Latent membrane proteins from EBV differentially target cellular pathways to accelerate MYC-induced lymphomagenesis.

MYC translocations in association with Epstein-Barr virus (EBV) infection are often observed in B-cell lymphomas. A subset of Burkitt lymphoma (BL) expresses EBV latent membrane proteins 1 and 2A (LMP1 and LMP2A) in addition to the typical restricted EBV latent gene expression. EBV-associated diffuse large B-cell lymphoma (DLBCL) typically exhibits latency type II or III and expresses LMP1. Here, we investigate the role of LMP1 in MYC-driven lymphomagenesis in our murine model. λ-MYC mice develop tumors having a "starry sky" appearance and have abnormal p53 expression that is also observed in human BL. LMP2A/λ-MYC double-transgenic mice develop tumors significantly faster than mice only expressing MYC. Similar to LMP2A/λ-MYC mice, LMP1/λ-MYC mice also have accelerated MYC-driven lymphomagenesis. As observed in LMP2A/λ-MYC mice, p27kip1 was degraded in LMP1/λ-MYC pretumor and tumor B cells. Coexpression of LMP1 and LMP2A resulted in the enhancement of B cell proliferation. In contrast to LMP2A, the inhibition of Syk or cyclin-dependant kinase (CDK)4/6 activity did not effectively inhibit LMP1-mediated MYC lymphomagenesis. Also, in contrast to LMP2A, LMP1 did not lessen abnormal p53 expression in λ-MYC tumors. To investigate the significance of LMP1 expression in human BL development, we reanalyzed RNA sequencing (RNA-Seq) data of primary human BL from previous studies. Interestingly, p53 mutations were less observed in LMP1-expressing BL, although they were not significantly changed by EBV infection, indicating LMP1 may lessen p53 mutations in human primary BL. This suggests that LMP1 effects in EBV-associated human BL vary from what we observe in our murine model. Finally, our studies suggest a novel pathogenic role of LMP1 in lymphomagenesis.

COVID-19 and oral diseases: Assessing manifestations of a new pathogen in oral infections.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is a recently identified virus responsible for life-threatening coronavirus disease 19 (COVID-19). The SARS-CoV-2 infected subjects can be asymptomatic or symptomatic; the later may present a wide spectrum of clinical manifestations. However, the impact of SARS-CoV-2 on oral diseases remain poorly studied. Detection of SARS-CoV-2 in saliva indicates existence of virus in the oral cavity. Recent studies demonstrating the expression of ACE-2, a SARS-CoV-2 entry receptor, in oral tissues further strengthens this observation. Cytokine storm in severe COVID-19 patients and copious secretion of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α) in multiple symptomatic oral pathologies including periodontitis and periapical periodontitis suggests that inflammatory microenvironment is a hallmark of both COVID-19 and oral diseases. Hyperinflammation may provide conducive microenvironment for the growth of local oral pathogens or opportunistic microbes and exert detrimental impact on the oral tissue integrity. Multiple case reports have indicated uncharacterized oral lesions, symptomatic irreversible pulpitis, higher plaque index, necrotizing/desquamative gingivitis in COVID-19 patients suggesting that SARS-CoV-2 may worsen the manifestations of oral infections. However, the underlying factors and pathways remain elusive. Here we summarize current literature and suggest mechanisms for viral pathogenesis of oral dental pathology derived from oral microbiome and oral mucosa-dental tissue interactions. Longitudinal studies will reveal how the virus impairs disease progression and resolution post-therapy. Some relationships we suggest provide the basis for novel monitoring and treatment of oral viral disease in the era of SARS-CoV-2 pandemic, promoting evidence-based dentistry guidelines to diagnose virus-infected patients to improve oral health.

Covid-19 and oral diseases: Crosstalk, synergy or association?

The coronavirus disease 2019 (Covid-19) is a viral infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that clinically affects multiple organs of the human body. Cells in the oral cavity express viral entry receptor angiotensin-converting enzyme 2 that allows viral replication and may cause tissue inflammation and destruction. Recent studies have reported that Covid-19 patients present oral manifestations with multiple clinical aspects. In this review, we aim to summarise main signs and symptoms of Covid-19 in the oral cavity, its possible association with oral diseases, and the plausible underlying mechanisms of hyperinflammation reflecting crosstalk between Covid-19 and oral diseases. Ulcers, blisters, necrotising gingivitis, opportunistic coinfections, salivary gland alterations, white and erythematous plaques and gustatory dysfunction were the most reported clinical oral manifestations in patients with Covid-19. In general, the lesions appear concomitant with the loss of smell and taste. Multiple reports show evidences of necrotic/ulcerative gingiva, oral blisters and hypergrowth of opportunistic oral pathogens. SARS-CoV-2 exhibits tropism for endothelial cells and Covid-19-mediated endotheliitis can not only promote inflammation in oral tissues but can also facilitate virus spread. In addition, elevated levels of proinflammatory mediators in patients with Covid-19 and oral infectious disease can impair tissue homeostasis and cause delayed disease resolution. This suggests potential crosstalk of immune-mediated pathways underlying pathogenesis. Interestingly, few reports suggest recurrent herpetic lesions and higher bacterial growth in Covid-19 subjects, indicating SARS-CoV-2 and oral virus/bacteria interaction. Larger cohort studies comparing SARS-CoV-2 negative and positive subjects will reveal oral manifestation of the virus on oral health and its role in exacerbating oral infection.

