Oral SARS-CoV-2 host responses predict the early COVID-19 disease course.

Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19-29aa, RMSD 1-1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

Oral SARS-CoV-2 host responses predict the early COVID-19 disease course.

Objectives: Oral fluids provide ready detection of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to determine relationships between oral virus, oral anti-SARS-CoV-2-specific antibodies, and symptoms. Methods: Saliva/throat wash (saliva/TW) were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+, subjects (n=47). SARS-CoV-2 RT-qPCR, N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR targeting viral subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. Statistical analyses were performed. Results: At baseline, LFA-detected N-antigen was immunoblot-confirmed in 82% of TW. However, only 3/17 were saliva/TW qPCR+. Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three negative subjects suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19-29aa, RMSD 1-1.5 Angstroms). At entry, symptomatic subjects demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (75%/63%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral IgG correlated 100% with NP+PCR status. Cough and fatigue severity (p=0.0008 and 0.016), and presence of nausea, weakness, and composite upper respiratory symptoms (p=0.005, 0.037 and 0.017) were negatively associated with oral IgM. Female oral IgM levels were higher than male (p=0.056). Conclusion: Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms, early Ig responses, and gender during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

Oral SARS-CoV-2 host responses predict the early COVID-19 disease course.

Objectives: Oral fluids provide ready detection of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to determine relationships between oral virus, oral anti-SARS-CoV-2-specific antibodies, and symptoms. Methods: Saliva/throat wash (saliva/TW) were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+, subjects (n=47). SARS-CoV-2 RT-qPCR, N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR targeting viral subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. Statistical analyses were performed. Results: At baseline, LFA-detected N-antigen was immunoblot-confirmed in 82% of TW. However, only 3/17 were saliva/TW qPCR+. Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three negative subjects suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19-29aa, RMSD 1-1.5 Angstroms). At entry, symptomatic subjects demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (75%/63%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral IgG correlated 100% with NP+PCR status. Cough and fatigue severity (p=0.0008 and 0.016), and presence of nausea, weakness, and composite upper respiratory symptoms (p=0.005, 0.037 and 0.017) were negatively associated with oral IgM. Female oral IgM levels were higher than male (p=0.056). Conclusion: Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms, early Ig responses, and gender during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

HIVtat Alters Epithelial Differentiation State and Increases HPV16 Infectivity in Oral Keratinocytes.

Human Papillomavirus (HPV)-associated oral disease has increased during the era of HIV antiretroviral therapy. HPV and HIV proteins may be co-present at mucosal surfaces. Recent published studies have determined that HIVtat is secreted in the saliva and has been detected in oral mucosa even in the context of antiretroviral therapy. We hypothesized that HIVtat promoted oral HPV pathogenesis. Clinical HPV16 cloned episomes were introduced into differentiated oral epithelial cells (OKF6tert1). HIVtat mediated transactivation, DNA damage, oxidative stress, and effects on cellular differentiation were assessed. Detection of keratin 10 and of loricrin confirmed terminal differentiation. Sodium butyrate-treated (NaB) cells demonstrated an eight-fold increase in cross-linked involucrin, suggesting full terminal differentiation. HIVtat modulated this differentiation both in the presence and absence of NaB. Later viral events, including E6* and E1^E4 gene expression were assessed. HIVtat mediated relief of repressed L1 expression that mapped to a known inhibitory region (nucleotides 5561-6820). Viruses from HIVtat co-expressing cells exhibited robust de novo HPV16 infection. In conclusion, a novel oral keratinocyte monolayer system supported replication of an HPV16 clinical isolate where direct HIVtat and oral HPV interactions enhanced HPV de novo infection.

Differentiated Oral Epithelial Cells Support the HPV Life Cycle.

