Dengue Virus Infection: A Tale of Viral Exploitations and Host Responses

Abstract

Dengue is a mosquito-borne viral disease (arboviral) caused by the Dengue virus. It is one of the prominent public health problems in tropical and subtropical regions with no effective vaccines. Every year around 400 million people get infected by the Dengue virus, with a mortality rate of about 20% among the patients with severe dengue. The Dengue virus belongs to the Flaviviridae family, and it is an enveloped virus with positive-sense single-stranded RNA as the genetic material. Studies of the infection cycle of this virus revealed potential host targets important for the virus replication cycle. Here in this review article, we will be discussing different stages of the Dengue virus infection cycle inside mammalian host cells and how host proteins are exploited by the virus in the course of infection as well as how the host counteracts the virus by eliciting different antiviral responses.

Keywords: Antibody Dependent Enhancement (ADE); Dengue genome; Dengue virus; apoptosis; autophagy; cytokine storm; host immune response; inflammation; viral pathogenesis; viral replication.

Figure 1

Structural organization of Dengue virus genome: The viral genome consists of 5′UTR, an ORF, and 3′UTR. The ORF encoding polyprotein serves as a template for the translation of 3 structural proteins (C (Capsid), PrM (Pre-Membrane), and E (Envelope)) and 7 Non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). The representation of 5′UTR depicts Stem-loop structures (SLA and SLB) flanked by oligo U, wherein SLB is followed by initiator AUG and the hairpin structure (cHP) in the C encoding region. The following complementary sequence at 5′UTR is essential for genome circularization, which in turn is important for genome replication. The 3′UTR consists of variable sequences, followed by dumbbell structures DB1 and DB2. The approaching conserved sequence CS1 and stem loop (SL) at 3′UTR play a pivotal role in genome conformational changes facilitating replication.

Figure 2

Replication cycle of Dengue virus: The replication cycle begins with entry into the host cell that initiates from binding of the virus to host cell receptors. (1) The DENV bind can host various receptors to enter via receptor-mediated endocytosis, while alternative routes of entry are described too. Most studied and reported, clathrin-mediated endocytosis is described hereafter. (2) The clathrin-coated pit serves as the site of internalization, and the virus is endocytosed into a perforated bubble coated with clathrin. (3) Further, the clathrin is released, initiating endosomal processing. (4) The early endosome is marked by presence of the Rab5 protein (green solid spheres) where pH is around 6.5, which proceeds towards endosome maturation. (5) The mature endosome is marked by Rab7 (red solid spheres) and at a lower pH, i.e., around 5.5, this acidic pH leads to various conformational changes. (6) The conformational changes cause fusion of the viral envelope and host membrane. (7) The disassembly of the virus results in the release of the encapsidated viral genome (capsid-bound RNA) into the cytoplasm. (The mechanism of uncoating the viral genome is unclear and thus not described in the figure.) (8) The viral RNA serves as a template for translation and replication in the ER. The special sight for replication, formed by convolution of the ER membrane in the presence of NS proteins is called the Replication complex. (9) The replicated genome and translated viral proteins are assembled, forming an immature virus particle in the ER. (10) This immature virus undergoes furin-mediated maturation in the Trans-Golgi Network (TGN). (11) The mature virus is then exocytosed from the infected cell, completing the infection cycle.

Figure 3

Inflammatory responses during Dengue virus infection: Dengue virus entry into the target macrophage cell triggers the inflammatory cascade. A. Macrophage activation leads to the increase in the vasoactive factors to induce permeability changes in the EC membrane ultimately leading to vascular leakage. B. Neutrophils, the major effectors in the inflammation process, are recruited by cytokines released from macrophages to migrate to the nearby tissues. Neutrophils under Dengue attack are capable of exhibiting NETosis, a phenomenon marked by the release of traps or NETs to capture the Dengue virus. Apart from NETosis, the neutrophils degranulate, an event that contributes to the penetration of the blood-brain-barrier to cause neuroinflammation. Certain miRNAs, miR-126, miR-155, and miR-221, which are potential rescuers of the EC permeability changes and inflammation, respectively, are being considered as therapeutic targets in order to reduce the damaging effects of inflammation in the Dengue patients.

Figure 4

Immune cells involved in the host responses to the Dengue virus: After the mosquito bite injects the Dengue virus into the skin, A. The Langerhans cells and the Dendritic cells (DCs), the primary cells that encounter the Dengue virus, come into play. B. The DCs recruit the NK cells to the site, whereas, C. The macrophages recruit the neutrophils. D. Macrophages infected with DENV present the processed antigen to the T cell, which in turn interacts with the B cell to aid in B cell development. E. Different types of T cells are activated to elicit various cell-mediated immune responses against the virus. F. A gush of cytokines are secreted in an event called the “cytokine storm”, out of which the overproduction of IL-10 leads to the inhibition of IFN responses as well as vascular leakage. G. The humoral immunity, represented by the B-cell-mediated antibody production from the plasma cells, as a natural immune response to any pathogen, induces ADE in the DENV infection. H. The ADE of the DENV entry into the macrophage via phagocytosis leads to an altered transcriptome of the cell, ultimately leading to TLR suppression, enhanced Th2 responses, along with the dysregulation of vesicular transport and the host translation process.

Figure 5

Regulation of cell death via apoptosis during Dengue virus infection: The Dengue virus is capable of 1. Inducing apoptosis as well as 2. Inhibiting apoptosis in target cells, depending upon its requirement. The different mechanisms of apoptosis induction are: A. Induction of the mitochondria-based apoptosis by the pro-survival protein, p53, to assemble the apoptosome for Caspase-3, 9 activation. B. The NFκB-TNF-α mediated induction of cell apoptosis. C. The Sphingosine-kinase-2 mediated induction of apoptosome formation. D. ROS-induced DNA damage that ultimately leads to the death of the cell. E. Induction of ER stress to activate the UPR responses in the ER of the cell. All these mechanisms result in morphological changes in the cell indicative of apoptosis-mediated cell death. The DENV also allows the survival of the cell so as to continue parasitizing it. The anti-apoptotic mechanisms include: i. Increased Akt phosphorylation to inhibit the ER-stress-induced UPR responses in the cell and ii. Enrichment of cytoplasmic calcium ions to inhibit the apoptosome formation.

Figure 6

Regulation of cell death via autophagy during Dengue virus infection: The entry of Dengue virus induces autophagy in the infected cell. The mechanisms of autophagy post-DENV infection are: A. Induction of ER stress to trigger the UPR responses, out of which 2 pathways, involving IRE1α and PERK, respectively, lead to the formation of autophagosomes in the cell. B. Autophagosome formation is also triggered by the involvement of ATM kinase to trigger the PERK pathway for autophagosome formation. C. AMPK, a kinase involved in lipophagy, is also exploited by the DENV to modulate the metabolic status of the cell. D. Apart from the common steps of autophagy, the DENV employs the proteasomal degradation of p62 to enhance its replication. The autophagosome in step 1 fuses with the lysosome to form the 2. Autolysosome structures which ultimately lead to 3. Autophagy and 4. Protein degradation.