Publications by authors named "James M Pipas"

65 Publications

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

J Virol 2021 02 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

Stability and detection of nucleic acid from viruses and hosts in controlled mosquito blood feeds.

PLoS One 2020 11;15(6):e0231061. Epub 2020 Jun 11.

Health Futures, Microsoft Research, Redmond, Washington, United States of America.

Monitoring the presence and spread of pathogens in the environment is of critical importance. Rapid detection of infectious disease outbreaks and prediction of their spread can facilitate early responses of health agencies and reduce the severity of outbreaks. Current sampling methods are sorely limited by available personnel and throughput. For instance, xenosurveillance utilizes captured arthropod vectors, such as mosquitoes, as sampling tools to access blood from a wide variety of vertebrate hosts. Next generation sequencing (NGS) of nucleic acid from individual blooded mosquitoes can be used to identify mosquito and host species, and microorganisms including pathogens circulating within either host. However, there are practical challenges to collecting and processing mosquitoes for xenosurveillance, such as the rapid metabolization or decay of microorganisms within the mosquito midgut. This particularly affects pathogens that do not replicate in mosquitoes, preventing their detection by NGS or other methods. Accordingly, we performed a series of experiments to establish the windows of detection for DNA or RNA from human blood and/or viruses present in mosquito blood meals. Our results will contribute to the development of xenosurveillance techniques with respect to optimal timing of sample collection and NGS processing and will also aid trap design by demonstrating the stabilizing effect of temperature control on viral genome recovery from blood-fed mosquitoes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231061PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289426PMC
August 2020

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

DNA Tumor Viruses and Their Contributions to Molecular Biology.

Authors:
James M Pipas

J Virol 2019 05 17;93(9). Epub 2019 Apr 17.

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

This summer marks the 51st anniversary of the DNA tumor virus meetings. Scientists from around the world will gather in Trieste, Italy, to report their latest results and to agree or disagree on the current concepts that define our understanding of this diverse class of viruses. This article offers a brief history of the impact the study of these viruses has had on molecular and cancer biology and discusses obstacles and opportunities for future progress.
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http://dx.doi.org/10.1128/JVI.01524-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475781PMC
May 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

Evolution on the Biophysical Fitness Landscape of an RNA Virus.

Mol Biol Evol 2018 10;35(10):2390-2400

Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA.

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.
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http://dx.doi.org/10.1093/molbev/msy131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188569PMC
October 2018

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

Dosage-dependent copy number gains in E2f1 and E2f3 drive hepatocellular carcinoma.

J Clin Invest 2017 Mar 30;127(3):830-842. Epub 2017 Jan 30.

Disruption of the retinoblastoma (RB) tumor suppressor pathway, either through genetic mutation of upstream regulatory components or mutation of RB1 itself, is believed to be a required event in cancer. However, genetic alterations in the RB-regulated E2F family of transcription factors are infrequent, casting doubt on a direct role for E2Fs in driving cancer. In this work, a mutation analysis of human cancer revealed subtle but impactful copy number gains in E2F1 and E2F3 in hepatocellular carcinoma (HCC). Using a series of loss- and gain-of-function alleles to dial E2F transcriptional output, we have shown that copy number gains in E2f1 or E2f3b resulted in dosage-dependent spontaneous HCC in mice without the involvement of additional organs. Conversely, germ-line loss of E2f1 or E2f3b, but not E2f3a, protected mice against HCC. Combinatorial mapping of chromatin occupancy and transcriptome profiling identified an E2F1- and E2F3B-driven transcriptional program that was associated with development and progression of HCC. These findings demonstrate a direct and cell-autonomous role for E2F activators in human cancer.
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http://dx.doi.org/10.1172/JCI87583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5330731PMC
March 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

E2f8 mediates tumor suppression in postnatal liver development.

J Clin Invest 2016 08 25;126(8):2955-69. Epub 2016 Jul 25.