Human Papillomavirus (HPV) associated oral disease continues to increase, both in the context of immune competence and of immune suppression. There are few models of oral HPV infection and current models are laborious. We hypothesized that differentiated oral epithelial cells could support the HPV life cycle. Clinical HPV16 cloned episomes were introduced into differentiated oral epithelial cells (OKF6tert1). Viral and cellular gene expression was assessed in the presence or absence of sodium butyrate, a differentiating agent that moved the cells to full terminal differentiation. Detection of keratin 10, cross-linked involucrin, and loricrin in the presence and absence of sodium butyrate confirmed terminal differentiation. Increasing sodium butyrate concentrations in the absence of HPV, were associated with decreased suprabasal markers and increased terminal differentiation markers. However, in the presence of HPV and of increasing sodium butyrate concentrations, both mitotic and suprabasal markers were increased and the terminal differentiation marker, loricrin, decreased. In this unique differentiated state, early and late viral gene products were detected including spliced mRNAs for E6*, E1^E4, and L1. E7 and L1 proteins were detected. The ratio of late (E1^E4) to early (E6/E7) transcripts in HPV16+ OKF6tert1 cells was distinct compared to HPV16+ C33a cells. Consistent with permissive HPV replication, DNA damage responses (phospho-chk2, gamma-H2AX), HPV E2-dependent LCR transactivation, and DNase-resistant particles were detected and visualized by transmission electron microscopy. In sum, monolayers of differentiated immortalized oral epithelial cells supported the full HPV life cycle. HPV may optimize the differentiation state of oral epithelial cells to facilitate its replication.

Interferon activated gene 204 protects against bone loss in experimental periodontitis.

BACKGROUND: Periodontal destruction can be the result of different known and yet-to-be-discovered biological pathways. Recent human genetic association studies have implicated interferon-gamma inducible protein 16 (IFI16) and absent in melanoma 2 (AIM2) with high periodontal interleukin (IL)-1ß levels and more destructive disease, but mechanistic evidence is lacking. Here, we sought to experimentally validate these observational associations and better understand IFI16 and AIM2's roles in periodontitis. METHODS: Periodontitis was induced in Ifi204-/- (IFI16 murine homolog) and Aim2-/- mice using the ligature model. Chimeric mice were created to identify the main source cells of Ifi204 in the periodontium. IFI16-silenced human endothelial cells were treated with periodontal pathogens in vitro. Periodontal tissues from Ifi204-/- mice were evaluated for alveolar bone (micro-CT), cell inflammatory infiltration (MPO+ staining), Il1b (qRT-PCR), and osteoclast numbers (cathepsin K+ staining). RESULTS: Ifi204-deficient mice> exhibited >20% higher alveolar bone loss than wild-type (WT) (P < 0.05), while no significant difference was found in Aim2-/- mice. Ifi204's effect on bone loss was primarily mediated by a nonbone marrow source and was independent of Aim2. Ifi204-deficient mice had greater neutrophil/macrophage trafficking into gingival tissues regardless of periodontitis development compared to WT. In human endothelial cells, IFI16 decreased the chemokine response to periodontal pathogens. In murine periodontitis, Ifi204 depletion elevated gingival Il1b and increased osteoclast numbers at diseased sites (P < 0.05). CONCLUSIONS: These findings support IFI16's role as a novel regulator of inflammatory cell trafficking to the periodontium that protects against bone loss and offers potential targets for the development of new periodontal disease biomarkers and therapeutics.

GWAS for Interleukin-1ß levels in gingival crevicular fluid identifies IL37 variants in periodontal inflammation.

There is no agnostic GWAS evidence for the genetic control of IL-1ß expression in periodontal disease. Here we report a GWAS for "high" gingival crevicular fluid IL-1ß expression among 4910 European-American adults and identify association signals in the IL37 locus. rs3811046 at this locus (p = 3.3 × 10-22) is associated with severe chronic periodontitis (OR = 1.50; 95% CI = 1.12-2.00), 10-year incident tooth loss (≥3 teeth: RR = 1.33; 95% CI = 1.09-1.62) and aggressive periodontitis (OR = 1.12; 95% CI = 1.01-1.26) in an independent sample of 4927 German/Dutch adults. The minor allele at rs3811046 is associated with increased expression of IL-1ß in periodontal tissue. In RAW macrophages, PBMCs and transgenic mice, the IL37 variant increases expression of IL-1ß and IL-6, inducing more severe periodontal disease, while IL-37 protein production is impaired and shows reduced cleavage by caspase-1. A second variant in the IL37 locus (rs2708943, p = 4.2 × 10-7) associates with attenuated IL37 mRNA expression. Overall, we demonstrate that IL37 variants modulate the inflammatory cascade in periodontal disease.