Rewiring of B cell receptor signaling by Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

Epstein-Barr Virus gH/gL and Kaposi's Sarcoma-Associated Herpesvirus gH/gL Bind to Different Sites on EphA2 To Trigger Fusion.

Both Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses and are important in a variety of malignancies. Eph family receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV and EBV. Previous studies identified five conserved residues (ELEFN50-54) in the N-terminal domain of KSHV gH that are critical for Eph binding and KSHV infection. However, the specific domains of EBV gH/gL important for EphA2 binding are not well described. We found that the KSHV gH (ELEFN50-54) motif is important for higher KSHV fusion and that EBV gH/gL does not utilize a similar motif for fusion activity. We previously identified that an EBV gL N-glycosylation mutant (gL-N69L/S71V) was hyperfusogenic in epithelial cells but not in B cells. To determine whether this glycosylation site may be the binding region for EphA2, we compared the EphA2 binding activity of EBV gH/gL and the EBV gH/gL-N69L/S71V mutant. We found that EBV gH/gL-N69L/S71V had higher binding affinity for EphA2, indicating that the EBV gL N-glycosylation site might be responsible for inhibiting the binding of gH/gL to EphA2. Loss of N-glycosylation at this site may remove steric hindrance that reduces EBV gH/gL binding to EphA2. In addition, the mutations located in the large groove of EBV gH/gL (R152A and G49C) also have decreased binding with EphA2. Taken together, our data indicate that the binding site of EphA2 on EBV gH/gL is at least in part proximal to the EBV gL glycosylation site, which in part accounts for differences in EphA2 binding affinity by KSHV.IMPORTANCE Virus entry into target cells is the first step for virus infection. Understanding the overall entry mechanism, including the binding mechanism of specific virus glycoproteins with cellular receptors, can be useful for the design of small molecule inhibitors and vaccine development. Recently, EphA2 was identified as an important entry receptor for both KSHV and EBV. In the present study, we investigated the required binding sites within EphA2 and EBV gH/gL that mediate the interaction of these two proteins allowing entry into epithelial cells and found that it differed in compared to the interaction of KSHV gH/gL with EphA2. Our discoveries may uncover new potential interventional strategies that block EBV and KSHV infection of target epithelial cells.

Viral MicroRNAs: Interfering the Interferon Signaling.

Interferons are secreted cytokines with potent antiviral, antitumor and immunomodulatory functions. As the first line of defense against viruses, this pathway restricts virus infection and spread. On the contrary, viruses have evolved ingenious strategies to evade host immune responses including the interferon pathway. Multiple families of viruses, in particular, DNA viruses, encode microRNA (miR) that are small, non-protein coding, regulatory RNAs. Virus-derived miRNAs (v-miR) function by targeting host and virus-encoded transcripts and are critical in shaping host-pathogen interaction. The role of v-miRs in viral pathogenesis is emerging as demonstrated by their function in subverting host defense mechanisms and regulating fundamental biological processes such as cell survival, proliferation, modulation of viral life-cycle phase. In this review, we will discuss the role of v-miRs in the suppression of host genes involved in the viral nucleic acid detection, JAK-STAT pathway, and cytokine-mediated antiviral gene activation to favor viral replication and persistence. This information has yielded new insights into our understanding of how v-miRs promote viral evasion of host immunity and likely provide novel antiviral therapeutic targets.

