Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

The role of preoperative immune cell metrics in renal cell carcinoma with a tumor thrombus.

INTRODUCTION: The objective of this study was to stratify preoperative immune cell counts by cancer specific outcomes in patients with renal cell carcinoma (RCC) and a tumor thrombus after radical nephrectomy with tumor thrombectomy. METHODS: Patients with a diagnosis of RCC with tumor thrombus that underwent radical nephrectomy with thrombectomy across an international consortium of seven institutions were included. Patients who were metastatic at diagnosis and those who received preoperative medical treatment were also included. Retrospective chart review was performed to collect demographic information, past medical history, preoperative lab work, surgical pathology, and follow up data. Neutrophil counts, lymphocyte counts, monocyte counts, neutrophil to lymphocyte ratios (NLR), lymphocyte to monocyte ratios (LMR), and neutrophil to monocyte ratios (NMR) were compared against cancer-specific outcomes using independent samples t-test, Pearson's bivariate correlation, and analysis of variance. RESULTS: One hundred forty-four patients were included in the study, including nine patients who were metastatic at the time of surgery. Absolute lymphocyte count preoperatively was greater in patients who died from RCC compared to those who did not (2 vs 1.4; p < 0.001). Patients with tumor pathology showing perirenal fat invasion had a greater neutrophil count compared to those who did not (7.5 vs 5.5; p = 0.010). Patients with metastatic RCC had a lower LMR compared to those without metastases after surgery (2.5 vs 3.2; p = 0.041). Tumor size, both preoperatively and on gross specimen, had an interaction with multiple immune cell metrics (p < 0.05). CONCLUSIONS: Preoperative immune metrics have clinical utility in predicting cancer-specific outcomes for patients with RCC and a tumor thrombus. Additional study is needed to determine the added value of preoperative serum immune cell data to established prognostic risk calculators for this patient population.

Increased spatial coupling of integrin and collagen IV in the immunoresistant clear cell renal cell carcinoma tumor microenvironment.

Background: Immunotherapy (IO) has improved survival for patients with advanced clear cell renal cell carcinoma (ccRCC), but resistance to therapy develops in most patients. We use cellular-resolution spatial transcriptomics in patients with IO naïve and IO exposed primary ccRCC tumors to better understand IO resistance. Spatial molecular imaging (SMI) was obtained for tumor and adjacent stroma samples. Spatial gene set enrichment analysis (GSEA) and autocorrelation (coupling with high expression) of ligand-receptor transcript pairs were assessed. Multiplex immunofluorescence (mIF) validation was used for significant autocorrelative findings and the cancer genome atlas (TCGA) and the clinical proteomic tumor analysis consortium (CPTAC) databases were queried to assess bulk RNA expression and proteomic correlates. Results: 21 patient samples underwent SMI. Viable tumors following IO harbored more stromal CD8+ T cells and neutrophils than IO naïve tumors. YES1 was significantly upregulated in IO exposed tumor cells. The epithelial-mesenchymal transition pathway was enriched on spatial GSEA and the associated transcript pair COL4A1-ITGAV had significantly higher autocorrelation in the stroma. Fibroblasts, tumor cells, and endothelium had the relative highest expression. More integrin αV+ cells were seen in IO exposed stroma on mIF validation. Compared to other cancers in TCGA, ccRCC tumors have the highest expression of both COL4A1 and ITGAV. In CPTAC, collagen IV protein was more abundant in advanced stages of disease. Conclusions: On spatial transcriptomics, COL4A1 and ITGAV were more autocorrelated in IO-exposed stroma compared to IO-naïve tumors, with high expression amongst fibroblasts, tumor cells, and endothelium. Integrin represents a potential therapeutic target in IO treated ccRCC.

MmuPV1 E6 induces cell proliferation and other hallmarks of cancer.