E2F-mediated transcriptional repression of cell cycle-dependent gene expression is critical for the control of cellular proliferation, survival, and development. E2F signaling also interacts with transcriptional programs that are downstream of genetic predictors for cancer development, including hepatocellular carcinoma (HCC). Here, we evaluated the function of the atypical repressor genes E2f7 and E2f8 in adult liver physiology. Using several loss-of-function alleles in mice, we determined that combined deletion of E2f7 and E2f8 in hepatocytes leads to HCC. Temporal-specific ablation strategies revealed that E2f8's tumor suppressor role is critical during the first 2 weeks of life, which correspond to a highly proliferative stage of postnatal liver development. Disruption of E2F8's DNA binding activity phenocopied the effects of an E2f8 null allele and led to HCC. Finally, a profile of chromatin occupancy and gene expression in young and tumor-bearing mice identified a set of shared targets for E2F7 and E2F8 whose increased expression during early postnatal liver development is associated with HCC progression in mice. Increased expression of E2F8-specific target genes was also observed in human liver biopsies from HCC patients compared to healthy patients. In summary, these studies suggest that E2F8-mediated transcriptional repression is a critical tumor suppressor mechanism during postnatal liver development.
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http://dx.doi.org/10.1172/JCI85506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966321PMC
August 2016

The Ancient Evolutionary History of Polyomaviruses.

PLoS Pathog 2016 Apr 19;12(4):e1005574. Epub 2016 Apr 19.

School of Biological Sciences, University of Canterbury, Christchurch, New Zealand.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.
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http://dx.doi.org/10.1371/journal.ppat.1005574DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836724PMC
April 2016

Expression of the small T antigen of Lymphotropic Papovavirus is sufficient to transform primary mouse embryo fibroblasts.

Virology 2016 Jan 27;487:112-20. Epub 2015 Oct 27.

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

Polyomaviruses induce cell proliferation and transformation through different oncoproteins encoded within the early region (ER): large T antigen (LT), small T antigen (sT) and, in some cases, additional components. Each virus utilizes different mechanisms to achieve transformation. For instance, the LTs of Simian virus 40 (SV40), BK and/or JC virus can induce transformation; but Merkel Cell Polyomavirus (MCPyV) requires expression of sT. Lymphotropic Papovavirus (LPV) is closely related to Human Polyomavirus 9 (HuPyV9) and, under similar conditions, mice expressing LPV.ER exhibit higher rates of tumor formation than mice expressing SV40.ER. We have investigated the contributions of individual LPV.ER components to cell transformation. In contrast to SV40, LPV.ER transforms mouse embryonic fibroblasts (MEFs), but expression of LPV LT is insufficient to transform MEFs. Furthermore, LPV sT induces immortalization and transformation of MEFs. Thus, in the case of LPV, sT is the main mediator of oncogenesis.
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http://dx.doi.org/10.1016/j.virol.2015.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679568PMC
January 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

Rapid, targeted and culture-free viral infectivity assay in drop-based microfluidics.

Lab Chip 2015 Oct;15(19):3934-40

School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.

A key viral property is infectivity, and its accurate measurement is crucial for the understanding of viral evolution, disease and treatment. Currently viral infectivity is measured using plaque assays, which involve prolonged culturing of host cells, and whose measurement is unable to differentiate between specific strains and is prone to low number fluctuation. We developed a rapid, targeted and culture-free infectivity assay using high-throughput drop-based microfluidics. Single infectious viruses are incubated in a large number of picoliter drops with host cells for one viral replication cycle followed by in-drop gene-specific amplification to detect infection events. Using murine noroviruses (MNV) as a model system, we measure their infectivity and determine the efficacy of a neutralizing antibody for different variants of MNV. Our results are comparable to traditional plaque-based assays and plaque reduction neutralization tests. However, the fast, low-cost, highly accurate genomic-based assay promises to be a superior method for drug screening and isolation of resistant viral strains. Moreover our technique can be adapted to measuring the infectivity of other pathogens, such as bacteria and fungi.
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http://dx.doi.org/10.1039/c5lc00556fDOI Listing
October 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

Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics.

J Virol 2015 Aug 13;89(15):7722-34. Epub 2015 May 13.

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

Unlabelled: Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.

Importance: RNA viruses increase diversity and escape host immune barriers by genomic recombination. Studies using a number of viral systems indicate that recombination occurs via template switching by the virus-encoded RNA-dependent RNA polymerase (RdRP). However, factors that govern the frequency and positions of recombination in an infected organism remain largely unknown. This work leverages advances in the applied physics of drop-based microfluidics to isolate and sequence rare recombinants arising from the coinfection of mice with two distinct strains of murine norovirus. This study is the first to detect and analyze norovirus recombination in an animal model.
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http://dx.doi.org/10.1128/JVI.01137-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505660PMC
August 2015

The conserved core enzymatic activities and the distinct dynamics of polyomavirus large T antigens.