Ephrin Receptor A4 is a New Kaposi's Sarcoma-Associated Herpesvirus Virus Entry Receptor.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus associated with the development of Kaposi's sarcoma (KS). KSHV target cells include endothelial cells, B cells, monocytes, epithelial cells, dendritic cells, macrophages, and fibroblasts. KSHV entry into target cells is a complex multistep process and is initiated by the binding and interaction of viral envelope glycoproteins with the cellular receptors. In the current studies, we have found that EphA4 promotes KSHV glycoprotein H/glycoprotein L (gH/gL)-mediated fusion and infection better than does ephrin A2 (EphA2) in HEK293T cells, indicating that EphA4 is a new KSHV entry receptor. To confirm that epithelial cells express EphA2 and EphA4, we analyzed the expression of EphA2 and EphA4 in epithelial cells, endothelial cells, B cells, monocytes, fibroblasts using RNA sequencing (RNA-seq) data analysis of existing data sets. We found that these cell types broadly express both EphA2 and EphA4, with the exception of monocytes and B cells. To confirm EphA4 is important for KSHV fusion and infection, we generated EphA2 and EphA4 single- and double-knockout cells. We found that both EphA2 and EphA4 play a role in KSHV fusion and infection, since EphA2-EphA4 double-knockout cells had the greatest decrease in fusion activity and infection compared to single-knockout cells. Fusion and infection of KSHV were rescued in the EphA2-EphA4 double-knockout cells upon overexpression of EphA2 and/or EphA4. EphA2 binds to both Epstein-Barr virus (EBV) and KSHV gH/gL; however, EphA4 binds only to KSHV gH/gL. Taken together, our results identify EphA4 as a new entry receptor for KSHV.IMPORTANCE The overall entry mechanism for herpesviruses is not completely known, including those for the human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). To fully understand the herpesvirus entry process, functional receptors need to be identified. In the current study, we found that EphA4 can also function for a KSHV entry receptor along with EphA2. Interestingly, we found that EphA4 does not function as an entry receptor for EBV, whereas EphA2 does. The discovery of EphA4 as a KSHV entry receptor has important implications for KSHV pathogenesis in humans, may prove useful in understanding the unique pathogenesis of KSHV infection in humans, and may uncover new potential targets that can be used for the development of novel interventional strategies.

Ephrin receptor A2 is a functional entry receptor for Epstein-Barr virus.

Epstein-Barr virus (EBV) is an oncogenic virus that infects more than 90% of the world's population 1 . EBV predominantly infects human B cells and epithelial cells, which is initiated by fusion of the viral envelope with a host cellular membrane 2 . The mechanism of EBV entry into B cells has been well characterized 3 . However, the mechanism for epithelial cell entry remains elusive. Here, we show that the integrins αvß5, αvß6 and αvß8 do not function as entry and fusion receptors for epithelial cells, whereas Ephrin receptor tyrosine kinase A2 (EphA2) functions well for both. EphA2 overexpression significantly increased EBV infection of HEK293 cells. Using a virus-free cell-cell fusion assay, we found that EphA2 dramatically promoted EBV but not herpes simplex virus (HSV) fusion with HEK293 cells. EphA2 silencing using small hairpin RNA (shRNA) or knockout by CRISPR-Cas9 blocked fusion with epithelial cells. This inhibitory effect was rescued by the expression of EphA2. Antibody against EphA2 blocked epithelial cell infection. Using label-free surface plasmon resonance binding studies, we confirmed that EphA2 but not EphA4 specifically bound to EBV gHgL and this interaction is through the EphA2 extracellular domain (ECD). The discovery of EphA2 as an EBV epithelial cell receptor has important implications for EBV pathogenesis and may uncover new potential targets that can be used for the development of novel intervention strategies.

EBV latent membrane protein 2A orchestrates p27kip1 degradation via Cks1 to accelerate MYC-driven lymphoma in mice.

Epstein-Barr virus (EBV) establishes lifelong infection in B lymphocytes of most human hosts and is associated with several B lymphomas. During latent infection, EBV encodes latent membrane protein 2A (LMP2A) to promote the survival of B cells by mimicking host B-cell receptor signaling. By studying the roles of LMP2A during lymphoma development in vivo, we found that LMP2A mediates rapid MYC-driven lymphoma onset by allowing B cells to bypass MYC-induced apoptosis mediated by the p53 pathway in our transgenic mouse model. However, the mechanisms used by LMP2A to facilitate transformation remain elusive. In this study, we demonstrate a key role of LMP2A in promoting hyperproliferation of B cells by enhancing MYC expression and MYC-dependent degradation of the p27kip1 tumor suppressor. Loss of the adaptor protein cyclin-dependent kinase regulatory subunit 1 (Cks1), a cofactor of the SCFSkp2 ubiquitin ligase complex and a downstream target of MYC, increases p27kip1 expression during a premalignant stage. In mice that express LMP2A, Cks1 deficiency reduces spleen weights, restores B-cell follicle formation, impedes cell cycle progression of pretumor B cells, and eventually prolongs MYC-driven tumor onset. This study demonstrates that LMP2A uses the role of MYC in the cell cycle, particularly in the p27kip1 degradation process, to accelerate lymphomagenesis in vivo. Thus, our results reveal a novel mechanism of EBV in diverting the functions of MYC in malignant transformation and provide a rationale for targeting EBV's roles in cell cycle modulation.