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. Whether excessive lytic EBV infection increases the risk of EBV-induced cancers is not clear. A particular variant (Zp-V3) of the viral promoter driving expression of the EBV immediate-early BZLF1 (Z) protein that mediates lytic viral reactivation has been reported to be over-represented (relative to the prototype Zp-P form of the promoter) in certain EBV-positive malignancies, but no functional difference between the two promoter variants has been reported. Here we show that the malignancy-associated Zp-V3 variant (but not the Zp-P variant) contains a binding site for the cellular NFATc1 (nuclear factor of activated T cells c1) transcription factor that allows it to be activated by NFATc1-inducing stimuli such as B-cell receptor stimulation. Furthermore, we demonstrate that restoring this NFATc1-motif to the Zp-P variant in the context of the intact EBV genome greatly enhances lytic viral reactivation in response to the NFATc1-inducing stimuli. We also find that the Zp-V3 variant is over-represented in EBV-positive Burkitt lymphomas and gastric carcinomas, and in lymphoblastoid cell lines transformed by EBV-infected breast milk of Kenyan mothers that had malaria during pregnancy. These findings suggest that the Zp-V3 version of the EBV BZLF1 promoter increases the likelihood of EBV-induced malignancies by increasing lytic EBV infection.

When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs). We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV). Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE) located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK) and gingival epithelial (hGET) cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV’s natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for development of a lytic-induction therapy for treating some EBV-associated malignancies. Most adults throughout the world are infected with Epstein-Barr virus (EBV), a human herpesvirus frequently associated in a latent state with some cancers of epithelial and B-cell origin such as nasopharyngeal carcinoma and Burkitt lymphoma, respectively. To develop an oncolytic therapy for treating patients with EBV-associated cancers, we need a method to efficiently induce synthesis of lytic EBV proteins. The EBV protein encoded by its immediate-early BZLF1 gene usually mediates the switch into lytic viral infection. We show here that HIF-1α, a cellular transcription factor that accumulates in cells when deprived of normal levels of oxygen, can induce lytic EBV infection. HIF-1α mediates this switch by directly binding to a specific sequence located within the BZLF1 gene promoter, activating its expression. Importantly, we also show that deferoxamine, an FDA-approved drug that inhibits degradation of HIF-1α, can induce synthesis of lytic EBV proteins in some EBV-positive epithelial and lymphocytic cell lines. These findings indicate that HIF-1α-stabilizing drugs, administered in combination with nucleoside analogues such as ganciclovir, may be helpful as part of a lytic-induction therapy for treating some patients with EBV-positive malignancies.

Humans are infected with two distinct strains (Type 1 (T1) and Type 2 (T2)) of Epstein-Barr virus (EBV) that differ substantially in their EBNA2 and EBNA 3A/B/C latency genes and the ability to transform B cells in vitro. While most T1 EBV strains contain the “prototype” form of the BZLF1 immediate-early promoter (“Zp-P”), all T2 strains contain the “Zp-V3” variant, which contains an NFAT binding motif and is activated much more strongly by B-cell receptor signalling. Whether B cells infected with T2 EBV are more lytic than cells infected with T1 EBV is unknown. Here we show that B cells infected with T2 EBV strains (AG876 and BL5) have much more lytic protein expression compared to B cells infected with T1 EBV strains (M81, Akata, and Mutu) in both a cord blood-humanized (CBH) mouse model and EBV-transformed lymphoblastoid cell lines (LCLs). Although T2 LCLs grow more slowly than T1 LCLs, both EBV types induce B-cell lymphomas in CBH mice. T1 EBV strains (M81 and Akata) containing Zp-V3 are less lytic than T2 EBV strains, suggesting that Zp-V3 is not sufficient to confer a lytic phenotype. Instead, we find that T2 LCLs express much higher levels of activated NFATc1 and NFATc2, and that cyclosporine (an NFAT inhibitor) and knockdown of NFATc2 attenuate constitutive lytic infection in T2 LCLs. Both NFATc1 and NFATc2 induce lytic EBV gene expression when combined with activated CAMKIV (which is activated by calcium signaling and activates MEF2D) in Burkitt Akata cells. Together, these results suggest that B cells infected with T2 EBV are more lytic due to increased activity of the cellular NFATc1/c2 transcription factors in addition to the universal presence of the Zp-V3 form of BZLF1 promoter. EBV causes various human B cell lymphomas and infects B cells in latent or lytic forms. The two different EBV strains (Type 1 (T1) and Type 2 (T2)) are divergent in their EBNA2 and EBNA3A/B/C latency genes, and T1 EBV is more transforming than T2 EBV in vitro. Interestingly, all T2 strains contain the Zp-V3 form of the BZLF1 immediate-early promoter (which regulates lytic viral reactivation) while most T1 strains contain the Zp-P form; the Zp-V3 variant is more responsive to BCR stimulation due to the presence of an NFAT binding site. Here we show that T2 and T1 EBV both induce lymphomas in cord blood-humanized mice, but T2 EBV infected mice have increased lytic infection. Using in vitro generated LCLs, we find that both total and activated NFATc1 and NFATc2 are elevated in T2 EBV-infected LCLs compared to T1 LCLs, and demonstrate that enhanced NFAT activity is required for the lytic phenotype in T2 LCLs. Knockdown of BZLF1 gene expression decreases the growth rate of T2 LCLs. These results suggest that an increased ability to enter lytic infection may partially compensate for the decreased transforming phenotype of T2 EBV infection.

