etiology has been debated for some time. Previous studies have looked for the presence of certain specific viruses, but generally did not use the types of sensitive and unbiased approaches that are currently available. As part of the MAPP (Multidisciplinary Approach to the Study of Chronic Pelvic Pain) Research Network, we examined urine specimens from interstitial cystitis patients who provided specimens over time and also reported various symptoms at the time of urine collection. We first performed next-generation sequencing to look for the presence of viruses in urines, and detected two human polyomaviruses that are known to be excreted into urine, BKPyV and JCPyV. We were especially interested in BKPyV because it is a known cause of another bladder disease, hemorrhagic cystitis, in bone marrow transplant recipients. Further analysis of individual samples indicates a trend toward higher excretion of polyomaviruses in patients experiencing increased symptoms.
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http://dx.doi.org/10.1007/s11262-020-01767-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339973PMC
August 2020

Coding-Complete Genome Sequence of a Pollen-Associated Virus Belonging to the Family Recovered from a Japanese Apricot () Metagenome Data Set.

Microbiol Resour Announc 2019 Oct 3;8(40). Epub 2019 Oct 3.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

We report the coding-complete genome sequence of (JAPSV1), a virus belonging to the family, recovered from Japanese apricot () pollen that is closely related to (PLPAV). This discovery adds to the number of known pollen-associated viruses.
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http://dx.doi.org/10.1128/MRA.00881-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6776771PMC
October 2019

Detecting viral sequences in NGS data.

Curr Opin Virol 2019 12 26;39:41-48. Epub 2019 Aug 26.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA. Electronic address:

Next generation sequencing (NGS) technologies provide an increasingly important avenue for detecting known viruses, and for discovering novel viruses present in clinical or environmental samples. Several computational pipelines capable of identifying and classifying viral sequences in NGS data have been developed and used to search for viruses in human or animal samples, microbiomes, and in various environments. In this review we summarize the different approaches used to determine viral presence in sequence data. Strategies for avoiding confounding factors such as physical contamination and computational artifacts that lead to false virus identification are discussed. The application of these methodologies to cancer data sets has led to important insights on viruses both as drivers of and biomarkers for specific tumors.
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http://dx.doi.org/10.1016/j.coviro.2019.07.010DOI Listing
December 2019

Human polyomavirus BKV infection of endothelial cells results in interferon pathway induction and persistence.

PLoS Pathog 2019 01 8;15(1):e1007505. Epub 2019 Jan 8.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

Polyomavirus BKV is highly prevalent among humans. The virus establishes an asymptomatic persistent infection in the urinary system in healthy people, but uncontrolled productive infection of the virus in immunocompromised patients can lead to serious diseases. In spite of its high prevalence, our knowledge regarding key aspects of BKV polyomavirus infection remains incomplete. To determine tissue and cell type tropism of the virus, primary human epithelial cells, endothelial cells and fibroblasts isolated from the respiratory and urinary systems were tested. Results from this study demonstrated that all 9 different types of human cells were infectable by BKV polyomavirus but showed differential cellular responses. In microvascular endothelial cells from the lung and the bladder, BKV persistent infection led to prolonged viral protein expression, low yield of infectious progeny and delayed cell death, in contrast with infection in renal proximal tubular epithelial cells, a widely used cell culture model for studying productive infection of this virus. Transcriptomic profiling revealed the activation of interferon signaling and induction of multiple interferon stimulated genes in infected microvascular endothelial cells. Further investigation demonstrated production of IFNβ and secretion of chemokine CXCL10 by infected endothelial cells. Activation of IRF3 and STAT1 in infected endothelial cells was also confirmed. In contrast, renal proximal tubular epithelial cells failed to mount an interferon response and underwent progressive cell death. These results demonstrated that microvascular endothelial cells are able to activate interferon signaling in response to polyomavirus BKV infection. This raises the possibility that endothelial cells might provide initial immune defense against BKV infection. Our results shed light on the persistence of and immunity against infection by BKV polyomavirus.
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http://dx.doi.org/10.1371/journal.ppat.1007505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338385PMC
January 2019

Draft Genome Sequence of a Novel Rhabdovirus Isolated from Deinocerites Mosquitoes.

