Serum IgG antibodies to HPV6 L1, E2, E4, E6, and E7 proteins among children prospectively followed-up for three years.

BACKGROUND: Current knowledge implicates that human papillomavirus (HPV) infection can be acquired at early age. However, the role of HPV-specific passive immunization from mother to neonate is nearly unexplored, especially against the HPV early proteins. We analyzed IgG antibodies against HPV6 early (E2, E4, E6, E7) and late (L1) proteins in children prospectively followed-up for three years. METHODS: A total of 272 children and their mothers from the Finnish Family HPV Study were included in these analyses. Serum samples were obtained from pregnant mothers at their third trimester and from newborn/infants at 1-, 2-, 6-, 12-, 24-, and 36-month visits after birth. Antibodies to the early and late proteins were analyzed by multiplex serology based on glutathione S-transferase fusion protein capture to fluorescent beads. RESULTS: Maternal antibodies to all tested HPV6 proteins were transferred to neonates, concordance between maternal and neonates' antibody levels being highly significant (p<0.001). Seropositivity of HPV6 L1 in the neonates declined during the first six months of life, whereas changes in the E-protein antibodies were less obvious. After the maternal antibodies have vanished, seroconversion to HPV6 L1 at 12 months (median) and to the HPV6 E-proteins between 23-35 months was observed. CONCLUSION: IgG antibodies against HPV6 E- and L-proteins are transferred from mothers to their children. Seroconversion against HPV6 L1, E2, E4, E6, and E7 does occur in early childhood, as a sign of acquired HPV6 infection by vertical or horizontal transmission starting at 12 months of age.

Detection of Human cytomegalovirus UL55 Gene and IE/E Protein Expression in Colorectal Cancer Patients in Egypt.

BACKGROUND: A possible relation between Human cytomegalovirus (HCMV) and colorectal cancer (CRC) has been widely explored with an unclear role yet speculated. AIM: The study aimed at detecting HCMV UL55 gene, immediate early and early (IE/E) proteins in colorectal tumor tissues and adjacent non neoplastic tissues (ANNT). Also, it aimed to correlate HCMV presence with CRC clinicopathological features. SUBJECTS AND METHODS: A prospective study of 50 HCMV seropositive patients with resectable CRC were enrolled in the study. Demographic, clinical, and radiological findings were recorded. Pathological assessment was done. Paired CRC tumorous and ANNT were examined for HCMV UL55 by PCR and for IE/ E proteins by immunohistochemistry (IHC). RESULTS: 70% of CRC patients enrolled were females and 36% were elderly (> 60y). Adenocarcinoma was the prevalent histopathological type (92%) with Grade 2, higher stages, and nodal involvement accounting for (64%, 64% and 56%) respectively. HCMV detection was significantly higher in tumoral tissue versus ANNT by PCR and IHC (P < 0.001, P < 0.008) respectively. Moderate agreement was found between the two techniques (κ = 0.572, P < 0.001). Univariate analysis identified HCMV presence to be significantly higher in elderly patients, in tumors with higher stage and with nodal involvement (P = 0.041, P = 0.008, P = 0.018 respectively). In multivariate analysis, the latter two retained significance (P = 0.010, P = 0.008). CONCLUSION: CRC tumor tissues are more infected by HCMV than ANNT. A significant association of HCMV presence with a higher CRC tumor stage and nodal involvement in an age-dependent manner was detected. HCMV oncomodulatory and a disease progression role is suspected.

E protein binding at the Tcra enhancer promotes Tcra repertoire diversity.

