Pattern Of Viral Gene Expression Research Articles

Comprehensive examination on codon usage bias pattern of the Bovine Ephemeral fever virus

Bovine Ephemeral Fever Virus (BEFV) is a non-contagious virus that commonly infects cattle and water buffalo, reduces milk productivity, decreases the quality of beef, and causes an adverse economic impact on the global livestock industry. However, the evolution of BEFV is unclear, and uncertainty exists regarding its global geodynamics. Consequently, this study aims to comprehend the pattern of viral evolution and gene expression in the BEFV genes G, M, N, and P, including synonymous codons. Additionally, we performed recombination analyses, which exclusively detected recombination signals in the G- and P-genes. Subsequently, a phylogenetic tree was constructed to validate and support these findings. The codon usage bias results showed that the BEFV-selected genes were influenced by both natural and mutation pressure. Furthermore, nucleotide A is more abundant in all the selected genes. The eNC values, ranging from 42.99 to 47.10, revealed the presence of moderate codon usage bias, where gene P exhibited the highest and gene G had the lowest codon usage bias. The neutrality and PR-2 plots, specified codon usage patterns of the genes, are also being shaped by strong selectional pressure. This comprehensive analysis of BEFV genes (G, M, N, and P) sheds light on the molecular evolutionary patterns, co-adaptation, and different genes expression in diverse regions, facilitating the development of preventative programs and insights into viral pathogenesis and vaccine design. Communicated by Ramaswamy H. Sarma

Comprehensive Profiling of EBV Gene Expression and Promoter Methylation Reveals Latency II Viral Infection and Sporadic Abortive Lytic Activation in Peripheral T-Cell Lymphomas.

Epstein-Barr virus (EBV) latency patterns are well defined in EBV-associated epithelial, NK/T-cell, and B-cell malignancies, with links between latency stage and tumorigenesis deciphered in various studies. In vitro studies suggest that the oncogenic activity of EBV in T-cells might be somewhat different from that in EBV-tropic B lymphoid cells, prompting us to study this much less investigated viral gene expression pattern and its regulation in nine EBV+ peripheral T-cell lymphoma (PTCL) biopsies. Using frozen specimens, RT-PCR showed 6/7 cases with a latency II pattern of EBV gene expression. Analyses of EBNA1 promoter usage and CpG methylation status in these six cases showed that only Qp was used, while Cp, Wp, and Fp were all silent. However, the remaining case showed an exceptionally unique latency III type with lytic activation, as evidenced by EBV lytic clonality and confirmed by the full usage of Cp and Qp as well as weakly lytic Fp and Wp, fully unmethylated Cp and marginally unmethylated Wp. Further immunostaining of the eight cases revealed a few focally clustered LMP1+ cells in 7/8 cases, with rare isolated LMP1+ cells detected in another case. Double immunostaining confirmed that the LMP1+ cells were of the T-cell phenotype (CD3+). In 6/8 cases, sporadically scattered Zta+ cells were detected. Double staining of EBER-ISH with T-cell (CD45RO/UCHL1) or B-cell (CD20) markers confirmed that the vast majority of EBER+ cells were of the T-cell phenotype. Predominant type-A EBV variant and LMP1 30-bp deletion variant were present, with both F and f variants detected. In summary, the EBV gene expression pattern in PTCL was found to be mainly of latency II (BART+EBNA1(Qp)+LMP1+LMP2A+BZLF1+), similar to that previously reported in EBV-infected nasopharyngeal epithelial, NK/T-cell, and Hodgkin malignancies; however, fully lytic infection could also be detected in occasional cases. Rare cells with sporadic immediate-early gene expression were commonly detected in PTCL. These findings have implications for the future development of EBV-targeting therapeutics for this cancer.