Arch Biochem Biophys 2015 May 6;573:23-31. Epub 2015 Mar 6.

Department of Biological Sciences, University of Pittsburgh, 4249 Fifth Avenue, Pittsburgh, PA 15260, USA. Electronic address:

Several human polyomaviruses including JCV, BKV and TSV are associated with diseases, particularly in immunosuppressed patients. While the large T antigen (LT) encoded by the monkey polyomavirus SV40 is well studied, and possesses intrinsic ATPase and DNA helicase activities, the LTs of the human polyomaviruses are relatively uncharacterized. In order to evaluate whether these enzymatic activities, which are required for viral DNA replication, are conserved between polyomaviruses, we performed a comparative study using the LTs from JCV, TSV and SV40. The ATPase and DNA helicase activities and the interaction with the cellular tumor suppressor p53 were assayed for the purified Zn-ATPase domains of the three LTs. We found that all Zn-ATPases were active ATPases. The Zn-ATPase domains also functioned as DNA helicases, although the measured kinetic constants differed among the three proteins. In addition, when tested against four small molecule ATPase inhibitors, the Zn-ATPase domains of TSV was more resistant than that of SV40 and JCV. Our results show that, while LTs from JCV and TSV share the core ATPase and DNA helicase activities, they possess important functional differences that might translate into their respective abilities to infect and replicate in hosts.
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http://dx.doi.org/10.1016/j.abb.2015.02.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865250PMC
May 2015

Cellular transformation of mouse embryo fibroblasts in the absence of activator E2Fs.

J Virol 2015 May 25;89(9):5124-33. Epub 2015 Feb 25.

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

Unlabelled: The E2F family of transcription factors, broadly divided into activator and repressor E2Fs, regulates cell cycle genes. Current models indicate that activator E2Fs are necessary for cell cycle progression and tumorigenesis and are also required to mediate transformation induced by DNA tumor viruses. E2Fs are negatively regulated by the retinoblastoma (RB) family of tumor suppressor proteins, and virus-encoded oncogenes disrupt the RB-E2F repressor complexes. This results in the release of activator E2Fs and induction of E2F-dependent genes. In agreement, expression of large tumor T antigens (TAg) encoded by polyomaviruses in mammalian cells results in increased transcriptional levels of E2F target genes. In addition, tumorigenesis induced by transgenic expression of simian virus 40 (SV40) TAg in choroid plexus or intestinal villi requires at least one activator E2F. In contrast, we show that SV40 TAg-induced transformation in mouse embryonic fibroblasts is independent of activator E2Fs. This work, coupled with recent studies showing that proliferation in stem and progenitor cells is independent of activator E2Fs, suggests the presence of parallel pathways governing cell proliferation and tumorigenesis.

Importance: The RB-E2F pathway is altered in many cancers and is also targeted by DNA tumor viruses. Viral oncoprotein action on RBs results in the release of activator E2Fs and upregulation of E2F target genes; thus, activator E2Fs are considered essential for normal and tumorigenic cell proliferation. However, we have observed that SV40 large T antigen can induce cell proliferation and transformation in the absence of activator E2Fs. Our results also suggest that TAg action on pRBs regulates both E2F-dependent and -independent pathways that govern proliferation. Thus, specific cell proliferation pathways affected by RB alterations in cancer may be a factor in tumor behavior and response to therapy.
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http://dx.doi.org/10.1128/JVI.03578-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403490PMC
May 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

Polyomavirus T antigens activate an antiviral state.

Virology 2015 Feb 10;476:377-385. Epub 2015 Jan 10.

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

Ectopic expression of Simian Virus 40 (SV40) large T antigen (LT) in mouse embryonic fibroblasts (MEFs) increased levels of mRNAs encoding interferon stimulated genes (ISGs). The mechanism by which T antigen increases levels of ISGs in MEFs remains unclear. We present evidence that expression of T antigen from SV40, Human Polyomaviruses BK (BKV) or JC (JCV) upregulate production of ISGs in MEFs, and subsequently result in an antiviral state, as determined by inhibition of VSV or EMCV growth. The first 136 amino acids of LT are sufficient for these activities. Furthermore, increased ISG expression and induction of the antiviral state requires STAT1. Finally, the RB binding motif of LT is necessary for activation of STAT1. We conclude that the induction of the STAT1 mediated innate immune response in MEFs is a common feature shared by SV40, BKV and JCV.
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http://dx.doi.org/10.1016/j.virol.2014.12.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323618PMC
February 2015

Synthesis and structure–activity relationships of small molecule inhibitors of the simian virus 40 T antigen oncoprotein, an anti-polyomaviral target.

