Epstein-Barr virus replication within differentiated epithelia requires pRb sequestration of activator E2F transcription factors.

Epstein-Barr virus (EBV) co-infections with human papillomavirus (HPV) have been observed in oropharyngeal squamous cell carcinoma. Modeling EBV/HPV co-infection in organotypic epithelial raft cultures revealed that HPV16 E7 inhibited EBV productive replication through the facilitated degradation of the retinoblastoma protein pRb/p105. To further understand how pRb is required for EBV productive replication, we generated CRISPR-Cas9 pRb knockout (KO) normal oral keratinocytes (NOKs) in the context of wild-type and mutant K120E p53. EBV replication was examined in organotypic rafts as a physiological correlate for epithelial differentiation. In pRb KO rafts, EBV DNA copy number was statistically decreased compared to vector controls, regardless of p53 context. Loss of pRb did not affect EBV binding or internalization of calcium-treated NOKs or early infection of rafts. Rather, the block in EBV replication correlated with impaired immediate early gene expression. An EBV infection time course in rafts with mutant p53 demonstrated that pRb-positive basal cells were initially infected with delayed replication occurring in differentiated layers. Loss of pRb showed increased S-phase progression makers and elevated activator E2F activity in raft tissues. Complementation with a panel of pRb/E2F binding mutants showed that wild type or pRb∆685 mutant capable of E2F binding reduced S-phase marker gene expression, rescued EBV DNA replication, and restored BZLF1 expression in pRb KO rafts. However, pRb KO complemented with pRb661W mutant, unable to bind E2Fs, failed to rescue EBV replication in raft culture. These findings suggest that EBV productive replication in differentiated epithelium requires pRb inhibition of activator E2Fs to restrict S-phase progression.IMPORTANCEA subset of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma is co-positive for Epstein-Barr virus (EBV). Potential oncogenic viral interactions revealed that HPV16 E7 inhibited productive EBV replication within the differentiated epithelium. As E7 mediates the degradation of pRb, we aimed to establish how pRb is involved in EBV replication. In the context of differentiated epithelium using organotypic raft culture, we evaluated how the loss of pRb affects EBV lytic replication to better comprehend EBV contributions to carcinogenesis. In this study, ablation of pRb interfered with EBV replication at the level of immediate early gene expression. Loss of pRb increased activator E2Fs and associated S-phase gene expression throughout the differentiated epithelium. Complementation studies showed that wild type and pRb mutant capable of binding to E2F rescued EBV replication, while pRb mutant lacking E2F binding did not. Altogether, these studies support that in differentiated tissues, HPV16 E7-mediated degradation of pRb inhibits EBV replication through unregulated E2F activity.

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

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.

Lipin-1 restrains macrophage lipid synthesis to promote inflammation resolution.

Macrophages are critical to maintaining and restoring tissue homeostasis during inflammation. The lipid metabolic state of macrophages influences their function, but a deeper understanding of how lipid metabolism is regulated in pro-resolving macrophage responses is needed. Lipin-1 is a phosphatidic acid phosphatase with a transcriptional coregulatory activity (TC) that regulates lipid metabolism. We previously demonstrated that lipin-1 supports pro-resolving macrophage responses, and here, myeloid-associated lipin-1 is required for inflammation resolution, yet how lipin-1-regulated cellular mechanisms promote macrophage pro-resolution responses is unknown. We demonstrated that the loss of lipin-1 in macrophages led to increased free fatty acid, neutral lipid, and ceramide content and increased phosphorylation of acetyl-CoA carboxylase. The inhibition of the first step of lipid synthesis and transport of citrate from the mitochondria in macrophages reduced lipid content and restored efferocytosis and inflammation resolution in lipin-1mKO macrophages and mice. Our findings suggest macrophage-associated lipin-1 restrains lipid synthesis, promoting pro-resolving macrophage function in response to pro-resolving stimuli.

EBV Association with Lymphomas and Carcinomas in the Oral Compartment.

Epstein-Barr virus (EBV) is an oncogenic human herpesvirus infecting approximately 90% of the world's population. The oral cavity serves a central role in the life cycle, transmission, and pathogenesis of EBV. Transmitted to a new host via saliva, EBV circulates between cellular compartments within oral lymphoid tissues. Epithelial cells primarily support productive viral replication, while B lymphocytes support viral latency and reactivation. EBV infections are typically asymptomatic and benign; however, the latent virus is associated with multiple lymphomas and carcinomas arising in the oral cavity. EBV association with cancer is complex as histologically similar cancers often test negative for the virus. However, the presence of EBV is associated with distinct features in certain cancers. The intrinsic ability of EBV to immortalize B-lymphocytes, via manipulation of survival and growth signaling, further implicates the virus as an oncogenic cofactor. A distinct mutational profile and burden have been observed in EBV-positive compared to EBV-negative tumors, suggesting that viral infection can drive alternative pathways that converge on oncogenesis. Taken together, EBV is also an important prognostic biomarker that can direct alternative therapeutic approaches. Here, we discuss the prevalence of EBV in oral malignancies and the EBV-dependent mechanisms associated with tumorigenesis.