Dengue, Japanese encephalitis, West Nile and yellow fever belong to the Flavivirus genus, which is a member of the Flaviviridae family. They are human pathogens that cause large epidemics and tens of thousands of deaths annually in many parts of the world. The structural organization of these viruses and their associated structural proteins has provided insight into the molecular transitions that occur during the viral life cycle, such as assembly, budding, maturation and fusion. This review focuses mainly on structural studies of dengue virus.

A structural perspective of the flavivirus life cycle

The Ah receptor (AHR) is a ligand-activated transcription factor that mediates a pleiotropic response to environmental contaminants such as benzo(a)pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin. In an effort to gain in- sight into the physiological role of the AHR and to develop models useful in risk assessment, gene targeting was used to inactivate the murine Ahr gene by homologous recombination. Ahr 2/2 mice are viable and fertile but show a spectrum of hepatic defects that indicate a role for the AHR in normal liver growth and development. The Ahr 2/2 phenotype is most severe between 0-3 weeks of age and involves slowed early growth and hepatic defects, including reduced liver weight, transient microvesicular fatty metamorphosis, prolonged extramedul- lary hematopoiesis, and portal hypercellularity with thicken- ing and fibrosis.

Characterization of a murine Ahr null allele: Involvement of the Ah receptor in hepatic growth and development (dioxiny2,3,7,8-tetrachlorodibenzo-p-dioxinygene targetingyliver)

Virus-like particle (VLP) technology is a promising approach for the construction of novel vaccines, diagnostic tools, and gene therapy vectors. Initially, VLPs were primarily derived from non-enveloped icosahedral or helical viruses and proved to be viable vaccine candidates due to their effective presentation of epitopes in a native conformation. VLP technology has also been used to prepare chimeric VLPs decorated with genetically fused or chemically coupled epitope stretches selected from immunologically defined target proteins. However, structural constraints associated with the rigid geometrical architecture of icosahedral or helical VLPs pose challenges for the expression and presentation of large epitopes. Complex VLPs derived from non-symmetric enveloped viruses are increasingly being used to incorporate large epitopes and even full-length foreign proteins. Pleomorphic VLPs derived from influenza or other enveloped viruses can accommodate multiple full-length and/or chimeric proteins that can be rationally designed for multifunctional purposes, including multivalent vaccines. Therefore, a second generation of VLP carriers is represented by complex particles reconstructed from natural or chimeric structural proteins derived from complex enveloped viruses. Further development of safe and efficient VLP nanotechnology may require a rational combination of both approaches.

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Development of Virus-Like Particle Technology from Small Highly Symmetric to Large Complex Virus-Like Particle Structures

MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

MicroRNAs: Target Recognition and Regulatory Functions

Immunotherapy is being increasingly recognized as a key therapeutic modality to treat cancer and represents one of the most exciting treatments for the disease. Fighting cancer with immunotherapy has revolutionized treatment for some patients and therapies targeting the immune checkpoint molecules such as CTLA-4 and PD-1 have achieved durable responses in melanoma, renal cancer, Hodgkin's diseases and lung cancer. However, the success rate of these treatments has been low and a large number of cancers, including colorectal cancer remain largely refractory to CTLA-4 and PD-1 blockade. This has provided impetus to identify other co-inhibitory receptors that could be exploited to enhance response rates of current immunotherapeutic agents and achieve responses to the cancers that are refectory to immunotherapy. Tim-3 is a co-inhibitory receptor that is expressed on IFN-g-producing T cells, FoxP3+ Treg cells and innate immune cells (macrophages and dendritic cells) where it has been shown to suppress their responses upon interaction with their ligand(s). Tim-3 has gained prominence as a potential candidate for cancer immunotherapy, where it has been shown that in vivo blockade of Tim-3 with other check-point inhibitors enhances anti-tumor immunity and suppresses tumor growth in several preclinical tumor models. This review discusses the recent findings on Tim-3, the role it plays in regulating immune responses in different cell types and the rationale for targeting Tim-3 for effective cancer immunotherapy.