The E6 protein encoded by the murine papillomavirus (MmuPV1) is essential for MmuPV1-induced skin disease. Our previous work has identified a number of cellular interacting partners of MmuPV1 E6 and E7 through affinity purification/mass spectrometry analysis. These studies revealed that MmuPV1 E6 potently inhibits keratinocyte differentiation through multiple molecular mechanisms including inhibition of NOTCH and TGF-ß signaling. Here, we report that MmuPV1 E6 has additional important oncogenic activities when expressed in its natural host cells, mouse keratinocytes, including increasing proliferation, overcoming density-mediated growth arrest, and proliferation under conditions of limited supply of growth factors. Unbiased proteomic/transcriptomic analyses of mouse keratinocytes expressing MmuPV1 E6 substantiated its effect on these cellular processes and divulged that some of these effects may be mediated in part through it upregulating E2F activity. Our analyses also revealed that MmuPV1 E6 may alter other cancer hallmarks including evasion of growth suppressors, inhibition of immune response, resistance to cell death, and alterations in DNA damage response. Collectively, our results suggest that MmuPV1 E6 is a major driver of multiple hallmarks of cancer in MmuPV1's natural host cells, mouse keratinocytes.IMPORTANCEThe Mus musculus papillomavirus 1 (MmuPV1) E6 and E7 proteins are required for MmuPV1-induced disease. Our understanding of the activities of MmuPV1 E6 has been based on affinity purification/mass spectrometry studies where cellular interacting partners of MmuPV1 E6 were identified, and these studies revealed that MmuPV1 E6 can inhibit keratinocyte differentiation through multiple mechanisms. We report that MmuPV1 E6 encodes additional activities including the induction of proliferation, resistance to density-mediated growth arrest, and decreased dependence on exogenous growth factors. Proteomic and transcriptomic analyses provided evidence that MmuPV1 E6 increases the expression and steady state levels of a number of cellular proteins that promote cellular proliferation and other hallmarks of cancer. These results indicate that MmuPV1 E6 is a major driver of MmuPV1-induced pathogenesis.

Type 1 and Type 2 Epstein-Barr viruses induce proliferation, and inhibit differentiation, in infected telomerase-immortalized normal oral keratinocytes.

Differentiated epithelial cells are an important source of infectious EBV virions in human saliva, and latent Epstein-Barr virus (EBV) infection is strongly associated with the epithelial cell tumor, nasopharyngeal carcinoma (NPC). However, it has been difficult to model how EBV contributes to NPC, since EBV has not been shown to enhance proliferation of epithelial cells in monolayer culture in vitro and is not stably maintained in epithelial cells without antibiotic selection. In addition, although there are two major types of EBV (type 1 (T1) and type 2 (T2)), it is currently unknown whether T1 and T2 EBV behave differently in epithelial cells. Here we inserted a G418 resistance gene into the T2 EBV strain, AG876, allowing us to compare the phenotypes of T1 Akata virus versus T2 AG876 virus in a telomerase-immortalized normal oral keratinocyte cell line (NOKs) using a variety of different methods, including RNA-seq analysis, proliferation assays, immunoblot analyses, and air-liquid interface culture. We show that both T1 Akata virus infection and T2 AG876 virus infection of NOKs induce cellular proliferation, and inhibit spontaneous differentiation, in comparison to the uninfected cells when cells are grown without supplemental growth factors in monolayer culture. T1 EBV and T2 EBV also have a similar ability to induce epithelial-to-mesenchymal (EMT) transition and activate canonical and non-canonical NF-κB signaling in infected NOKs. In contrast to our recent results in EBV-infected lymphoblastoid cells (in which T2 EBV infection is much more lytic than T1 EBV infection), we find that NOKs infected with T1 and T2 EBV respond similarly to lytic inducing agents such as TPA treatment or differentiation. These results suggest that T1 and T2 EBV have similar phenotypes in infected epithelial cells, with both EBV types enhancing cellular proliferation and inhibiting differentiation when growth factors are limiting.