Genome Announc 2018 May 10;6(19). Epub 2018 May 10.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

We present a draft genome of a novel rhabdovirus, called Grenada mosquito rhabdovirus 1 (GMRV1), with homology to Wuhan mosquito virus 9 (WMV9) (NCBI reference sequence NC_031303), isolated from mosquitoes. The genome has a length of 14,420 nucleotides and encodes five open reading frames.
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http://dx.doi.org/10.1128/genomeA.01438-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946037PMC
May 2018

Complete Genome Sequence of Pittsburgh Sewage-Associated Virus 1.

Genome Announc 2018 Mar 15;6(11). Epub 2018 Mar 15.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

We present the complete genome sequence of a virus found in raw sewage collected in Pittsburgh, PA, USA. Pittsburgh sewage-associated virus 1 (PSAV1) encodes one large open reading frame with conserved domains typically found in the order of viruses. PSAV1 is closely related to virus 2 (BV2).
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http://dx.doi.org/10.1128/genomeA.01460-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854781PMC
March 2018

Identification of Head and Neck Cancer Subtypes Based on Human Papillomavirus Presence and E2F-Regulated Gene Expression.

mSphere 2018 Jan-Feb;3(1). Epub 2018 Jan 10.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Human papillomavirus (HPV) is present in a subset of head and neck squamous cell carcinomas (HNSCCs). The cell cycle regulatory Rb-E2F pathway is a major target of HPV and is perturbed by these viruses in cell culture and animal models, as well as in human tumors. In this study, we examined differences in the Rb-E2F pathway displayed by HPV-positive (HPV) and HPV-negative (HPV) HNSCC tumors. We created a computational approach that effectively categorizes gene expression as unchanged, downregulated, or upregulated by comparing the gene's mRNA levels in the tumor to the corresponding mRNA levels across normal tissue samples. Our findings suggest that there are three major HNSCC subtypes, defined by differences in the presence of HPV and in E2F-regulated gene expression. Most HPV HNSCC tumors show upregulation of E2F-regulated genes, which is consistent with inactivation of Rb by the virus-encoded E7 protein. In contrast, many HPV HNSCCs show little or no change in the Rb-E2F pathway. However, we also identified a set of tumors that show alterations in the Rb-E2F pathway in the absence of HPV. Thus, one class of HPV HNSCCs arise without significant alterations of the Rb-E2F pathway, while a second class of tumors appear to deregulate this pathway independently of the presence of HPV. Cancer is a complex disease that can be caused by a multitude of factors. HNSCC is complicated because some of these cancers are clearly associated with HPV, while others have no viral involvement. Determining the pathways that are commonly altered in both types of HNSCC, as well as those that are unique to viral and nonviral tumors, is important for a basic understanding of how these cancers arise and progress and critical to the development of targeted therapies. In this work, we show that all HPV-associated tumors have increased expression of E2F target genes, indicating that the tumor suppressor function of Rb is blocked. Importantly, Rb is also inhibited in a subset of nonviral tumors, suggesting that mutations present in these cancers mimic the action of the HPV E6 and E7 oncogenes.
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http://dx.doi.org/10.1128/mSphere.00580-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760753PMC
January 2018

Viral sequences in human cancer.

Virology 2018 01 5;513:208-216. Epub 2017 Nov 5.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA. Electronic address:

We have developed a virus detection and discovery computational pipeline, Pickaxe, and applied it to NGS databases provided by The Cancer Genome Atlas (TCGA). We analyzed a collection of whole genome (WGS), exome (WXS), and RNA (RNA-Seq) sequencing libraries from 3052 participants across 22 different cancers. NGS data from nearly all tumor and normal tissues examined contained contaminating viral sequences. Intensive computational and manual efforts are required to remove these artifacts. We found that several different types of cancers harbored Herpesviruses including EBV, CMV, HHV1, HHV2, HHV6 and HHV7. In addition to the reported associations of Hepatitis B and C virus (HBV & HCV) with liver cancer, and Human papillomaviruses (HPV) with cervical cancer and a subset of head and neck cancers, we found additional cases of HPV integrated in a small number of bladder cancers. Gene expression and mutational profiles suggest that HPV drives tumorigenesis in these cases.
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http://dx.doi.org/10.1016/j.virol.2017.10.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828528PMC
January 2018

Multi-modality analysis supports APOBEC as a major source of mutations in head and neck squamous cell carcinoma.

Oral Oncol 2017 11 13;74:8-14. Epub 2017 Sep 13.

Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, Chevy Chase, MD, 20815, USA.

Objectives: The mutagenic processes underlying head and neck squamous cell carcinoma (HNSCC) are poorly understood. Pan-cancer mutational signature analyses have identified a signature for APOBEC, a cytosine deaminase, in a subset of cancers, including HNSCC. The role of APOBEC activity in HNSCC remains poorly understood. Therefore, we sought to determine the role of APOBEC in HNSCC pathogenesis.

Material And Methods: Utilizing bioinformatic approaches we explored the role of APOBEC mediated mutations in tumor exomes, transcriptomes and germline exomes from 511HNSCC patients in the TCGA.

Results: 58% of HNSCC were statistically enriched for the APOBEC signature. APOBEC3A expression had the highest correlation coefficient with APOBEC mutation rate. Gene specific motif analysis revealed a slight predominance of APOBEC3A mutations. Canonical pathway analysis demonstrated immune pathway upregulation in APOBEC mutation rich samples. Overall mutational burden was positively correlated with APOBEC enrichment.

Conclusions: APOBEC mediated mutations are highly prevalent in HNSCC. APOBEC3A is the most likely gene to be active in HPV+ HNSCC. APOBEC activity correlates with upregulation of immune signaling pathways, supporting the hypothesis that APOBEC activity could be activated as part of the innate immune response.
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http://dx.doi.org/10.1016/j.oraloncology.2017.09.002DOI Listing
November 2017

Complete Genome Sequence of a Polyomavirus Recovered from a Pomona Leaf-Nosed Bat (Hipposideros pomona) Metagenome Data Set.

Genome Announc 2017 Jan 19;5(3). Epub 2017 Jan 19.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

We report here the complete genome sequence of a polyomavirus found in a nasal/rectal metagenome of Hipposideros pomona (Pomona leaf-nosed bat). Interestingly, the genetic organization and phylogenetic relationships of the new virus suggest greater similarity to recently discovered fish-associated polyomaviruses rather than to polyomavirus species previously observed in bats.
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http://dx.doi.org/10.1128/genomeA.01053-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5255915PMC
January 2017

Merkel Cell Polyomavirus Exhibits Dominant Control of the Tumor Genome and Transcriptome in Virus-Associated Merkel Cell Carcinoma.

mBio 2017 01 3;8(1). Epub 2017 Jan 3.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA

Merkel cell polyomavirus is the primary etiological agent of the aggressive skin cancer Merkel cell carcinoma (MCC). Recent studies have revealed that UV radiation is the primary mechanism for somatic mutagenesis in nonviral forms of MCC. Here, we analyze the whole transcriptomes and genomes of primary MCC tumors. Our study reveals that virus-associated tumors have minimally altered genomes compared to non-virus-associated tumors, which are dominated by UV-mediated mutations. Although virus-associated tumors contain relatively small mutation burdens, they exhibit a distinct mutation signature with observable transcriptionally biased kataegic events. In addition, viral integration sites overlap focal genome amplifications in virus-associated tumors, suggesting a potential mechanism for these events. Collectively, our studies indicate that Merkel cell polyomavirus is capable of hijacking cellular processes and driving tumorigenesis to the same severity as tens of thousands of somatic genome alterations.

Importance: A variety of mutagenic processes that shape the evolution of tumors are critical determinants of disease outcome. Here, we sequenced the entire genome of virus-positive and virus-negative primary Merkel cell carcinomas (MCCs), revealing distinct mutation spectra and corresponding expression profiles. Our studies highlight the strong effect that Merkel cell polyomavirus has on the divergent development of viral MCC compared to the somatic alterations that typically drive nonviral tumorigenesis. A more comprehensive understanding of the distinct mutagenic processes operative in viral and nonviral MCCs has implications for the effective treatment of these tumors.
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http://dx.doi.org/10.1128/mBio.02079-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210499PMC
January 2017

Transcriptome regulation and chromatin occupancy by E2F3 and MYC in mice.

Sci Data 2016 Feb 16;3:160008. Epub 2016 Feb 16.

Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine, Columbus, Ohio 43210, USA.