Distinct BK polyomavirus non-coding control region (NCCR) variants in oral fluids of HIV- associated Salivary Gland Disease patients.

HIV-associated Salivary Gland Disease (HIVSGD) is among the most common salivary gland-associated complications in HIV positive individuals and was associated with the small DNA tumorvirus BK polyomavirus (BKPyV). The BKPyV non-coding control region (NCCR) is the main determinant of viral replication and rearranges readily. This study analyzed the BKPyV NCCR architecture and viral loads of 35 immunosuppressed individuals. Throatwash samples from subjects diagnosed with HIVSGD and urine samples from transplant patients were BKPyV positive and yielded BKPyV NCCR sequences. 94.7% of the BKPyV HIVSGD NCCRs carried a rearranged OPQPQQS block arrangement, suggesting a distinct architecture among this sample set. BKPyV from HIV positive individuals without HIVSGD harbored NCCR block sequences that were distinct from OPQPQQS. Cloned HIVSGD BKPyV isolates displayed active promoters and efficient replication capability in human salivary gland cells. The unique HIVSGD NCCR architecture may represent a potentially significant oral-tropic BKPyV substrain.

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics.

Oncoviruses cause tremendous global cancer burden. For several DNA tumor viruses, human genome integration is consistently associated with cancer development. However, genomic features associated with tumor viral integration are poorly understood. We sought to define genomic determinants for 1897 loci prone to hosting human papillomavirus (HPV), hepatitis B virus (HBV) or Merkel cell polyomavirus (MCPyV). These were compared to HIV, whose enzyme-mediated integration is well understood. A comprehensive catalog of integration sites was constructed from the literature and experimentally-determined HPV integration sites. Features were scored in eight categories (genes, expression, open chromatin, histone modifications, methylation, protein binding, chromatin segmentation and repeats) and compared to random loci. Random forest models determined loci classification and feature selection. HPV and HBV integrants were not fragile site associated. MCPyV preferred integration near sensory perception genes. Unique signatures of integration-associated predictive genomic features were detected. Importantly, repeats, actively-transcribed regions and histone modifications were common tumor viral integration signatures.

Correlation of transcription of MALAT-1, a novel noncoding RNA, with deregulated expression of tumor suppressor p53 in small DNA tumor virus models.

Although metastasis-associated lung adenocarcinoma transcript (MALAT)-1 is known to be consistently upregulated in several epithelial malignancies, little is known about its function or regulation. We therefore examined the relationship between MALAT-1 expression and candidate modulators such as DNA tumor virus oncoproteins human papillomavirus (HPV)-16 E6 and E7, BK virus T antigen (BKVTAg), mouse polyoma virus middle T antigen (MPVmTAg) and tumor suppressor genes p53 and pRb. Using suppressive subtractive hybridization (SSH) and real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays, MALAT-1 was shown to be increased in viral oncongene-expressing salivary gland biopsies from humans and mice. The results also indicated that MALAT-1 transcripts and promoter activity were increased in vitro when viral oncongene-expressing plasmids were introduced into different cell types. These same viral oncogenes in addition to increasing MALAT-1 transcription have also been shown to inhibit p53 and/or pRb function. In p53 mutant or inactive cell lines MALAT-1 was also shown to be highly upregulated. We hypothesize that there is a correlation between MALAT-1 over-expression and p53 deregulation. In conclusion, we show that disruption of p53, by both polyoma and papilloma oncoproteins appear to play an important role in the up-regulation of MALAT-1. MALAT-1 might therefore represent a biomarker for p53 deregulation within malignancies.