Calibrated Regression Models Based on the Risk of Clinical Nodal Metastasis Should be Used as Decision Aids for Prostate Cancer Staging to Reduce Unnecessary Imaging.

INTRODUCTION: Radionuclide imaging will change the role of computed tomography and magnetic resonance imaging (CT/MRI) for prostate cancer (CaP) staging. Current guidelines recommend abdominopelvic imaging for new cases of CaP categorized as unfavorable intermediate risk (UIR) or higher. We assessed the performance characteristics of CT/MRI based on the National Comprehensive Cancer Network (NCCN) guidelines and developed a model that predicts cN1 disease using conventional imaging. PATIENTS AND METHODS: We selected patients in the National Cancer Database diagnosed with CaP from 2010 to 2016 with available age, prostate specific antigen, clinical locoregional staging, biopsy Gleason grading, and core information. Multivariate logistic regression (MLR) was used on a undersampled training dataset using cN1 as the outcome. Performance characteristics were compared to those of the three most recent versions of the NCCN guidelines. RESULTS: A total of 443,640 men were included, and 2.5% had cN1 disease. Using CT/MRI only, the current NCCN guidelines have a sensitivity of 99%, and the number needed to image (NNI) is 24. At the same sensitivity, the cN1 risk was 1.6% using the MLR. The NNI for UIR alone is 341. Using the MLR model and a threshold of 10%, the PPV is 10.3% and 64% of CTs/MRIs could be saved at a cost of missing 6% of cN1 patients (or 0.15% of all patients). CONCLUSION: The NCCN guidelines are sensitive for detecting cN1 with CT/MRI, however, the number needed to image is 24. Obtaining CT/MRI for nodal staging when patients have a cN1 risk of 10% would reduce total imaging while still remaining sensitive. As novel PET tracers becomes increasingly used for initial CaP staging, well calibrated prediction models trained on the outcome of interest should be developed as decision aids for obtaining imaging.

Optimal LentiCRISPR-Based System for Sequential CRISPR/Cas9 Screens.

The advent of genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening has advanced the understanding of molecular systems within cells. Here, we demonstrate the utility of sequentially performed CRISPR knockout screens that use an existing library to explore a biological question across the human genome, and then the remaining cells are used to examine each gene candidate against one common gene of interest. We call this approach "Many vs One" CRISPR screening, made possible by a modified 7SK promoter in place of the U6 promoter to drive expression of a single guide RNA. Inserting this novel 7SK promoter into the ubiquitously used lentiCRISPRv2 backbone is crucial, because it overcomes the need for a substantial increase in CRISPR library coverage during screening, sample processing, and next generation sequencing. This new 7SK vector equals the original lentiCRISPRv2 in lentiviral titer, knockout efficiency, and ease of use.

Stress Keratin 17 Expression in Head and Neck Cancer Contributes to Immune Evasion and Resistance to Immune-Checkpoint Blockade.

PURPOSE: We investigated whether in human head and neck squamous cell carcinoma (HNSCC) high levels of expression of stress keratin 17 (K17) are associated with poor survival and resistance to immunotherapy. EXPERIMENTAL DESIGN: We investigated the role of K17 in regulating both the tumor microenvironment and immune responsiveness of HNSCC using a syngeneic mouse HNSCC model, MOC2. MOC2 gives rise to immunologically cold tumors that are resistant to immune-checkpoint blockade (ICB). We engineered multiple, independent K17 knockout (KO) MOC2 cell lines and monitored their growth and response to ICB. We also measured K17 expression in human HNSCC of patients undergoing ICB. RESULTS: MOC2 tumors were found to express K17 at high levels. When knocked out for K17 (K17KO MOC2), these cells formed tumors that grew slowly or spontaneously regressed and had a high CD8+ T-cell infiltrate in immunocompetent syngeneic C57BL/6 mice compared with parental MOC2 tumors. This phenotype was reversed when we depleted mice for T cells. Whereas parental MOC2 tumors were resistant to ICB treatment, K17KO MOC2 tumors that did not spontaneously regress were eliminated upon ICB treatment. In a cohort of patients with HNSCC receiving pembrolizumab, high K17 expression correlated with poor response. Single-cell RNA-sequencing analysis revealed broad differences in the immune landscape of K17KO MOC2 tumors compared with parental MOC2 tumors, including differences in multiple lymphoid and myeloid cell types. CONCLUSIONS: We demonstrate that K17 expression in HNSCC contributes to immune evasion and resistance to ICB treatment by broadly altering immune landscapes of tumors.