E2F3 and MYC are transcription factors that control cellular proliferation. To study their mechanism of action in the context of a regenerating tissue, we isolated both proliferating (crypts) and non-dividing (villi) cells from wild-type and Rb depleted small intestines of mice and performed ChIP-exo-seq (chromatin immunoprecipitation combined with lambda exonuclease digestion followed by high-throughput sequencing). The genome-wide chromatin occupancy of E2F3 and MYC was determined by mapping sequence reads to the genome and predicting preferred binding sites (peaks). Binding sites could be accurately identified within small regions of only 24 bp-28 bp long, highlighting the precision to which binding peaks can be identified by ChIP-exo-seq. Forty randomly selected E2F3- and MYC-specific binding sites were validated by ChIP-PCR. In addition, we also presented gene expression data sets from wild type, Rb-, E2f3- and Myc-depleted crypts and villi within this manuscript. These represent comprehensive and validated datasets that can be integrated to identify putative direct targets of E2F3 and MYC involved in the control of cellular proliferation in normal and Rb-deficient small intestines.
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http://dx.doi.org/10.1038/sdata.2016.8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755127PMC
February 2016

Whole-Genome Sequencing of a Single Viral Species from a Highly Heterogeneous Sample.

Angew Chem Int Ed Engl 2015 Nov 28;54(47):13985-8. Epub 2015 Aug 28.

Department of Physics, School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138 (USA).

Metagenomic studies suggest that only a small fraction of the viruses that exist in nature have been identified and studied. Characterization of unknown viral genomes is hindered by the many genomes populating any virus sample. A new method is reported that integrates drop-based microfluidics and computational analysis to enable the purification of any single viral species from a complex mixed virus sample and the retrieval of complete genome sequences. By using this platform, the genome sequence of a 5243 bp dsDNA virus that was spiked into wastewater was retrieved with greater than 96% sequence coverage and more than 99.8% sequence identity. This method holds great potential for virus discovery since it allows enrichment and sequencing of previously undescribed viruses as well as known viruses.
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http://dx.doi.org/10.1002/anie.201507047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715630PMC
November 2015

Artifact-Free Quantification and Sequencing of Rare Recombinant Viruses by Using Drop-Based Microfluidics.

Chembiochem 2015 Oct 7;16(15):2167-71. Epub 2015 Sep 7.

School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Pierce 231, Cambridge, MA, 02138, USA.

Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high-throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact-free estimate of in vitro recombination rate between murine norovirus strains MNV-1 and WU20 co-infecting a cell (P(rec) = 3.3 × 10(-4) ± 2 × 10(-5) ) for a 1205 nt region. Our approach represents a time- and cost-effective improvement over current methods, and can be adapted for genomic studies requiring artifact- and bias-free selective amplification, such as microbial pathogens, or rare cancer cells.
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http://dx.doi.org/10.1002/cbic.201500384DOI Listing
October 2015

Redeployment of Myc and E2f1-3 drives Rb-deficient cell cycles.

Nat Cell Biol 2015 Aug 20;17(8):1036-48. Epub 2015 Jul 20.

1] Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA [2] Department of Molecular Genetics, College of Biological Sciences, The Ohio State University, Columbus, Ohio 43210, USA [3] Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210, USA.

Robust mechanisms to control cell proliferation have evolved to maintain the integrity of organ architecture. Here, we investigated how two critical proliferative pathways, Myc and E2f, are integrated to control cell cycles in normal and Rb-deficient cells using a murine intestinal model. We show that Myc and E2f1-3 have little impact on normal G1-S transitions. Instead, they synergistically control an S-G2 transcriptional program required for normal cell divisions and maintaining crypt-villus integrity. Surprisingly, Rb deficiency results in the Myc-dependent accumulation of E2f3 protein and chromatin repositioning of both Myc and E2f3, leading to the 'super activation' of a G1-S transcriptional program, ectopic S phase entry and rampant cell proliferation. These findings reveal that Rb-deficient cells hijack and redeploy Myc and E2f3 from an S-G2 program essential for normal cell cycles to a G1-S program that re-engages ectopic cell cycles, exposing an unanticipated addiction of Rb-null cells on Myc.
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http://dx.doi.org/10.1038/ncb3210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526313PMC
August 2015

HeLa nucleic acid contamination in the cancer genome atlas leads to the misidentification of human papillomavirus 18.