Association of p16(INK4a) overexpression with improved outcomes in young patients with squamous cell cancers of the oral tongue.

BACKGROUND: The aim of this study was to examine biomolecular profiles in a cohort of young adults with squamous cell cancers (SCCs) of the oral tongue. METHODS: We identified all patients aged 18 to 39 years diagnosed with SCC of the oral tongue at our institution. Immunohistochemical (IHC) staining was performed for p16(INK4a) , epidermal growth factor receptor (EGFR), phosphorylated-EGFR (pEGFR), p53, and ERCC1. Human papillomavirus (HPV) testing was performed using in situ hybridization (ISH) and polymerase chain reaction (PCR). Biomarker expression and HPV status were correlated with outcomes. RESULTS: We identified 25 patients with sufficient tumor samples. Median age at diagnosis was 30 years (range, 20-39 years). p16(INK4a) overexpression was observed in 11 of 25 patients, whereas HPV-16 positivity was observed in none of the tumor samples by ISH and 2 of the tumor samples by PCR. p16(INK4a) positivity was correlated with improved relapse-free survival (hazard ratio [HR] = 0.23, p = .01) and overall survival (HR = 0.28, p = .05). Neither EGFR, pEGFR, p53, nor excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) expression correlated with outcome on univariate analysis. CONCLUSIONS: p16(INK4a) overexpression was common and was a marker of favorable prognosis. p16(INK4a) overexpression was not a reliable predictor of HPV positivity in our cohort.

Detection and quantitation of HPV in genital and oral tissues and fluids by real time PCR.

BACKGROUND: Human papillomaviruses (HPVs) remain a serious world health problem due to their association with anogenital/oral cancers and warts. While over 100 HPV types have been identified, a subset is associated with malignancy. HPV16 and 18 are the most prevalent oncogenic types, while HPV6 and 11 are most commonly responsible for anogenital warts. While other quantitative PCR (qPCR) assays detect oncogenic HPV, there is no single tube assay distinguishing the most frequent oncogenic types and the most common types found in warts. RESULTS: A Sybr Green-based qPCR assay was developed utilizing degenerate primers to the highly conserved HPV E1 theoretically detecting any HPV type. A single tube multiplex qPCR assay was also developed using type-specific primer pairs and TaqMan probes that allowed for detection and quantitation of HPV6,11,16,18. Each HPV type was detected over a range from 2 x 10(1) to 2 x 10(6)copies/reaction providing a reliable method of quantitating type-specific HPV in 140 anogenital/cutaneous/oral benign and malignant specimens. 35 oncogenic and low risk alpha genus HPV types were detected. Concordance was detected in previously typed specimens. Comparisons to the gold standard detected an overall sensitivity of 89% (95% CI: 77% - 96%) and specificity of 90% (95%CI: 52% - 98%). CONCLUSION: There was good agreement between the ability of the qPCR assays described here to identify HPV types in malignancies previously typed using standard methods. These novel qPCR assays will allow rapid detection and quantitation of HPVs to assess their role in viral pathogenesis.

Concurrent human papillomavirus-associated tonsillar carcinoma in 2 couples.

We describe 2 nonsmoking, nondrinking couples who developed human papillomavirus (HPV)-associated tonsillar cancer within 12 months of each other. After histopathologic evaluation, HPV L1, E2, E6, and LCR regions were amplified, and phylogenetic analysis of amplimers was determined. Quantitative polymerase chain reaction targeting HPV-16/18 L1 and E7 regions and P16 immunohistochemistry were performed. Tissues were HPV-16 positive, with distinct intercouple nucleotide differences and multiple unique intracouple similarities identified. Diffuse nuclear P16 expression was detected in tumor cells. This report demonstrates matching HPV-16 strains in 2 couples with concurrent development of tonsillar carcinoma who did not have other risk factors, revealing the potential infectious nature of oropharyngeal cancer.

Methylation-dependent binding of the epstein-barr virus BZLF1 protein to viral promoters.

The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection.