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

Accurate Quantification of Overlapping Herpesvirus Transcripts from RNA Sequencing Data.

Herpesviruses employ extensive bidirectional transcription of overlapping genes to overcome length constraints on their gene product repertoire. As a consequence, many lytic transcripts cannot be measured individually by reverse transcription-quantitative PCR (RT-qPCR) or conventional RNA sequencing (RNA-seq) analysis. A. G. Bruce, S. Barcy, T. DiMaio, E. Gan, et al. (Pathogens 6:11, 2017, https://doi.org/10.3390/pathogens6010011) have proposed an approximation method using unique coding sequences (UCDS) to estimate lytic gene abundance from Kaposi's sarcoma-associated herpesvirus (KSHV) RNA-seq data. Although UCDS has been widely employed, its accuracy, to our knowledge, has never been rigorously validated for any herpesvirus. In this study, we use cap analysis of gene expression sequencing (CAGE-seq) as a gold-standard to determine the accuracy of UCDS for estimating Epstein-Barr virus (EBV) lytic gene expression levels from RNA-seq data. We also introduce the Unique TranScript (UTS) method, which, like UCDS, estimates transcript abundance from changes in mean RNA-seq read depth. UTS is distinguished by its use of empirically determined 5' and 3' transcript ends rather than coding sequence annotations. Compared to conventional read assignment, both UCDS and UTS improved the accuracy of quantitation of overlapping genes, with UTS giving the most-accurate results. The UTS method discards fewer reads and may be advantageous for experiments with less sequencing depth. UTS is compatible with any aligner and, unlike isoform-aware alignment methods, can be implemented on a laptop computer. Our findings demonstrate that the accuracy achieved by complex and expensive techniques such as CAGE-seq can be approximated using conventional short-read RNA-seq data when read assignment methods address transcript overlap. Although our study focuses on EBV transcription, the UTS method should be applicable across all herpesviruses as well as to other genomes with extensively overlapping transcriptomes. IMPORTANCE Many viruses employ extensively overlapping transcript structures. This complexity makes it difficult to quantify gene expression by using conventional methods, including RNA-seq. Although high-throughput techniques that overcome these limitations exist, they are complex, expensive, and scarce in the herpesvirus literature relative to short-read RNA-seq. Here, using Epstein-Barr virus (EBV) as a model, we demonstrate that conventional RNA-seq analysis methods fail to accurately quantify the abundances of many overlapping transcripts. We further show that the previously described Unique CoDing Sequence (UCDS) method and our Unique TranScript (UTS) method greatly improve the accuracy of EBV lytic gene measurements obtained from RNA-seq data. The UTS method has the advantages of discarding fewer reads and being implementable on a laptop computer. Although this study focuses on EBV, the UCDS and UTS methods should be applicable across herpesviruses and for other viruses that make extensive use of overlapping transcription.

Staged Fowler Stephens, Who Requires a Third Procedure?