J Virol 2015 Apr 28;89(8):4051-7. Epub 2015 Jan 28.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

Unlabelled: We searched The Cancer Genome Atlas (TCGA) database for viruses by comparing non-human reads present in transcriptome sequencing (RNA-Seq) and whole-exome sequencing (WXS) data to viral sequence databases. Human papillomavirus 18 (HPV18) is an etiologic agent of cervical cancer, and as expected, we found robust expression of HPV18 genes in cervical cancer samples. In agreement with previous studies, we also found HPV18 transcripts in non-cervical cancer samples, including those from the colon, rectum, and normal kidney. However, in each of these cases, HPV18 gene expression was low, and single-nucleotide variants and positions of genomic alignments matched the integrated portion of HPV18 present in HeLa cells. Chimeric reads that match a known virus-cell junction of HPV18 integrated in HeLa cells were also present in some samples. We hypothesize that HPV18 sequences in these non-cervical samples are due to nucleic acid contamination from HeLa cells. This finding highlights the problems that contamination presents in computational virus detection pipelines.

Importance: Viruses associated with cancer can be detected by searching tumor sequence databases. Several studies involving searches of the TCGA database have reported the presence of HPV18, a known cause of cervical cancer, in a small number of additional cancers, including those of the rectum, kidney, and colon. We have determined that the sequences related to HPV18 in non-cervical samples are due to nucleic acid contamination from HeLa cells. To our knowledge, this is the first report of the misidentification of viruses in next-generation sequencing data of tumors due to contamination with a cancer cell line. These results raise awareness of the difficulty of accurately identifying viruses in human sequence databases.
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http://dx.doi.org/10.1128/JVI.03365-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4442357PMC
April 2015

A retinoblastoma allele that is mutated at its common E2F interaction site inhibits cell proliferation in gene-targeted mice.

Mol Cell Biol 2014 Jun 24;34(11):2029-45. Epub 2014 Mar 24.

London Regional Cancer Program, Western University, London, Ontario, Canada.

The retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1(ΔG)) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1(ΔG/ΔG) mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1(ΔG/ΔG) MEFs display a magnitude of E2F target gene derepression similar to that of Rb1(-/-) cells, even though Rb1(ΔG/ΔG) cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1(ΔG/ΔG) MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G1 to arrest proliferation. Some Rb1(ΔG/ΔG) mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.
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http://dx.doi.org/10.1128/MCB.01589-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4019062PMC
June 2014

Raw sewage harbors diverse viral populations.

mBio 2011 4;2(5). Epub 2011 Oct 4.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Unlabelled: At this time, about 3,000 different viruses are recognized, but metagenomic studies suggest that these viruses are a small fraction of the viruses that exist in nature. We have explored viral diversity by deep sequencing nucleic acids obtained from virion populations enriched from raw sewage. We identified 234 known viruses, including 17 that infect humans. Plant, insect, and algal viruses as well as bacteriophages were also present. These viruses represented 26 taxonomic families and included viruses with single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), positive-sense ssRNA [ssRNA(+)], and dsRNA genomes. Novel viruses that could be placed in specific taxa represented 51 different families, making untreated wastewater the most diverse viral metagenome (genetic material recovered directly from environmental samples) examined thus far. However, the vast majority of sequence reads bore little or no sequence relation to known viruses and thus could not be placed into specific taxa. These results show that the vast majority of the viruses on Earth have not yet been characterized. Untreated wastewater provides a rich matrix for identifying novel viruses and for studying virus diversity.

Importance: At this time, virology is focused on the study of a relatively small number of viral species. Specific viruses are studied either because they are easily propagated in the laboratory or because they are associated with disease. The lack of knowledge of the size and characteristics of the viral universe and the diversity of viral genomes is a roadblock to understanding important issues, such as the origin of emerging pathogens and the extent of gene exchange among viruses. Untreated wastewater is an ideal system for assessing viral diversity because virion populations from large numbers of individuals are deposited and because raw sewage itself provides a rich environment for the growth of diverse host species and thus their viruses. These studies suggest that the viral universe is far more vast and diverse than previously suspected.
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http://dx.doi.org/10.1128/mBio.00180-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3187576PMC
January 2012

Induction of interferon-stimulated genes by Simian virus 40 T antigens.

Virology 2010 Oct 7;406(2):202-11. Epub 2010 Aug 7.

Department of Biological Sciences, University of Pittsburgh, 559 Crawford Hall, Pittsburgh, PA 15260, USA.

