Uveal melanoma immunogenomics predict immunotherapy resistance and susceptibility.

Immune checkpoint inhibition has shown success in treating metastatic cutaneous melanoma but has limited efficacy against metastatic uveal melanoma, a rare variant arising from the immune privileged eye. To better understand this resistance, we comprehensively profile 100 human uveal melanoma metastases using clinicogenomics, transcriptomics, and tumor infiltrating lymphocyte potency assessment. We find that over half of these metastases harbor tumor infiltrating lymphocytes with potent autologous tumor specificity, despite low mutational burden and resistance to prior immunotherapies. However, we observe strikingly low intratumoral T cell receptor clonality within the tumor microenvironment even after prior immunotherapies. To harness these quiescent tumor infiltrating lymphocytes, we develop a transcriptomic biomarker to enable in vivo identification and ex vivo liberation to counter their growth suppression. Finally, we demonstrate that adoptive transfer of these transcriptomically selected tumor infiltrating lymphocytes can promote tumor immunity in patients with metastatic uveal melanoma when other immunotherapies are incapable.

Cultured Renal Proximal Tubular Epithelial Cells Resemble a Stressed/Damaged Kidney While Supporting BK Virus Infection.

BK virus (BKV; human polyomavirus 1) infections are asymptomatic in most individuals, and the virus persists throughout life without harm. However, BKV is a threat to transplant patients and those with immunosuppressive disorders. Under these circumstances, the virus can replicate robustly in proximal tubule epithelial cells (PT). Cultured renal proximal tubule epithelial cells (RPTE) are permissive to BKV and have been used extensively to characterize different aspects of BKV infection. Recently, lines of hTERT-immortalized RPTE have become available, and preliminary studies indicate they support BKV infection as well. Our results indicate that BKV infection leads to a similar response in primary and immortalized RPTE. In addition, we examined the patterns of global gene expression of primary and immortalized RPTE and compared them with uncultured PT freshly dissociated from human kidney. As expected, PT isolated from the healthy kidney express a number of differentiation-specific genes that are associated with kidney function. However, the expression of most of these genes is absent or repressed in cultured RPTE. Rather, cultured RPTE exhibit a gene expression profile indicative of a stressed or injured kidney. Inoculation of cultured RPTE with BKV results in the suppression of many genes associated with kidney stress. In summary, this study demonstrated similar global gene expression patterns and responses to BKV infection between primary and immortalized RPTE. Moreover, results from bulk transcriptome sequencing (RNA-seq) and SCT experiments revealed distinct transcriptomic signatures representing cell injury and stress in primary RPTE in contrast to the uncultured, freshly dissociated PT from human kidney. IMPORTANCE Cultured primary human cells provide powerful tools for the study of viral infectious cycles and host virus interactions. In the case of BKV-associated nephropathy, viral replication occurs primarily in the proximal tubule epithelia in the kidney. Consequently, cultured primary and immortalized renal proximal tubule epithelial cells (RPTE) are widely used to study BKV infection. In this work, using bulk and single-cell transcriptomics, we found that primary and immortalized RPTE responded similarly to BKV infection. However, both uninfected primary and immortalized RPTE have gene expression profiles that are markedly different from healthy proximal tubule epithelia isolated directly from human kidney without culture. Cultured RPTE are in a gene expression state indicative of an injured or stressed kidney. These results raise the possibility that BKV replicates preferentially in injured or stressed kidney epithelial cells during nephropathy.

The pollen virome of wild plants and its association with variation in floral traits and land use.

Pollen is a unique vehicle for viral spread. Pollen-associated viruses hitchhike on or within pollen grains and are transported to other plants by pollinators. They are deposited on flowers and have a direct pathway into the plant and next generation via seeds. To discover the diversity of pollen-associated viruses and identify contributing landscape and floral features, we perform a species-level metagenomic survey of pollen from wild, visually asymptomatic plants, located in one of four regions in the United States of America varying in land use. We identify many known and novel pollen-associated viruses, half belonging to the Bromoviridae, Partitiviridae, and Secoviridae viral families, but many families are represented. Across the regions, species harbor more viruses when surrounded by less natural and more human-modified environments than the reverse, but we note that other region-level differences may also covary with this. When examining the novel connection between virus richness and floral traits, we find that species with multiple, bilaterally symmetric flowers and smaller, spikier pollen harbored more viruses than those with opposite traits. The association of viral diversity with floral traits highlights the need to incorporate plant-pollinator interactions as a driver of pollen-associated virus transport into the study of plant-viral interactions.

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

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.IMPORTANCE 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.

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

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.

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

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.

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

We report the coding-complete genome sequence of Japanese apricot pollen-associated secovirus 1 (JAPSV1), a virus belonging to the Secoviridae family, recovered from Japanese apricot (Prunus mume) pollen that is closely related to Peach leaf pitting-associated virus (PLPAV). This discovery adds to the number of known pollen-associated viruses.

Detecting viral sequences in NGS data.

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.

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

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 IFNß 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.

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

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 Deinocerites mosquitoes. The genome has a length of 14,420 nucleotides and encodes five open reading frames.

Complete Genome Sequence of Pittsburgh Sewage-Associated Virus 1.

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 Picornavirales order of viruses. PSAV1 is closely related to Biomphalaria virus 2 (BV2).

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

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. IMPORTANCE 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.

Viral sequences in human cancer.

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.