PURPOSE OF REVIEW: The two-stage Fowler-Stephens orchiopexy is a well-described surgical approach for most pediatric urologists tackling the high intra-abdominal testis. Testicular ascent is a well-reported outcome of the surgery and a problem that could be fixed with a repeat procedure. The purpose of this review is to determine the rate of subsequent testicular ascent and repeat surgery after a two-stage Fowler-Stephens using best available evidence. RECENT FINDINGS/RESULT: We selected 16 studies that pertained to our topic. 0-13% of testicles were deemed to be in an inappropriate position on follow-up (6-37 months). The definition of appropriate postoperative position varied between studies and rates of repeat procedures were sparsely available. Available data reports a repeat orchiopexy rate of 2.6-7.5% after two-stage Fowler-Stephens. Two-stage Fowler-Stephens orchiopexy is a highly successful surgical technique to manage high intra-abdominal testes. Rates of testicular ascent are difficult to ascertain due to varying definitions and small sample sizes. While repeat procedures are typically indicated, the actual rate of repeat orchiopexy is rarely reported and long-term outcomes following a third procedure is sparse.

Epstein-Barr virus nuclear antigen 3C (EBNA3C) interacts with the metabolism sensing C-terminal binding protein (CtBP) repressor to upregulate host genes.

Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16INK4A and possibly p14ARF is central to EBNA3C's role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1α and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1α or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation.

Misaligned Incentives in Benign Prostatic Enlargement Surgery: More Complex and Efficacious Procedures Are Earning Fewer Relative Value Units.

Background: Relative value units (RVUs) are the measure of value used in US Medicare reimbursement. Medicare determines physician work RVUs (wRVUs) from the Relative Value Update Committee (RUC) for a procedure based on operative time, technical skill and effort, mental effort and judgment, and stress. In theory, work RVUs should account for the complexity and operative time involved in a procedure. The aim of this study was to assess whether major procedures for treatment of benign prostatic enlargement (BPE) are fairly compensated based on complexity and operative time in the RVU system and compare them with the intended reimbursement. Methods: The American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) database and the Centers for Medicare and Medicaid Services (CMS) Medicare Physician Fee Schedule were queried from 2015 to 2017. Single, current, procedural terminology codes associated with BPE treatments were included: transurethral resection of the prostate (TURP), photovaporization of the prostate (PVP), holmium laser enucleation of the prostate (HoLEP), retropubic simple prostatectomy (RSP), and suprapubic simple prostatectomy (SSP). The CMS operative times and the NSQIP real data were used in turn to calculate separate values for wRVUs per hour (wRVUs/hr) of operative time. The wRVUs/hr derived from CMS operative times represent RUC-estimated wRVUs/hr and wRVUs/hr derived from NSQIP represent actual wRVUs/hr. Results: A total of 27,664 cases were included from the NSQIP dataset. Median wRVU was 15.3 (interquartile range [IQR] 12.2-15.3), median operative time 50 minutes (IQR 33-74), and median wRVUs/hr 17.0 (IQR 11.6-26.2). RUC-estimated wRVUs/hr were TURP 12.2, PVP 12.2, RSP 9, SSP 9.3, and HoLEP 7.3. The actual wRVUs/hr were TURP 19.1, PVP 15.5, RSP 10.2, HoLEP 9.4, and SSP 7.6. Conclusions: Laser enucleation and simple prostatectomy are highly complex and efficacious procedures for treating BPE, yet the current payment schedule assigns these procedures the least amount of wRVUs/hr. Financial incentives for performing BPE surgeries are clearly misaligned.

EBNA2-deleted Epstein-Barr virus (EBV) isolate, P3HR1, causes Hodgkin-like lymphomas and diffuse large B cell lymphomas with type II and Wp-restricted latency types in humanized mice.