Bioorg Med Chem 2014 Nov;22(22):6490-6502

Department of Biological Science, University of Pittsburgh, Pittsburgh, PA 15260.

Polyomavirus infections are common and relatively benign in the general human population but can become pathogenic in immunosuppressed patients. Because most treatments for polyomavirusassociated diseases nonspecifically target DNA replication, existing treatments for polyomavirus infection possess undesirable side effects. However, all polyomaviruses express Large Tumor Antigen (T Ag), which is unique to this virus family and may serve as a therapeutic target. Previous screening of pyrimidinone–peptoid hybrid compounds identified MAL2-11B and a MAL2-11B tetrazole derivative as inhibitors of viral replication and T Ag ATPase activity (IC50 of ~20-50 μM. To improve upon this scaffold and to develop a structure–activity relationship for this new class of antiviral agents, several iterative series of MAL2-11B derivatives were synthesized. The replacement of a flexible methylene chain linker with a benzyl group or, alternatively, the addition of an ortho-methyl substituent on the biphenyl side chain in MAL2-11B yielded an IC50 of 50 μM, which retained antiviral activity. After combining both structural motifs, a new lead compound was identified that inhibited T Ag ATPase activity with an IC50 of 50 μM. We suggest that the knowledge gained from the structure–activity relationship and a further refinement cycle of the MAL2-11B scaffold will provide a specific, novel therapeutic treatment option for polyomavirus infections and their associated diseases.
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http://dx.doi.org/10.1016/j.bmc.2014.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293281PMC
November 2014

SummonChimera infers integrated viral genomes with nucleotide precision from NGS data.

BMC Bioinformatics 2014 Oct 22;15:348. Epub 2014 Oct 22.

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

Background: Viral integration into a host genome is defined by two chimeric junctions that join viral and host DNA. Recently, computational tools have been developed that utilize NGS data to detect chimeric junctions. These methods identify individual viral-host junctions but do not associate chimeric pairs as an integration event. Without knowing the chimeric boundaries of an integration, its genetic content cannot be determined.

Results: Summonchimera is a Perl program that associates chimera pairs to infer the complete viral genomic integration event to the nucleotide level within single or paired-end NGS data. SummonChimera integration prediction was verified on a set of single-end IonTorrent reads from a purified Salmonella bacterium with an integrated bacteriophage. Furthermore, SummonChimera predicted integrations from experimentally verified Hepatitis B Virus chimeras within a paired-end Whole Genome Sequencing hepatocellular carcinoma tumor database.

Conclusions: SummonChimera identified all experimentally verified chimeras detected by current computational methods. Further, SummonChimera integration inference precisely predicted bacteriophage integration. The application of SummonChimera to cancer NGS accurately identifies deletion of host and viral sequence during integration. The precise nucleotide determination of an integration allows prediction of viral and cellular gene transcription patterns.
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http://dx.doi.org/10.1186/s12859-014-0348-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4210586PMC
October 2014

Removal of a small C-terminal region of JCV and SV40 large T antigens has differential effects on transformation.

Virology 2014 Nov 16;468-470:47-56. Epub 2014 Aug 16.

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

The large T antigen (LT) protein of JCV and SV40 polyomaviruses is required to induce tumors in rodents and transform cells in culture. When both LTs are compared side-by-side in cell culture assays, SV40 shows a more robust transformation phenotype even though the LT sequences are highly conserved. A complete understanding of SV40׳s enhanced transforming capabilities relative to JCV is lacking. When the least conserved region of the LT proteins, the variable linker and host range region (VHR), was removed, changes in T antigen expression and cellular p53 post-translational modifications occurred, but interaction with the pRB pathway was unaffected. Transformation assessed by growth in low serum was reduced after VHR truncation of the SV40, but not the JCV, T antigen. Conversely, anchorage independent transformation was enhanced only by truncation of the JCV VHR. This is the first report to link the SV40 or JCV VHR region to transformation potential.
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http://dx.doi.org/10.1016/j.virol.2014.07.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253650PMC
November 2014