V(D)J recombination of antigen receptor loci is a highly developmentally regulated process. During T lymphocyte development, recombination of the Tcra gene occurs in CD4+CD8+ double positive (DP) thymocytes and requires the Tcra enhancer (Eα). E proteins are known regulators of DP thymocyte development and have three identified binding sites in Eα. To understand the contribution of E proteins to Eα function, mutants lacking one or two of the respective binding sites were generated. The double-binding site mutant displayed a partial block at the positive selection stage of αß T cell development. Further investigation revealed loss of germline transcription within the Tcra locus at the Jα array, along with dysregulated primary and impaired secondary Vα-Jα rearrangement. Eα E protein binding increases Tcra locus accessibility and regulates TCRα recombination, thus directly promoting Tcra repertoire diversity.

LYL1 facilitates AETFC assembly and gene activation by recruiting CARM1 in t(8;21) AML.

Transcription factors (TFs) play critical roles in hematopoiesis, and their aberrant expression can lead to various types of leukemia. The t(8;21) leukemogenic fusion protein AML1-ETO (AE) is the most common fusion protein in acute myeloid leukemia and can enhance hematopoietic stem cell renewal while blocking differentiation. A key question in understanding AE-mediated leukemia is what determines the choice of AE to activate self-renewal genes or repress differentiation genes. Toward the resolution of this problem, we earlier showed that AE resides in the stable AETFC complex and that its components colocalize on up- or down-regulated target genes and are essential for leukemogenesis. In the current study, using biochemical and genomic approaches, we show that AE-containing complexes are heterogeneous, and that assembly of the larger AETFC (containing AE, CBFß, HEB, E2A, LYL1, LMO2, and LDB1) requires LYL1. Furthermore, we provide strong evidence that the LYL1-containing AETFC preferentially binds to active enhancers and promotes AE-dependent gene activation. Moreover, we show that coactivator CARM1 interacts with AETFC and facilitates gene activation by AETFC. Collectively, this study describes a role of oncoprotein LYL1 in AETFC assembly and gene activation by recruiting CARM1 to chromatin for AML cell survival.

Signaling networks controlling ID and E protein activity in T cell differentiation and function.

E and inhibitor of DNA binding (ID) proteins are involved in various cellular developmental processes and effector activities in T cells. Recent findings indicate that E and ID proteins are not only responsible for regulating thymic T cell development but also modulate the differentiation, function, and fate of peripheral T cells in multiple immune compartments. Based on the well-established E and ID protein axis (E-ID axis), it has been recognized that ID proteins interfere with the dimerization of E proteins, thus restricting their transcriptional activities. Given this close molecular relationship, the extent of expression or stability of these two protein families can dynamically affect the expression of specific target genes involved in multiple aspects of T cell biology. Therefore, it is essential to understand the endogenous proteins or extrinsic signaling pathways that can influence the dynamics of the E-ID axis in a cell-specific and context-dependent manner. Here, we provide an overview of E and ID proteins and the functional outcomes of the E-ID axis in the activation and function of multiple peripheral T cell subsets, including effector and memory T cell populations. Further, we review the mechanisms by which endogenous proteins and signaling pathways alter the E-ID axis in various T cell subsets influencing T cell function and fate at steady-state and in pathological settings. A comprehensive understanding of the functions of E and ID proteins in T cell biology can be instrumental in T cell-specific targeting of the E-ID axis to develop novel therapeutic modalities in the context of autoimmunity and cancer.

Shifting gears: Id3 enables recruitment of E proteins to new targets during T cell development and differentiation.