EBV transforms B cells in vitro and causes human B-cell lymphomas including classical Hodgkin lymphoma (CHL), Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). The EBV latency protein, EBNA2, transcriptionally activates the promoters of all latent viral protein-coding genes expressed in type III EBV latency and is essential for EBV's ability to transform B cells in vitro. However, EBNA2 is not expressed in EBV-infected CHLs and BLs in humans. EBV-positive CHLs have type II latency and are largely driven by the EBV LMP1/LMP2A proteins, while EBV-positive BLs, which usually have type I latency are largely driven by c-Myc translocations, and only express the EBNA1 protein and viral non-coding RNAs. Approximately 15% of human BLs contain naturally occurring EBNA2-deleted viruses that support a form of viral latency known as Wp-restricted (expressing the EBNA-LP, EBNA3A/3B/3C, EBNA1 and BHRF1 proteins), but whether Wp-restricted latency and/or EBNA2-deleted EBV can induce lymphomas in humanized mice, or in the absence of c-Myc translocations, is unknown. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL induces EBV-positive B-cell lymphomas in a subset of infected cord blood-humanized (CBH) mice. Furthermore, we find that P3HR1-infected lymphoma cells support two different viral latency types and phenotypes that are mutually exclusive: 1) Large (often multinucleated), CD30-positive, CD45-negative cells reminiscent of the Reed-Sternberg (RS) cells in CHL that express high levels of LMP1 but not EBNA-LP (consistent with type II viral latency); and 2) smaller monomorphic CD30-negative DLBCL-like cells that express EBNA-LP and EBNA3A but not LMP1 (consistent with Wp-restricted latency). These results reveal that EBNA2 is not absolutely required for EBV to form tumors in CBH mice and suggest that P3HR1 virus can be used to model EBV positive lymphomas with both Wp-restricted and type II latency in vivo.

An EBNA3A-Mutated Epstein-Barr Virus Retains the Capacity for Lymphomagenesis in a Cord Blood-Humanized Mouse Model.

Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice.IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

Stress keratin 17 enhances papillomavirus infection-induced disease by downregulating T cell recruitment.

High-risk human papillomaviruses (HPVs) cause 5% of human cancers. Despite the availability of HPV vaccines, there remains a strong urgency to find ways to treat persistent HPV infections, as current HPV vaccines are not therapeutic for individuals already infected. We used a mouse papillomavirus infection model to characterize virus-host interactions. We found that mouse papillomavirus (MmuPV1) suppresses host immune responses via overexpression of stress keratins. In mice deficient for stress keratin K17 (K17KO), we observed rapid regression of papillomas dependent on T cells. Cellular genes involved in immune response were differentially expressed in the papillomas arising on the K17KO mice correlating with increased numbers of infiltrating CD8+ T cells and upregulation of IFNγ-related genes, including CXCL9 and CXCL10, prior to complete regression. Blocking the receptor for CXCL9/CXCL10 prevented early regression. Our data provide a novel mechanism by which papillomavirus-infected cells evade host immunity and defines new therapeutic targets for treating persistent papillomavirus infections.

Tales from the Trips: A Qualitative Study of Timely Recognition, Treatment, and Transfer of Emergency Department Patients with Acute Ischemic Stroke.

BACKGROUND AND OBJECTIVES: Acute Ischemic stroke (AIS) is a time-sensitive emergency and patients frequently present to, and are transferred from emergency departments (EDs). We sought to evaluate potential factors, particularly organizational, that may influence the timeliness of interfacility transfer for ED patients with AIS. METHODS: We conducted semistructured interviews at 3 EDs that routinely transfer AIS patients. A structured interview guide was developed and piloted prior to use. Staff were asked about perceived facilitators and barriers to timely and high quality emergency care for patients with AIS who require transfer. Each interview was audio recorded, transcribed, coded, and analyzed using an iterative inductive-deductive approach to build a list of themes and subthemes, and identify supporting quotes. RESULTS: We interviewed 45 ED staff (administrative staff, nurses, and physicians) involved in acute stroke care. We identified 4 major themes influencing the execution of interfacility transfers of AIS patients: (1) processes, (2) historical experiences; (3) communication; and (4) resources. Pre-existing protocols that standardized processes (eg, autoacceptance protocols) and reduced unnecessary communication, combined with direct communication with the neurology team at the comprehensive stroke center, and the flexibility and availability of human and physical resources (eg, staff and equipment) were commonly cited as facilitators. Lack of communication of clinical and operational outcomes back to transferring ED staff was viewed as a lost opportunity for process improvement, interorganization relationship building, and professional satisfaction. CONCLUSIONS: ED staff view the interfacility transfer of AIS patients as highly complex with multiple opportunities for delay. Coordination through the use of protocols and communication pre- and post-transfer represented opportunities to facilitate transfers. Staff and clinicians at transferring facilities identified multiple opportunities to enhance existing processes and ongoing communication quality among facilities involved in the acute management of patients with AIS.