Single cell atlas for 11 non-model mammals, reptiles and birds

The availability of viral entry factors is a prerequisite for the cross-species transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Large-scale single-cell screening of animal cells could reveal the expression patterns of viral entry genes in different hosts. However, such exploration for SARS-CoV-2 remains limited. Here, we perform single-nucleus RNA sequencing for 11 non-model species, including pets (cat, dog, hamster, and lizard), livestock (goat and rabbit), poultry (duck and pigeon), and wildlife (pangolin, tiger, and deer), and investigated the co-expression of ACE2 and TMPRSS2. Furthermore, cross-species analysis of the lung cell atlas of the studied mammals, reptiles, and birds reveals core developmental programs, critical connectomes, and conserved regulatory circuits among these evolutionarily distant species. Overall, our work provides a compendium of gene expression profiles for non-model animals, which could be employed to identify potential SARS-CoV-2 target cells and putative zoonotic reservoirs.

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

The Ends Dictate the Means: Promoter Switching in Herpesvirus Gene Expression.

Herpesvirus gene expression is dynamic and complex, with distinct complements of viral genes expressed at specific times in different infection contexts. These complex patterns of viral gene expression arise in part from the integration of multiple cellular and viral signals that affect the transcription of viral genes. The use of alternative promoters provides an increased level of control, allowing different promoters to direct the transcription of the same gene in response to distinct temporal and contextual cues. While once considered rare, herpesvirus alternative promoter usage was recently found to be far more pervasive and impactful than previously thought. Here we review several examples of promoter switching in herpesviruses and discuss the functional consequences on the transcriptional and post-transcriptional regulation of viral gene expression.

Cas9-targeted nanopore sequencing reveals epigenetic heterogeneity after de novo assembly of native full-length hepatitis B virus genomes.

Hepatitis B virus (HBV) contains a 3.2 kb DNA genome and causes acute and chronic hepatitis. HBV infection is a global health problem, with 350 million chronically infected people at increased risk of developing liver disease and hepatocellular carcinoma (HCC). Methylation of HBV DNA in a CpG context (5mCpG) can alter the expression patterns of viral genes related to infection and cellular transformation. Moreover, it may also provide clues as to why certain infections are cleared or persist with or without progression to cancer. The detection of 5mCpG often requires techniques that damage DNA or introduce bias through a myriad of limitations. Therefore, we developed a method for the detection of 5mCpG on the HBV genome that does not rely on bisulfite conversion or PCR. With Cas9-guided RNPs to specifically target the HBV genome, we enriched in HBV DNA from primary human hepatocytes (PHHs) infected with different HBV genotypes, as well as enriching in HBV from infected patient liver tissue, followed by sequencing with Oxford Nanopore Technologies MinION. Detection of 5mCpG by nanopore sequencing was benchmarked with bisulfite-quantitative methyl-specific qPCR (BS-qMSP). The 5mCpG levels in HBV determined by BS-qMSP and nanopore sequencing were highly correlated. Our nanopore sequencing approach achieved a coverage of ~2000× of HBV depending on infection efficiency, sufficient coverage to perform a de novo assembly and detect small fluctuations in HBV methylation, providing the first de novo assembly of native HBV DNA, as well as the first landscape of 5mCpG from native HBV sequences. Moreover, by capturing entire HBV genomes, we explored the epigenetic heterogeneity of HBV in infected patients and identified four epigenetically distinct clusters based on methylation profiles. This method is a novel approach that enables the enrichment of viral DNA in a mixture of nucleic acid material from different species and will serve as a valuable tool for infectious disease monitoring.

Defective Epstein-Barr Virus Genomes and Atypical Viral Gene Expression in B-Cell Lines Derived from Multiple Myeloma Patients.