Shifting levels of E proteins and Id factors are pivotal in T cell commitment and differentiation, both in the thymus and in the periphery. Id2 and Id3 are two different factors that prevent E proteins from binding to their target gene cis-regulatory sequences and inducing gene expression. Although they use the same mechanism to suppress E protein activity, Id2 and Id3 play very different roles in T cell development and CD4 T cell differentiation. Id2 imposes an irreversible choice in early T cell precursors between innate and adaptive lineages, which can be thought of as a railway switch that directs T cells down one path or another. By contrast, Id3 acts in a transient fashion downstream of extracellular signals such as T cell receptor (TCR) signaling. TCR-dependent Id3 upregulation results in the dislodging of E proteins from their target sites while chromatin remodeling occurs. After the cessation of Id3 expression, E proteins can reassemble in the context of a new genomic landscape and molecular context that allows induction of different E protein target genes. To describe this mode of action, we have developed the "Clutch" model of differentiation. In this model, Id3 upregulation in response to TCR signaling acts as a clutch that stops E protein activity ("clutch in") long enough to allow shifting of the genomic landscape into a different "gear", resulting in accessibility to different E protein target genes once Id3 decreases ("clutch out") and E proteins can form new complexes on the DNA. While TCR signal strength and cytokine signaling play a role in both peripheral and thymic lineage decisions, the remodeling of chromatin and E protein target genes appears to be more heavily influenced by the cytokine milieu in the periphery, whereas the outcome of Id3 activity during T cell development in the thymus appears to depend more on the TCR signal strength. Thus, while the Clutch model applies to both CD4 T cell differentiation and T cell developmental transitions within the thymus, changes in chromatin accessibility are modulated by biased inputs in these different environments. New emerging technologies should enable a better understanding of the molecular events that happen during these transitions, and how they fit into the gene regulatory networks that drive T cell development and differentiation.

Transcriptional dynamics and epigenetic regulation of E and ID protein encoding genes during human T cell development.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards αß and γδ T cells. Although many of the mechanisms that control αß- and γδ-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of γδ T cells. In contrast, in αß T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a cross-species and cross-developmental level.

ILC Differentiation in the Thymus.

The thymus provides a microenvironment conducive to the differentiation of innate lymphoid cells (ILCs), supplying IL-7 as well as Notch ligands. Early T cell precursors also express a number of obligatory transcription factors essential for ILC differentiation. Therefore, the thymus could be a powerhouse for ILC production. However, coordinated regulation by transcription factors and T cell receptor signaling events ensure that T cell production is the dominating output of the thymus. One group of the key regulators are the basic helix-loop-helix E protein transcription factors and their inhibitors, Id proteins. When E protein activities are downregulated, T cell development is blocked and massive ILC2 production occurs in the thymus. Normally, the thymus indeed generates a small number of ILCs, mostly group 2 ILCs (ILC2s). It has been shown in vitro that ILC2s can be differentiated from multipotent early T cell progenitors (ETPs) as well as committed T cell precursors. Moreover, thymus-derived ILC precursors have been found in the blood of adult mice. They then home to peripheral tissues and undergo differentiation into distinct ILC subsets. These ILC precursors may replenish tissue ILC pools in steady state or on demand in pathophysiological conditions. Collectively, emerging evidence suggests that the thymus plays an underappreciated role in ILC homeostasis.

Helix-Loop-Helix Proteins in Adaptive Immune Development.

The E/ID protein axis is instrumental for defining the developmental progression and functions of hematopoietic cells. The E proteins are dimeric transcription factors that activate gene expression programs and coordinate changes in chromatin organization. Id proteins are antagonists of E protein activity. Relative levels of E/Id proteins are modulated throughout hematopoietic development to enable the progression of hematopoietic stem cells into multiple adaptive and innate immune lineages including natural killer cells, B cells and T cells. In early progenitors, the E proteins promote commitment to the T and B cell lineages by orchestrating lineage specific programs of gene expression and regulating VDJ recombination of antigen receptor loci. In mature B cells, the E/Id protein axis functions to promote class switch recombination and somatic hypermutation. E protein activity further regulates differentiation into distinct CD4+ and CD8+ T cells subsets and instructs mature T cell immune responses. In this review, we discuss how the E/Id proteins define the adaptive immune system lineages, focusing on their role in directing developmental gene programs.

Mechanistic role of HPV-associated early proteins in cervical cancer: Molecular pathways and targeted therapeutic strategies.