Simian virus 40 (SV40) large T antigen (TAg) is a multifunctional oncoprotein essential for productive viral infection and for cellular transformation. We have used microarray analysis to examine the global changes in cellular gene expression induced by wild-type T antigen (TAg(wt)) and TAg-mutants in mouse embryo fibroblasts (MEFs). The expression profile of approximately 800 cellular genes was altered by TAg(wt) and a truncated TAg (TAg(N136)), including many genes that influence cell cycle, DNA-replication, transcription, chromatin structure and DNA repair. Unexpectedly, we found a significant number of immune response genes upregulated by TAg(wt) including many interferon-stimulated genes (ISGs) such as ISG56, OAS, Rsad2, Ifi27 and Mx1. Additionally, we also observed activation of STAT1 by TAg(wt). Our genetic studies using several TAg-mutants reveal an unexplored function of TAg and indicate that the LXCXE motif and p53 binding are required for the upregulation of ISGs.
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http://dx.doi.org/10.1016/j.virol.2010.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2939315PMC
October 2010

E2f1-3 switch from activators in progenitor cells to repressors in differentiating cells.

Nature 2009 Dec;462(7275):930-4

Department of Molecular Virology, Immunology and Medical Genetics, College of Medicine, The Ohio State University, Columbus, Ohio 43210, USA.

In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.
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http://dx.doi.org/10.1038/nature08677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806193PMC
December 2009

Simian virus 40 T-antigen-mediated gene regulation in enterocytes is controlled primarily by the Rb-E2F pathway.

J Virol 2009 Sep 1;83(18):9521-31. Epub 2009 Jul 1.

Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.

Simian virus 40 large T antigen (TAg) contributes to cell transformation, in part, by targeting two well-characterized tumor suppressors, pRb and p53. TAg expression affects the transcriptional circuits controlled by Rb and by p53. We have performed a microarray analysis to examine the global change in gene expression induced by wild-type TAg (TAg(wt)) and TAg mutants, in an effort to link changes in gene expression to specific transforming functions. For this analysis we have used enterocytes from the mouse small intestine expressing TAg. Expression of TAg(wt) in the mouse intestine results in hyperplasia and dysplasia. Our analysis indicates that practically all gene expression regulated by TAg in enterocytes is dependent upon its binding and inactivation of the Rb family proteins. To further dissect the role of the Rb family in the induction of intestinal hyperplasia, we have screened several lines of transgenic mice expressing a truncated TAg (TAg(N136)), which is able to interfere with the Rb pathway but lacks the functions associated with the carboxy terminus of the protein. This analysis confirmed the pivotal association between the Rb pathway and the induction of intestinal hyperplasia and revealed that upregulation of p53 target genes is not associated with the tumorigenic phenotype. Furthermore, we found that TAg(N136) was sufficient to induce intestinal hyperplasia, although the appearance of dysplasia was significantly delayed.
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http://dx.doi.org/10.1128/JVI.00583-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2738269PMC
September 2009

Cell-type specific regulation of gene expression by simian virus 40 T antigens.

Virology 2009 Mar 8;386(1):183-91. Epub 2009 Feb 8.

Department of Biological Sciences, 559 Crawford Hall, University of Pittsburgh Pittsburgh, Pennsylvania 15260, USA.

SV40 transforms cells through the action of two oncoproteins, large T antigen and small t antigen. Small t antigen targets phosphatase PP2A, while large T antigen stimulates cell proliferation and survival by action on multiple proteins, including the tumor suppressors Rb and p53. Large T antigen also binds components of the transcription initiation complex and several transcription factors. We examined global gene expression in SV40-transformed mouse embryo fibroblasts, and in enterocytes obtained from transgenic mice. SV40 transformation alters the expression of approximately 800 cellular genes in both systems. Much of this regulation is observed in both MEFs and enterocytes and is consistent with T antigen action on the Rb-E2F pathway. However, the regulation of many genes is cell-type specific, suggesting that unique signaling pathways are activated in different cell types upon transformation, and that the consequences of SV40 transformation depends on the type of cell targeted.
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http://dx.doi.org/10.1016/j.virol.2008.12.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2737743PMC
March 2009

Complete nucleotide sequence of polyomavirus SA12.

J Virol 2005 Oct;79(20):13094-104

Department of Biological Sciences, University of Pittsburgh, PA 15260, USA.

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.
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http://dx.doi.org/10.1128/JVI.79.20.13094-13104.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1235855PMC
October 2005