Epstein-Barr virus (EBV) is a human gammaherpesvirus that is causally associated with various lymphomas and carcinomas. Although EBV is not typically associated with multiple myeloma (MM), it can be found in some B-cell lines derived from MM patients. Here, we analyzed two EBV-positive MM-patient-derived cell lines, IM9 and ARH77, and found defective viral genomes and atypical viral gene expression patterns. We performed transcriptome sequencing to characterize the viral and cellular properties of the two EBV-positive cell lines, compared to the canonical MM cell line 8226. Principal-component analyses indicated that IM9 and ARH77 clustered together and distinct from 8226. Immunological Genome Project analysis designated these cells as stem cell and bone marrow derived. IM9 and ARH77 displayed atypical viral gene expression, including leaky lytic cycle gene expression with an absence of lytic DNA amplification. Genome sequencing revealed that the EBV genomes in ARH77 contain large deletions, while IM9 has copy number losses in multiple EBV loci. Both IM9 and ARH77 showed EBV genome heterogeneity, suggesting cells harboring multiple and variant viral genomes. We identified atypical high-level expression of lytic genes BLRF1 and BLRF2. We demonstrated that short hairpin RNA (shRNA) depletion of BLRF2 altered viral and host gene expression, including a reduction in lytic gene activation and DNA amplification. These findings demonstrate that aberrant viral genomes and lytic gene expression persist in rare B cells derived from MM tumors, and they suggest that EBV may contribute to the etiology of MM. IMPORTANCE EBV is an oncogenic herpesvirus, but its mechanisms of oncogenesis are not fully understood. A role for EBV in MM has not yet been established. We analyzed EBV-positive B-cell lines derived from MM patients and found that the cells harbored defective viral genomes with aberrant viral gene expression patterns and cell gene signatures for bone marrow-derived lymphoid stem cells. These findings suggest that aberrant EBV latent infection may contribute to the etiology of MM.

Adult T-cell leukemia-lymphoma as a viral disease: Subtypes based on viral aspects.

Adult T‐cell leukemia‐lymphoma (ATL) is caused by human T‐cell leukemia virus type 1 (HTLV‐1) infection. Among HTLV‐1 encoded genes, HTLV‐1 bZIP factor (HBZ) and tax are critical for the leukemogenesis of ATL. Adult T‐cell leukemia‐lymphoma needs a long latent period before onset, indicating that both viral genes and alterations (genetic and epigenetic) of the host genome play important roles for leukemogenesis. Viral genes influence genetic and epigenetic changes of the host genome, indicating that the virus is of primary importance in leukemogenesis. HBZ is expressed in all ATL cases, whereas Tax expression is heterogeneous among ATL cases. Different patterns of viral gene expression in tumors are also observed for Epstein‐Barr virus. We propose three subtypes of ATL cases based on Tax expression: high, intermittent, and lost expression. HBZ is detected in all ATL cases. Approximately 25% of all ATL cases lost Tax expression at infection of HTLV‐1, indicating that HBZ is the only viral gene responsible for leukemogenesis in addition to genetic and epigenetic changes of the host genes in these ATL cases. The host immune responses to Tax are also implicated in the heterogeneity of ATL. Thus, ATL is a heterogeneous disease in terms of its viral gene expression, which is important for pathogenesis of this intractable lymphomatous neoplasm.

Use of Cap Analysis Gene Expression to detect human papillomavirus promoter activity patterns at different disease stages

Transcription of human papillomavirus (HPV) genes proceeds unidirectionally from multiple promoters. Direct profiling of transcription start sites (TSSs) by Cap Analysis Gene Expression (CAGE) is a powerful strategy for examining individual HPV promoter activity. The objective of this study was to evaluate alterations of viral promoter activity during infection using CAGE technology. We used CAGE-based sequencing of 46 primary cervical samples, and quantitatively evaluated TSS patterns in the HPV transcriptome at a single-nucleotide resolution. TSS patterns were classified into two types: early promoter-dominant type (Type A) and late promoter-dominant type (Type B). The Type B pattern was more frequently found in CIN1 and CIN2 lesions than in CIN3 and cancer samples. We detected transcriptomes from multiple HPV types in five samples. Interestingly, in each sample, the TSS patterns of both HPV types were the same. The viral gene expression pattern was determined by the differentiation status of the epithelial cells, regardless of HPV type. We performed unbiased analyses of TSSs across the HPV genome in clinical samples. Visualising TSS pattern dynamics, including TSS shifts, provides new insights into how HPV infection status relates to disease state.

Single cell heterogeneity in influenza A virus gene expression shapes the innate antiviral response to infection.