PURPOSE: Cervical cancer (CC), one of the major causes of death of women throughout the world is primarily caused due to Human Papilloma Virus (HPV) 16 and 18. The early region (E) oncoproteins of HPV are associated with the etiopathogenesis and contribute to the progression of cancer. The present article comprehensively discussed the structural organization and biological functions of all E proteins of HPV and their contribution to progression of CC with an intent to decipher the pathological hallmarks and their relationship. Additionally, the role of E proteins in reference to therapeutics will also be presented. METHODS: A systematic search has been carried out for articles published in PubMed database by using combinations of different keywords with Boolean operators (AND, OR, NOT) including cervical cancer, HPV, E proteins, and signaling. RESULTS: From the analysis of literature review, its apparent that E proteins are the major contributor to disease progression. E1, E2, and E4 forms are mainly associated with viral integration, replication, and transcription whereas E6 and E7 act as an oncoprotein and are associated with the progression of cancer. E5 regulates cell proliferation, apoptosis, and facilitates the activity of E6 and E7. Additionally, E proteins were observed associated with numerous cell signaling pathways including PI3K/AKT, Wnt, Notch and reasonably contribute to the initiation of malignancy, cell proliferation, metastasis, and drug resistance. Knowing the role and interplay of each protein in initiation to progression of CC, their therapeutic significance has been elucidated. The present study observations demonstrate that E6 and E7 are the major cause of HPV-mediated CC progression. E1, E2, and E5 also act as a backbone for E6 and E7 and most of the current approaches have targeted E6 and E7 mediated action only. CONCLUSION: The present review illustrates the structural organization as well as function and regulation of all early proteins of HPV and their association with several cellular signaling pathways. The observations provide clue on the regulatory aspect of these proteins in initiation to progression and reasonably represent that targeting these proteins could be a novel therapeutic strategy for CC. In particular, its seemingly appears that inhibition of the activity of E6 and E7 oncoproteins may be a better selective target to delay the progression of CC. The review reaffirms the role of E proteins and encourages future studies on developing diagnostics, and most importantly therapeutics strategies targeting E6 and E7 oncoproteins to tackle CC related morbidity and mortality.

Homodimeric and Heterodimeric Interactions among Vertebrate Basic Helix-Loop-Helix Transcription Factors.

The basic helix-loop-helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell's fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed.

Up-regulated DNA-binding inhibitor Id3 promotes differentiation of regulatory T cell to influence antiviral immunity in chronic hepatitis B virus infection.

AIM: Elevated Treg is relevant to persistent HBV infection, and the regulatory mechanism of Treg levels remains unclear. E proteins are important transcriptional regulators and could be antagonized by inhibitors of DNA-binding (Id) 1-4. We aim to clarify the role of Ids during HBV infection. MAIN METHODS: Changes of Ids and their relationship with Treg were investigated in both HBV transfection model and hepatitis B patients. Significance of Ids was studied by in vitro Treg differentiation induction with Id inhibited or over-expressed. The role of inflammatory cytokines for Id was studied by co-culture. RNA-Seq was conducted to explore the differentially expressed genes in Id-overexpressed CD4 T cells upon Treg differentiation induction conditions. KEY FINDINGS: Id-overexpressed mice attenuated virus clearance in HBV transfection model. In the HBV transfection mouse model, Tregs were up-regulated, with Id3 increased in Treg as well. Clinically, circulating Tregs in chronic hepatitis B (CHB) patients were elevated, and elevated Id3 transcriptional levels were positively correlated with Tregs. IL-1ß could up-regulate Id3 in Treg cells induced in vitro. RNA-Seq revealed that increased Id could cause a series of signaling pathway changes during Treg differentiation. SIGNIFICANCE: Id3 is elevated during HBV infection to ease Treg differentiation, and the antiviral immunity is influenced that make the infection to develop into chronic state.

Type 2 innate lymphoid cells from Id1 transgenic mice alleviate skin manifestations of graft-versus-host disease.

