Probes for INS

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

In 2017, the World Health Organization (WHO) confirmed a new entity, Epstein Barr virus (EBV) + Diffuse large B cell lymphoma (DLBCL), not otherwise specified (NOS). Traces of EBV transcripts were described in lymphomas, including DLBCL, that were diagnosed as EBV negative by conventional methods. The aim of this study was to detect viral genome by qPCR, as well as LMP1 and EBNA2 transcripts, with a more sensitive method in DLBCL cases from Argentina. Fourteen cases originally considered as EBV negative expressed LMP1 and/or EBNA2 transcripts. In addition, LMP1 and/or EBNA2 transcripts were also observed in bystander cells. However, EBERs+ cells cases by conventional ISH showed higher numbers of cells with LMP1 transcripts and LMP1 protein. In the cases that were EBERS- in tumor cells but with expression of LMP1 and/or EBNA2 transcripts, the viral load was below the limit of detection. This study provides further evidence that EBV could be detected in tumor cells by more sensitive methods. However, higher expression of the most important oncogenic protein, LMP1, as well as increased viral load, are only observed in cases with EBERs+ cells by conventional ISH, suggesting that traces of EBV might not display a key role in DLBCL pathogenesis.

Viral pathogens have been implicated in the development of certain cancers including human papillomavirus (HPV) in squamous cell carcinoma and Epstein-Barr virus (EBV) in Burkitt's lymphoma. The significance of viral pathogens in brain tumors is controversial, and human cytomegalovirus (HCMV) has been associated with glioblastoma (GBM) in some but not all studies, making the role of HCMV unclear. In this study we sought to determine if viral pathogen sequences could be identified in an unbiased manner from previously discarded, unmapped, non-human, next-generation sequencing (NGS) reads obtained from targeted oncology, panel-based sequencing of high grade gliomas (HGGs), including GBMs. Twenty one sequential HGG cases were analyzed by a targeted NGS clinical oncology panel containing 151 genes using DNA obtained from formalin-fixed, paraffin-embedded (FFPE) tissue. Sequencing reads that did not map to the human genome (average of 38,000 non-human reads/case (1.9%)) were filtered and low quality reads removed. Extracted high quality reads were then sequentially aligned to the National Center for Biotechnology Information (NCBI) non-redundant nucleotide (nt and nr) databases. Aligned reads were classified based on NCBI taxonomy database and all eukaryotic viral sequences were further classified into viral families. Two viral sequences (both herpesviruses), EBV and Roseolovirus were detected in 5/21 (24%) cases and in 1/21 (5%) cases, respectively. None of the cases had detectable HCMV. Of the five HGG cases with detectable EBV DNA, four had additional material for EBV in situ hybridization (ISH), all of which were negative for expressed viral sequence. Overall, a similar discovery approach using unmapped non-human NGS reads could be used to discover viral sequences in other cancer types.