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. The EBV protein, EBNA2, is required for the ability of EBV to transform B cells in vitro, but EBV-infected cells that express EBNA2 (and its downstream viral gene targets) are highly immunogenic and thus EBV-positive tumors in immunocompetent patients almost never express EBNA2. Currently, there are no model systems for studying how EBV causes B-cell lymphomas in the absence of EBNA2 expression in the context of the intact viral genome. Here we show that a naturally occurring EBNA2-deleted EBV strain (P3HR1) isolated from a human BL causes two different types of lymphomas in a cord blood-humanized mouse model. Some P3HR1-infected lymphoma cells phenotypically resemble the RS cell of CHL and express high levels of the viral LMP1 protein, a protein thought to drive EBV-positive human CHLs. The other P3HR1-induced lymphomas resemble DLBCLs and have a viral protein expression pattern consistent with Wp-restricted latency (EBNA-LP positive, LMP1 negative) similar to that occurring in human BLs infected with EBNA2-deleted EBV. These studies are the first to show that an EBNA2-deleted EBV can cause lymphomas in humanized mice with restricted forms of viral latency that resemble human CHLs and DLBCLs.

EBV causes human B-cell lymphomas and transforms B cells in vitro. EBNA3C, an EBV protein expressed in latently-infected cells, is required for EBV transformation of B cells in vitro. While EBNA3C undoubtedly plays a key role in allowing EBV to successfully infect B cells, many EBV+ lymphomas do not express this protein, suggesting that cellular mutations and/or signaling pathways may obviate the need for EBNA3C in vivo under certain conditions. EBNA3C collaborates with EBNA3A to repress expression of the CDKN2A-encoded tumor suppressors, p16 and p14, and EBNA3C-deleted EBV transforms B cells containing a p16 germline mutation in vitro. Here we have examined the phenotype of an EBNAC-deleted virus (Δ3C EBV) in a cord blood-humanized mouse model (CBH). We found that the Δ3C virus induced fewer lymphomas (occurring with a delayed onset) in comparison to the wild-type (WT) control virus, although a subset (10/26) of Δ3C-infected CBH mice eventually developed invasive diffuse large B cell lymphomas with type III latency. Both WT and Δ3C viruses induced B-cell lymphomas with restricted B-cell populations and heterogeneous T-cell infiltration. In comparison to WT-infected tumors, Δ3C-infected tumors had greatly increased p16 levels, and RNA-seq analysis revealed a decrease in E2F target gene expression. However, we found that Δ3C-infected tumors expressed c-Myc and cyclin E at similar levels compared to WT-infected tumors, allowing cells to at least partially bypass p16-mediated cell cycle inhibition. The anti-apoptotic proteins, BCL2 and IRF4, were expressed in Δ3C-infected tumors, likely helping cells avoid c-Myc-induced apoptosis. Unexpectedly, Δ3C-infected tumors had increased T-cell infiltration, increased expression of T-cell chemokines (CCL5, CCL20 and CCL22) and enhanced type I interferon response in comparison to WT tumors. Together, these results reveal that EBNA3C contributes to, but is not essential for, EBV-induced lymphomagenesis in CBH mice, and suggest potentially important immunologic roles of EBNA3C in vivo. Epstein-Barr virus is associated with multiple human B-cell malignancies and transforms B cells in vitro into immortalized cell lines. The EBV protein, EBNA3C, inhibits expression of the tumor suppressor protein, p16, and is required for EBV transformation of B cells in vitro. Here, we show that an EBNA3C-deleted virus (Δ3C) has a reduced ability (in comparison to the wild-type control virus) to cause B-cell lymphomas in a cord blood-humanized mouse model, although a subset of infected animals develop lymphomas at later time points. These tumors had high-level expression of the cell cycle inhibitor, p16, yet also expressed the cell cycle activators, c-Myc and cyclin E. Tumors also expressed pro-survival proteins, including BCL2 and IRF4. Unexpectedly, Δ3C tumors had increased T-cell infiltration, enhanced expression of several different T-cell chemokines, and a RNA-seq signature suggestive of an increased type 1 Interferon response. These results reveal that EBNA3C promotes EBV-induced lymphomas in the cord blood-humanized mouse model (but is not absolutely required), and suggest that EBNA3C may have immune modulatory effects in vivo.

Human papillomaviruses (HPVs) cause a substantial amount of human disease from benign disease such as warts to malignant cancers including cervical carcinoma, head and neck cancer, and non-melanoma skin cancer. Our ability to model HPV-induced malignant disease has been impeded by species specific barriers and pre-clinical animal models have been challenging to develop. The recent discovery of a murine papillomavirus, MmuPV1, that infects laboratory mice and causes the same range of malignancies caused by HPVs provides the papillomavirus field the opportunity to test mechanistic hypotheses in a genetically manipulatable laboratory animal species in the context of natural infections. The E6 and E7 proteins encoded by high-risk HPVs, which are the HPV genotypes associated with human cancers, are multifunctional proteins that contribute to HPV-induced cancers in multiple ways. In this review, we describe the known activities of the MmuPV1-encoded E6 and E7 proteins and how those activities relate to the activities of HPV E6 and E7 oncoproteins encoded by mucosal and cutaneous high-risk HPV genotypes.