BACKGROUND: Acute graft-versus-host disease (aGVHD) is one of the most common causes of morbidity for patients undergoing allogeneic stem cell transplantation. There is preliminary evidence that activated Group 2 innate lymphoid cells (ILC2s) from wild type (WT) mice reduces the lethality of aGVHD and is effective in treating lower gastrointestinal (GI) tract manifestations of aGVHD. This raises the prospect that ILC2s may be used for cell-based therapy of aGVHD but vigorous investigation is necessary to assess their impacts on different aspects of aGVHD. Genetically engineered mice which either express Id1 protein (Id1tg/tg), an inhibitor of E protein transcription factors or have E protein genes knocked out (dKO) in the thymus produce massive numbers of ILC2s, thus allowing extensive evaluation of ILC2s. We investigated whether these ILC2s have protective effects in aGVHD as WT ILC2s do using an established mouse model of aGVHD. RESULTS: bone marrow transplant was performed by irradiating BALB/c strain of recipient mice and transplanting with bone marrow and T cells from the MHC-disparate C57BL/6 strain. We isolated ILC2s from Id1tg/tg and dKO mice and co-transplanted them to study their effects. Our results confirm that activated ILC2s have a protective role in aGVHD, but the effects varied depending on the origin of ILC2s. Co-transplantation of ILC2s from Id1tg/tg mice were beneficial in aGVHD and are especially helpful in ameliorating the skin manifestations of aGVHD. However, ILC2s from dKO mice were less effective at the protection and behaved differently depending on if the cells were isolated from dKO mice were pre-treated with IL-25 in vivo. CONCLUSION: These findings support the notion that thymus-derived ILC2s from Id1tg/tg mice are protective against aGVHD, with a significant improvement of skin lesions and they behave differently from dKO mice in the setting of aGVHD.

The E protein-TCF1 axis controls γδ T cell development and effector fate.

TCF1 plays a critical role in T lineage commitment and the development of αß lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

Interleukin-6 (IL-6) Activates the NOTCH1 Signaling Pathway Through E-Proteins in Endometriotic Lesions.

CONTEXT: NOTCH signaling is activated in endometriotic lesions, but the exact mechanisms remains unclear. IL-6, which is increased in the peritoneal fluid of women with endometriosis, induces NOTCH1 through E-proteins including E2A and HEB in cancer. OBJECTIVE: To study the role of E-proteins in inducing NOTCH1 expression under the regulation of IL-6 in endometriosis. SETTING AND DESIGN: The expression of E-proteins and NOTCH1 was first investigated in endometrium of women with endometriosis and the baboon model of endometriosis. Regulation of E-proteins and NOTCH1 expression was examined after IL-6 stimulation and siRNA mediated inhibition of E2A or/and HEB in human endometriotic epithelial cells (12Z) in vitro, and subsequently following IL-6 treatment in the mouse model of endometriosis in vivo. RESULTS: E2A, HEB, and NOTCH1 were significantly upregulated in glandular epithelium (GE) of ectopic endometrium compared to eutopic endometrium in both women and the baboon model. IL-6 treatment upregulated the expression of NOTCH1 together with E2A and HEB in 12Z cells. Small interfering RNA inhibition of E2A and HEB or HEB alone decreased NOTCH1 expression. Binding efficiency of both E2A and HEB was significantly higher at the binding sites on the human NOTCH1 promoter after IL-6 treatment. Finally, IL-6 treatment resulted in a significantly increased number of endometriotic lesions along with increased expression of E2A, HEB, and NOTCH1 in GE of the lesions compared with the vehicle group in an endometriosis mouse model. CONCLUSIONS: IL-6 induced NOTCH1 expression is mediated by E-proteins in the ectopic GE cells, which may promote endometriotic lesion development.

Evaluation of immunogenicity and protective efficacy of a CpG-adjuvanted DNA vaccine against Tembusu virus.