Viral infection outcomes are governed by the complex and dynamic interplay between the infecting virus population and the host response. It is increasingly clear that both viral and host cell populations are highly heterogeneous, but little is known about how this heterogeneity influences infection dynamics or viral pathogenicity. To dissect the interactions between influenza A virus (IAV) and host cell heterogeneity, we examined the combined host and viral transcriptomes of thousands of individual cells, each infected with a single IAV virion. We observed complex patterns of viral gene expression and the existence of multiple distinct host transcriptional responses to infection at the single cell level. We show that human H1N1 and H3N2 strains differ significantly in patterns of both viral and host anti-viral gene transcriptional heterogeneity at the single cell level. Our analyses also reveal that semi-infectious particles that fail to express the viral NS can play a dominant role in triggering the innate anti-viral response to infection. Altogether, these data reveal how patterns of viral population heterogeneity can serve as a major determinant of antiviral gene activation.

Immunosuppressive FK506 treatment leads to more frequent EBV-associated lymphoproliferative disease in humanized mice.

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication after organ transplantation frequently associated with the Epstein-Barr virus (EBV). Immunosuppressive treatment is thought to allow the expansion of EBV-infected B cells, which often express all eight oncogenic EBV latent proteins. Here, we assessed whether HLA-A2 transgenic humanized NSG mice treated with the immunosuppressant FK506 could be used to model EBV-PTLD. We found that FK506 treatment of EBV-infected mice led to an elevated viral burden, more frequent tumor formation and diminished EBV-induced T cell responses, indicative of reduced EBV-specific immune control. EBV latency III and lymphoproliferation-associated cellular transcripts were up-regulated in B cells from immunosuppressed animals, akin to the viral and host gene expression pattern found in EBV-PTLD. Utilizing an unbiased gene expression profiling approach, we identified genes differentially expressed in B cells of EBV-infected animals with and without FK506 treatment. Upon investigating the most promising candidates, we validated sCD30 as a marker of uncontrolled EBV proliferation in both humanized mice and in pediatric patients with EBV-PTLD. High levels of sCD30 have been previously associated with EBV-PTLD in patients. As such, we believe that humanized mice can indeed model aspects of EBV-PTLD development and may prove useful for the safety assessment of immunomodulatory therapies.

Human papillomavirus type 16 causes a defined subset of conjunctival in situ squamous cell carcinomas

Squamous cell carcinoma of the conjunctiva is associated with a number of risk factors, including HIV infection, iatrogenic immunosuppression and atopy. In addition, several studies have suggested an involvement of HPV, based on the presence of viral DNA, but did not establish whether there was active infection or evidence of causal disease association. In this manuscript, 31 cases of conjunctival in situ squamous cell carcinoma were classified as HPV DNA-positive or -negative, before being analysed by immunohistochemistry to establish the distribution of viral and cellular biomarkers of HPV gene expression. Our panel included p16INK4a, TP53 and MCM, but also the virally encoded E4 gene product, which is abundantly expressed during productive infection. Subsequent in situ detection of HPV mRNA using an RNAscope approach confirmed that early HPV gene expression was occurring in the majority of cases of HPV DNA-positive conjunctival in situ squamous cell carcinoma, with all of these cases occurring in the atopic group. Viral gene expression correlated with TP53 loss, p16INK4a elevation, and extensive MCM expression, in line with our general understanding of E6 and E7’s role during transforming infection at other epithelial sites. A characteristic E4 expression pattern was detected in only one case. HPV mRNA was not detected in lower grades of dysplasia, and was not observed in cases that were HPV DNA-negative. Our study demonstrates an active involvement of HPV in the development of a subset of conjunctival in situ squamous cell carcinoma. No high-risk HPV types were detected other than HPV16. It appears that the conjunctiva is a vulnerable epithelial site for HPV-associated transformation. These cancers are defined by their pattern of viral gene expression, and by the distribution of surrogate markers of HPV infection.

High-Throughput Sequence Analysis of Peripheral T-Cell Lymphomas Indicates Subtype-Specific Viral Gene Expression Patterns and Immune Cell Microenvironments.