Oropharyngeal carcinoma in non-smokers and non-drinkers: a role for HPV.

Incidence of oropharyngeal squamous cell carcinoma (OSCC) increased 3% annually from 1973 to 2001. OSCC's can be attributed to tobacco and alcohol, but 25% are unlinked to typical risks. Case-control studies on HPV detection in non-smoking/non-drinking (NS/ND) OSCC patients have not previously been performed. The primary objective of this study was to determine whether high-risk HPV infection was significantly associated with development of oral squamous malignancy in non-smokers/non-drinkers. A chart review of 802 OSCC patients from the UNC Pathology Archives (1995-2006) yielded 40 non-smoker/non-drinker subjects. Utilizing a case-control design, 18 cancer cases and 22 benign biopsy controls were consecutively identified. Biopsy tissue was subjected to (i) HPV-L1 consensus PCR and sequencing (ii) real-time PCR. Chi-square and logistic regression analysis was employed. Logistic regression analysis determined that cases were 6.1 (OR 95% CI, 1.3-28) times more likely to have HPV infection in their tumors than controls. High-risk HPV-DNA was readily detected in the tonsils and base of tongue (oropharynx) of 14/18 cases and 6/22 controls by both consensus and real-time PCR. Of high-risk HPV containing lesions, 85% (17/20) originated in the oropharynx (chi-square, p=0.03). High risk HPV was also detected in benign biopsies of the oropharynx in 30% (3/10) of individuals who had a previous oral cancer (chi-square, p=0.006). The infectious nature of OSCC in NS/ND was revealed by consistent detection of HPV, suggesting HPV's potential role in transforming oral epithelium, providing further evidence of the need to screen the oropharynx for HPV in NS/ND.

Roles of lytic viral infection and IL-6 in early versus late passage lymphoblastoid cell lines and EBV-associated lymphoproliferative disease.

Lytically infected EBV-positive lymphoblastoid cells enhance the growth of early-passage, but not late-passage, EBV-immortalized lymphoblastoid cell lines (LCLs) in SCID mice and have enhanced IL-6 secretion. Here, we have examined the importance of IL-6 for the growth of early-passage LCLs (EPL) in SCID mice, identified lytic EBV proteins that activate IL-6 production and compared viral and cellular differences between early versus late passage LCLs (LPL). IL-6 was required for efficient growth of EPL in SCID mice. The EBV immediate-early (IE) proteins, BRLF1 and BZLF1, each induced IL-6 secretion when transfected into 293 and BJAB cells. Interestingly, the combination of BZLF1 and the latent EBV protein, LMP-1, induced much more IL-6 expression in both 293 and BJAB cells than either protein alone. Both BZLF1 and BRLF1 also enhanced IL-10 production in 293 cells. In comparison to the EPL, LPL had much reduced expression of early lytic viral proteins and cellular IL-6. In contrast, expression of cellular IL-10 was similar in EPL versus LPL, while VEGF secretion was increased in late-passage LCLs. These results suggest that both BRLF1 and BZLF1 contribute to IL-6 secretion in lytically infected cells and that lytically infected cells may promote early lymphoproliferative disease in patients through enhanced IL-6 production.

BZLF1 activation of the methylated form of the BRLF1 immediate-early promoter is regulated by BZLF1 residue 186.

The Epstein-Barr virus (EBV) genome is highly methylated in latently infected cells. We recently reported that the EBV immediate-early (IE) protein BZLF1 (Z) preferentially binds to and activates transcription from the methylated form of the BRLF1 IE gene promoter (Rp). We now report that serine residue 186 in the Z DNA-binding domain plays an important role in the ability of Z to bind to and activate methylated Rp. A Z mutant containing an alanine residue at position 186 [Z(S186A)] was significantly defective in binding to methylated, as well as unmethylated, ZREs (Z-responsive elements) in Rp and was unable to activate lytic EBV gene transcription from the methylated or demethylated form of the viral genome. A Z mutant containing threonine at residue 186 [Z(S186T)] bound only to the methylated form of the ZRE-2 site in Rp and induced lytic EBV gene transcription from the methylated, but not demethylated, form of the viral genome. The defect in both of these mutants was primarily due to an inability to activate the Rp in the context of the viral genome. Finally, a Z mutant containing an aspartic acid at position 186 [Z(S186D)] did not bind to either the consensus AP-1 site or to the methylated or unmethylated Rp ZRE-2 site and did not induce lytic gene transcription. These results indicate that replacement of serine with threonine at residue 186 in the Z DNA-binding domain differentially affects its ability to reactivate the unmethylated, versus methylated, viral genome.