Tembusu virus (TMUV) is a contagious pathogen of waterfowl including ducks and geese, which causes symptoms of high fever, loss of appetite and reduced egg production. The development of an effective vaccine is important for the prevention and control of the disease. We evaluated a DNA vaccine based on a recombinant pre-membrane (prM) and envelope (E) protein, using CpG oligodeoxynucleotide (ODN) as an adjuvanted, and tested it for protection efficacy. BHK21 cells were transfected with the recombinant plasmid pVAX1-prM/E-CpG, and the antigenicity of the expressed protein was detected using an indirect immunofluorescence assay (IFA) and western blot assay. One-day-old ducklings were intramuscularly injected with 200 µg doses of pVAX1-prM/E-CpG or pVAX1-CpG, or PBS at ten day intervals. The neutralizing antibodies and cell-mediated immune responses elicited by the DNA vaccine were detected using serum neutralization tests (SNTs) and ELISAs. At 20 days old, the ducks were challenged with 103EID50 doses of TMUV SD/02 strain and observed for 15 days post challenge. After the second DNA vaccination and during the monitoring period, the levels of TMUV neutralizing antibodies increased in the pVAX1-prM/E-CpG vaccinated ducks. Vaccination with pVAX1-prM/E-CpG resulted in 100.0% protection of the ducks, whereas approximately 40% of ducks vaccinated with pVAX-CpG or PBS manifested clinical symptoms. Expressions of IFN-γ and IL-6 in the pVAX1-prM/E-CpG group were significantly increased (p <  0.01) compared with the control groups during the entire experimental period. The results revealed that a vaccine co-expressing prM and E, and using a CpG-ODN motif as an adjuvant, could elicit effective neutralizing antibody titers and provide efficient protection to ducks against TMUV infection.

Programming for T-lymphocyte fates: modularity and mechanisms.

T-cell development in mammals is a model for lineage choice and differentiation from multipotent stem cells. Although T-cell fate choice is promoted by signaling in the thymus through one dominant pathway, the Notch pathway, it entails a complex set of gene regulatory network and chromatin state changes even before the cells begin to express their signature feature, the clonal-specific T-cell receptors (TCRs) for antigen. This review distinguishes three developmental modules for T-cell development, which correspond to cell type specification, TCR expression and selection, and the assignment of cells to different effector types. The first is based on transcriptional regulatory network events, the second is dominated by somatic gene rearrangement and mutation and cell selection, and the third corresponds to establishing a poised state of latent regulator priming through an unknown mechanism. Interestingly, in different lineages, the third module can be deployed at variable times relative to the completion of the first two modules. This review focuses on the gene regulatory network and chromatin-based kinetic constraints that determine activities of transcription factors TCF1, GATA3, PU.1, Bcl11b, Runx1, and E proteins in the primary establishment of T-cell identity.

Cross-Protection Against Four Serotypes of Dengue Virus in Mice Conferred by a Zika DNA Vaccine.

Both Zika virus (ZIKV) and four serotypes of dengue virus (DENV1-4) are antigenically related mosquito-borne flaviviruses that co-circulate in overlapping geographic distributions. The considerable amino acid sequence homology and structural similarities between ZIKV and DENV1-4 may be responsible for the complicated immunological cross-reactivity observed for these viruses. Thus, a successful Zika vaccine needs to not only confer protection from ZIKV infection but must also be safe during secondary exposures with other flavivirus, especially DENVs. In this study, we used a Zika DNA vaccine candidate (pV-ZME) expressing the ZIKV premembrane and envelop proteins to immunize BALB/c mice and evaluated the potential cross-reactive immune responses to DENV1-4. We observed that three doses of the pV-ZME vaccine elicited the production of cross-reactive antibodies, cytokines and CD8+ T cell responses and generated cross-protection against DENV1-4. Our results demonstrate a novel approach for design and development of safe Zika and/or dengue vaccines.

The Roles of prM-E Proteins in Historical and Epidemic Zika Virus-mediated Infection and Neurocytotoxicity.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.