A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection.

Latent Epstein-Barr virus (EBV) infection contributes to both B-cell and epithelial-cell malignancies. However, whether lytic EBV infection also contributes to tumors is unclear, although the association between malaria infection and Burkitt lymphomas (BLs) may involve excessive lytic EBV replication. A particular variant of the viral promoter (Zp) that controls lytic EBV reactivation is over-represented, relative to its frequency in non-malignant tissue, in EBV-positive nasopharyngeal carcinomas and AIDS-related lymphomas. To date, no functional differences between the prototype Zp (Zp-P) and the cancer-associated variant (Zp-V3) have been identified. Here we show that a single nucleotide difference between the Zp-V3 and Zp-P promoters creates a binding site for the cellular transcription factor, NFATc1, in the Zp-V3 (but not Zp-P) variant, and greatly enhances Zp activity and lytic viral reactivation in response to NFATc1-inducing stimuli such as B-cell receptor activation and ionomycin. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV B95.8 strain genome greatly enhances lytic viral reactivation in response to the NFATc1-activating agent, ionomycin, and this effect is blocked by the NFAT inhibitory agent, cyclosporine, as well as NFATc1 siRNA. We also show that the Zp-V3 variant is over-represented in EBV-positive BLs and gastric cancers, and in EBV-transformed B-cell lines derived from EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These results demonstrate that the Zp-V3 enhances EBV lytic reactivation to physiologically-relevant stimuli, and suggest that increased lytic infection may contribute to the increased prevalence of this variant in EBV-associated malignancies.

CAGE-seq analysis of Epstein-Barr virus lytic gene transcription: 3 kinetic classes from 2 mechanisms.

Epstein-Barr virus (EBV) lytic replication proceeds through an ordered cascade of gene expression that integrates lytic DNA amplification and late gene transcription. We and others previously demonstrated that 6 EBV proteins that have orthologs in ß- and γ-, but not in α-herpesviruses, mediate late gene transcription in a lytic DNA replication-dependent manner. We proposed a model in which the ßγ gene-encoded viral pre-initiation complex (vPIC) mediates transcription from newly replicated viral DNA. While this model explains the dependence of late gene transcription on lytic DNA replication, it does not account for this dependence in α-herpesviruses nor for recent reports that some EBV late genes are transcribed independently of vPIC. To rigorously define which transcription start sites (TSS) are dependent on viral lytic DNA replication or the ßγ complex, we performed Cap Analysis of Gene Expression (CAGE)-seq on cells infected with wildtype EBV or EBV mutants defective for DNA replication, ßγ function, or lacking an origin of lytic replication (OriLyt). This approach identified 16 true-late, 32 early, and 16 TSS that are active at low levels early and are further upregulated in a DNA replication-dependent manner (leaky late). Almost all late gene transcription is vPIC-dependent, with BCRF1 (vIL10), BDLF2, and BDLF3 transcripts being notable exceptions. We present evidence that leaky late transcription is not due to a distinct mechanism, but results from superimposition of the early and late transcription mechanisms at the same promoter. Our results represent the most comprehensive characterization of EBV lytic gene expression kinetics reported to date and suggest that most, but not all EBV late genes are vPIC-dependent.