The Epstein-Barr virus protein BMRF1 activates gastrin transcription.

The Epstein-Barr virus (EBV) BMRF1 gene encodes an early lytic protein that functions not only as the viral DNA polymerase processivity factor but also as a transcriptional activator. BMRF1 has been previously shown to activate transcription of an EBV early promoter, BHLF1, though a GC-rich motif which binds to SP1 and ZBP-89, although the exact mechanism for this effect is not known (D. J. Law, S. A. Tarle, and J. L. Merchant, Mamm. Genome 9:165-167, 1998). Here we demonstrate that BMRF1 activates transcription of the cellular gastrin gene in telomerase-immortalized keratinocytes. Furthermore, BMRF1 activated a reporter gene construct driven by the gastrin promoter in a variety of cell types, and this effect was mediated by two SP1/ZBP-89 binding sites in the gastrin promoter. ZBP-89 has been previously shown to negatively regulate the gastrin promoter. However, ZBP-89 can function as either a negative or positive regulator of transcription, depending upon the promoter and perhaps other, as-yet-unidentified factors. BMRF1 increased the binding of ZBP-89 to the gastrin promoter, and a ZBP-89-GAL4 fusion protein was converted into a positive transcriptional regulator by cotransfection with BMRF1. BMRF1 also enhanced the transcriptional activity of an SP1-GAL4 fusion protein. These results suggest that BMRF1 activates target promoters through its effect on both the SP1 and ZBP-89 transcription factors. Furthermore, as the EBV genome is present in up to 10% of gastric cancers, and the different forms of gastrin are growth factors for gastrointestinal epithelium, our results suggest a mechanism by which lytic EBV infection could promote the growth of gastric cells.

The EBV lytic switch protein, Z, preferentially binds to and activates the methylated viral genome.

DNA methylation promotes gene silencing, yet the Epstein-Barr virus immediate-early protein, BZLF1 (Z), converts the virus from the latent to the lytic form of infection even when the viral genome is highly methylated. Here we show that methylation of CpG motifs in Z-responsive elements of the viral BRLF1 immediate-early promoter enhances Z binding to, and activation of, this promoter. Demethylation of the viral genome impairs Z activation of lytic viral genes. Z is the first transcription factor that preferentially binds to, and activates, a methylated promoter. These results identify an unexpected mechanism by which Epstein-Barr virus circumvents the inhibitory effects of viral genome methylation.

Lytic switch protein (ORF50) response element in the Kaposi's sarcoma-associated herpesvirus K8 promoter is located within but does not require a palindromic structure.

Kaposi's sarcoma-associated virus (KSHV) ORF50 protein induces lytic replication and activates the K8 promoter. We show that ORF50-induced and tetradecanoyl phorbol acetate (TPA) induced K8 transcripts initiated from the same start site. A newly identified palindrome (PAL2), containing a 12-bp response region required for ORF50-induced activation in lymphoid cells, was identified in the K8 promoter. Specific DNA binding of bacterially expressed ORF50 was not seen with the K8 promoter despite specific binding to the PAN promoter. The new palindrome shared homology with a previously described ORF50 response element (50RE(K8) and 50RE(57)). We demonstrate that the new 50RE(K8) (50RE(K8-PAL2)) is not the palindrome per se. Instead, the response element is buried within the right arm of the palindrome. We propose that the complexity of the K8 response elements reflects the complexity of mechanisms used by ORF50 during viral reactivation.