Tim‐3 and its role in regulating anti‐tumor immunity

T-cell immune responses modulated by T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) during Mycobacterium tuberculosis (Mtb) infection in humans remain poorly understood. Here, we found that active TB patients exhibited increases in numbers of Tim-3-expressing CD4+ and CD8+ T cells, which preferentially displayed polarized effector memory phenotypes. Consistent with effector phenotypes, Tim-3+CD4+ and Tim-3+CD8+ T-cell subsets showed greater effector functions for producing Th1/Th22 cytokines and CTL effector molecules than Tim-3− counterparts, and Tim-3-expressing T cells more apparently limited intracellular Mtb replication in macrophages. The increased effector functions for Tim-3-expressing T cells consisted with cellular activation signaling as Tim-3+CD4+ and Tim-3+CD8+ T-cell subsets expressed much higher levels of phosphorylated signaling molecules p38, stat3, stat5, and Erk1/2 than Tim-3- controls. Mechanistic experiments showed that siRNA silencing of Tim-3 or soluble Tim-3 treatment interfering with membrane Tim-3-ligand interaction reduced de novo production of IFN-γ and TNF-α by Tim-3-expressing T cells. Furthermore, stimulation of Tim-3 signaling pathways by antibody cross-linking of membrane Tim-3 augmented effector function of IFN-γ production by CD4+ and CD8+ T cells, suggesting that Tim-3 signaling helped to drive stronger effector functions in active TB patients. This study therefore uncovered a previously unknown mechanism for T-cell immune responses regulated by Tim-3, and findings may have implications for potential immune intervention in TB.

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Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

The envelope glycoprotein (E) of flavivirus is the major structural protein on the surface of the mature virions. The complexes of premembrane (prM) and E play important roles in virus assembly and fusion modulation and in potential immunity-inducing vaccines. In the present study, the cDNA encoding prM and E proteins of dengue virus type 2 (DENV-2) was subcloned into the pGAPZαA vector and further integrated into the genome of Pichia pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The high-level constitutive expression of recombinant E antigen was achieved in P. pastoris. Both the cell lysate and the culture supernatant, examined by electron microscopy, were found to contain DENV-2 virus-like particles (VLPs) with diameters of about 30 nm. After immunization of BALB/c mice, the VLPs exhibited similar efficacies as inactivated virus in terms of antibody induction and neutralization titer. These results suggest that recombinant DENV VLPs can be efficiently produced in the GAP promoter-based P. pastoris expression system. This system may be useful for the development of effective and economic dengue subunit vaccine.

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Recombinant dengue virus-like particles from Pichia pastoris: efficient production and immunological properties

The endothelial cell line ECV304, derived from human umbilical cord and identified to be susceptible to dengue virus type 2 (DEN-2) infection, was used to study the molecular mechanism of DEN-2 binding to endothelial cells. DEN-2 was found by virus overlay protein-binding assays (VOPBAs) to bind to three ECV304 cell membrane proteins with molecular masses of 29, 34 and 43 kDa. Only a single protein of 29 kDa was observed when VOPBAs were carried out using preparations of trypsin-treated ECV304 cells. Pre-incubation of live ECV304 cells in culture or cell membrane proteins in modified VOPBAs with the recombinant DEN-2 envelope glycoprotein (rEgp) inhibited DEN-2 infection and blocked virus binding to the three proteins identified. These results indicate that DEN-2 rEgp could bind to three proteins on the surface of ECV304 cells. This virus–cell interaction may be associated with the receptor complex specific for DEN-2 infection of endothelial cells.

Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides.

Selection and identification of B-cell epitope on NS1 protein of dengue virus type 2

Dengue virus (DENV) remains a major public health threat because no vaccine or drugs are available for the prevention and treatment of DENV infection, and the immunopathogenesis mechanisms of DENV infection are not fully understood. Cytotoxic molecules, such as granzyme B (GrzB), may be necessary to control viral infections. However, the exact role of GrzB during DENV infection and the mechanisms regulating GrzB expression during DENV infection are not clear. This study found that miR-27a*, miR-30e, and miR-378 were down-regulated in DENV-infected patients, and DENV infection in humans induced a significant up-regulation of GrzB in natural killer (NK) cells and CD8+ T cells. Further investigation indicated that NK cells, but not CD8+ T cells, were the major sources of GrzB, and miR-378, but not miR-27a* or miR-30e, suppressed GrzB expression in NK cells. Notably, we found that overexpression of miR-378 using a miR-378 agomir in DENV-infected mice inhibited GrzB expression and promoted DENV replication. These results suggest the critical importance of miR-378 in the regulation of GrzB expression and a protective role for GrzB in controlling DENV replication in vivo. Therefore, this study provides a new insight into the immunopathogenesis mechanism of DENV infection and a biological basis for the development of new therapeutic strategies to control DENV infection.

Danyun Fang

Papers

Tim-3-Expressing CD4+ and CD8+ T Cells in Human Tuberculosis (TB) Exhibit Polarized Effector Memory Phenotypes and Stronger Anti-TB Effector Functions

Recombinant dengue virus-like particles from Pichia pastoris: efficient production and immunological properties

Dengue virus type 2 infects human endothelial cells through binding of the viral envelope glycoprotein to cell surface polypeptides.

Selection and identification of B-cell epitope on NS1 protein of dengue virus type 2

Suppressed expression of miR-378 targeting gzmb in NK cells is required to control dengue virus infection.