Certain peripheral T-cell lymphomas (PTCLs) have been associated with viral infection, particularly infection with Epstein-Barr virus (EBV). However, a comprehensive virome analysis across PTCLs has not previously been reported. Here we utilized published whole-transcriptome RNA sequencing (RNA-seq) data sets from seven different PTCL studies and new RNA-seq data from our laboratory to screen for virus association, to analyze viral gene expression, and to assess B- and T-cell receptor diversity paradigms across PTCL subtypes. In addition to identifying EBV in angioimmunoblastic T-cell lymphoma (AITL) and extranodal NK/T-cell lymphoma (ENKTL), two PTCL subtypes with well-established EBV associations, we also detected EBV in several cases of anaplastic large-cell lymphoma (ALCL), and we found evidence of infection by the oncogenic viruses Kaposi's sarcoma-associated herpesvirus and human T-cell leukemia virus type 1 in isolated PTCL cases. In AITLs, EBV gene expression analysis showed expression of immediate early, early, and late lytic genes, suggesting either low-level lytic gene expression or productive infection in a subset of EBV-infected B-lymphocyte stromal cells. Deconvolution of immune cell subpopulations demonstrated a greater B-cell signal in AITLs than in other PTCL subtypes, consistent with a larger role for B-cell support in the pathogenesis of AITL. Reconstructed T-cell receptor (TCR) and B-cell receptor (BCR) repertoires demonstrated increased BCR diversity in AITLs, consistent with a possible EBV-driven polyclonal response. These findings indicate potential alternative roles for EBV in PTCLs, in addition to the canonical oncogenic mechanisms associated with EBV latent infection. Our findings also suggest the involvement of other viruses in PTCL pathogenesis and demonstrate immunological alterations associated with these cancers.IMPORTANCE In this study, we utilized next-generation sequencing data from 7 different studies of peripheral T-cell lymphoma (PTCL) patient samples to globally assess viral associations, provide insights into the contributions of EBV gene expression to the tumor phenotype, and assess the unique roles of EBV in modulating the immune cell tumor microenvironment. These studies revealed potential roles for EBV replication genes in some PTCL subtypes, the possible role of additional human tumor viruses in rare cases of PTCLs, and a role for EBV in providing a unique immune microenvironmental niche in one subtype of PTCLs. Together, these studies provide new insights into the understudied role of tumor viruses in PTCLs.

RNA-seq analysis of viral gene expression in the skin of Marek’s disease virus infected chickens

Marek’s disease virus (MDV), a highly cell-associated oncogenic avian α-herpesvirus, is the causative agent of malignant transformation of T cells in domestic chickens. The latently infected CD4+CD8− T cells carry the virus through the blood stream and establish lymphomas in the skin, visceral organs and peripheral nerves. The feather follicle epithelium (FFE) is the only anatomical site where fully infectious enveloped virions are produced and eventually disseminated into the environment to infect contact birds. Therefore, skin and FFE play a critical role as being the common source of re-infection of birds sharing the same habitat. The molecular mechanism involved in the replication and assembly of MDV in the FFE leading to the production and release of cell-free infectious virus particles is unknown and to date no viral or host gene has been implicated in the process. To examine alterations in the expression pattern of viral genes, we performed RNA-seq on the skin samples of Marek’s disease virus-infected susceptible chickens at 10, 20, and 30 days post infection. For comparative analysis of the expression patterns of viral genes between the skin and spleen of the MD-susceptible and resistant lines, Real-Time RT-PCR was employed. In total, RNA-seq based analysis identified 42 viral genes that were differentially expressed in the skin of infected birds. Majority of the identified genes are involved in DNA replication, capsid, tegument, and envelop formation. Comparative analysis between the skin and spleen of MD-susceptible and resistant chicken lines, revealed significantly higher expression of the genes in the skin of either lines than the spleen. Furthermore, much higher expression of the genes was observed in the skin of the susceptible line than the resistant line.

The pathology of epstein-barr virus lymphoproliferations.

The pathology of epstein-barr virus lymphoproliferations.

Revisiting the Tissue Microenvironment of Infectious Mononucleosis: Identification of EBV Infection in T Cells and Deep Characterization of Immune Profiles.

To aid understanding of primary EBV infection, we have performed an in depth analysis of EBV-infected cells and of local immune cells in tonsils from infectious mononucleosis (IM) patients. We show that EBV is present in approximately 50% of B-cells showing heterogeneous patterns of latent viral gene expression probably reflecting different stages of infection. While the vast majority of EBV+ cells are B-cells, around 9% express T-cell antigens, with a predominance of CD8+ over CD4+ cells. PD-L1 was expressed by a median of 14% of EBV+ cells. The numbers of EBER+PD-L1+ cells were directly correlated with the numbers of EBER+CD3+ and EBER+CD8+ cells suggesting a possible role for PD-L1 in EBV infection of T-cells. The microenvironment of IM tonsils was characterized by a predominance of M1-polarized macrophages over M2-polarized cells. However, at the T-cell level, a heterogeneous picture emerged with numerous Th1/cytotoxic cells accompanied and sometimes outnumbered by Th2/regulatory T-cells. Further, we observed a direct correlation between the numbers of Th2-like cells and EBV– B-cells. Also, a prevalence of cytotoxic T-cells over Th2-like cells was associated with an increased viral load. These observations point to contribution of B- and Th2-like cells to the control of primary EBV infection. 35% of CD8+ cells were differentiated CD8+TBET+ cells, frequently detected in post-capillary venules. An inverse correlation was observed between the numbers of CD8+TBET+ cells and viral load suggesting a pivotal role for these cells in the control of primary EBV infection. Our results provide the basis for a better understanding of immune reactions in EBV-associated tumors.

Epstein-Barr virus microRNAs regulate B cell receptor signal transduction and lytic reactivation.

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch.

Global Analysis of Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Gene Expression in the Midgut of the Lepidopteran Host Trichoplusia ni.

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a large double-stranded DNA (dsDNA) virus that encodes approximately 156 genes and is highly pathogenic to a variety of larval lepidopteran insects in nature. Oral infection of larval midgut cells is initiated by the occlusion-derived virus (ODV), while secondary infection of other tissues is mediated by the budded virus (BV). Global viral gene expression has been studied in detail in BV-infected cell cultures, but studies of ODV infection in the larval midgut are limited. In this study, we examined expression of the ∼156 AcMNPV genes in Trichoplusia ni midgut tissue using a transcriptomic approach. We analyzed expression profiles of viral genes in the midgut and compared them with profiles from a T. ni cell line (Tnms42). Several viral genes (p6.9, orf76, orf75, pp31, Ac-bro, odv-e25, and odv-ec27) had high expression levels in the midgut throughout the infection. Also, the expression of genes associated with occlusion bodies (polh and p10) appeared to be delayed in the midgut in comparison with the cell line. Comparisons of viral gene expression profiles revealed remarkable similarities between the midgut and cell line for most genes, although substantial differences were observed for some viral genes. These included genes associated with high level BV production (fp-25k), acceleration of systemic infection (v-fgf), and enhancement of viral movement (arif-1/orf20). These differential expression patterns appear to represent specific adaptations for virus infection and transmission through the polarized cells of the lepidopteran midgut.IMPORTANCE Baculoviruses such as AcMNPV are pathogens that are natural regulators of certain insect populations. Baculovirus infections are biphasic, with a primary phase initiated by oral infection of midgut epithelial cells by occlusion-derived virus (ODV) virions and a secondary phase in which other tissues are infected by budded-virus (BV) virions. While AcMNPV infections in cultured cells have been studied extensively, comparatively little is known regarding primary infection in the midgut. In these studies, we identified gene expression patterns associated with ODV-mediated infection of the midgut in Trichoplusia ni and compared those results with prior results from BV-infected cultured cells, which simulate secondary infection. These studies provide a detailed analysis of viral gene expression patterns in the midgut, which likely represent specific viral strategies to (i) overcome or avoid host defenses in the gut and (ii) rapidly move infection from the midgut, into the hemocoel to facilitate systemic infection.

Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2.

Epstein-Barr virus (EBV) associated Burkitt’s lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8 positive T-cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease.

Genomic characterization of human papillomavirus-positive and -negative human squamous cell cancer cell lines.

Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.