Non-Canonical Roles of Dengue Virus Non-Structural Proteins

Abstract

The Flaviviridae family comprises a number of human pathogens, which, although sharing structural and functional features, cause diseases with very different outcomes. This can be explained by the plurality of functions exerted by the few proteins coded by viral genomes, with some of these functions shared among members of a same family, but others being unique for each virus species. These non-canonical functions probably have evolved independently and may serve as the base to the development of specific therapies for each of those diseases. Here it is discussed what is currently known about the non-canonical roles of dengue virus (DENV) non-structural proteins (NSPs), which may account for some of the effects specifically observed in DENV infection, but not in other members of the Flaviviridae family. This review explores how DENV NSPs contributes to the physiopathology of dengue, evasion from host immunity, metabolic changes, and redistribution of cellular components during infection.

Keywords: dengue virus; immunity; metabolism; non-structural proteins; physiopathology.

Figure 1

Schematic representation of the flaviviruses’ polyprotein. Viral RNA encodes a polyprotein that is co- and post-translationally processed by host proteases (black scissors) or by the viral protease NS2B/NS3 (red scissors) to generate the structural (C, PrM, and E) and non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), represented in different colors.

Figure 2

Involvement of DENV NSPs in the evasion of host innate immune response. (A) Pathways of immune response affected by DENV NSPs. (1) Activation of the complement system is triggered by different pathways that converge to the cleavage of factor C3 by the protease C3 convertase. This enzyme formed by the association of two other cleavage products: C4b, a fragment of C4, and C2a, a fragment of C2. The cleavage of C4 and C2 may be catalyzed by two different pathways: the classical pathway, triggered by C1 binding to antigen-antibody complexes, or by the lectin pathway, in which a carbohydrate recognition receptor, such as mannose binding lectin (MBL), associates to a serine protease after binding to carbohydrates. One of the products of C3 cleavage, C3b, binds to C3 convertase changing its substrate specificity, so that the enzyme becomes a C5 convertase. The fragment C5b, generated from the cleavage of C5, binds to the infected cell membrane, initiating the assembly of a complex formed by C6, C7, C8, and C9, which promotes cell lysis. (2) Viral dsRNAs produced during the replication of RNA viruses are recognized by PRRs. Binding of dsRNA leads the cytosolic PPRs to associate with the mitochondrial antiviral signaling protein (MAVS) through its caspase-recruitment domain (CARD), recruiting the TANK-protein kinase 1 (TBK1) and IκB kinase-ε (IKKε). These kinases phosphorylate IRF-3, which forms homodimers or heterodimers with IRF-7, which, in turn, translocate to the nucleus, inducing the expression of type I IFN and pro-inflammatory cytokines. This pathway also includes the participation of the adaptor protein STING, which acts in mitochondrial-associated membrane (MAM) to mediate RIG-I downstream signaling. (3) Type I IFN-mediated antiviral responses occurs via the expression of several IFN-stimulated genes (ISGs). IFN binding to its heterodimeric IFN-α receptor (IFNAR1/2) activates Janus kinase 1 (JAK1) or tyrosine kinase 2 (TYK2), leading to the phosphorylation of the signal transducer and activator of transcription (STAT) proteins, which dimerize and translocate to the nucleus. STAT1–STAT2 heterodimer binds to IFN regulatory factor 9 (IRF9) and migrates to the nucleus, inducing the expression of ISGs through its binding to the IFN-stimulated response elements (ISRE). Type I and type II IFNs can also induce dimerization of STAT3, which translocate to the nucleus, where it binds to gamma-activated sequences (GAS), stimulating the production of both pro- and anti-inflammatory cytokines; (B) Participation of DENV NSPs in the evasion of the host immune response. (1) DENV NS1 inhibits complement activation by interacting with different components of the complement system, including C1 proenzyme, C1s, C4, C4b, and MBL. The formation of the complex C4-NS1-C1s/C1 results in degradation of C4, impairing the formation of C3 convertase. NS1 binding to MBL protects DENV against MBL-mediated virus neutralization by the lectin pathway of complement activation. (2) DENV NSPs impair the innate immune response mediated by viral dsRNA recognition. NS4B interacts with the CARD domain of MAVS, impairing its binding to the cytoplasmic PRRs. Moreover, this protein, by inducing the formation of convoluted membranes (CM) and promoting mitochondrial elongation, inhibits the translocation of PRRs to MAMs. NS2B/NS3 interacts with IKKε and cleaves STING and NS2A together with NS4B, inhibiting the phosphorylation of TBK1 and its substrate IRF3. These steps impair the activation of transcription factors IRF-3 and IRF-7. (3) DENV NSPs inhibit INF-stimulated signaling in different points. NS4B interacts with STAT1, blocking its phosphorylation, and NS5 mediates STAT2 degradation, so both proteins inhibit the expression of ISGs by interfering in ISRE activation. Additionally, NS1 interacts with STAT3, inhibiting the formation of its homodimers, thus preventing GAS-induced gene expression. Red arrows represent the events induced by NSPs, while dashed red arrows represent those ones that are blocked by NSPs. ER, endoplasmic reticulum; MT, mitochondria.

Figure 2

Involvement of DENV NSPs in the evasion of host innate immune response. (A) Pathways of immune response affected by DENV NSPs. (1) Activation of the complement system is triggered by different pathways that converge to the cleavage of factor C3 by the protease C3 convertase. This enzyme formed by the association of two other cleavage products: C4b, a fragment of C4, and C2a, a fragment of C2. The cleavage of C4 and C2 may be catalyzed by two different pathways: the classical pathway, triggered by C1 binding to antigen-antibody complexes, or by the lectin pathway, in which a carbohydrate recognition receptor, such as mannose binding lectin (MBL), associates to a serine protease after binding to carbohydrates. One of the products of C3 cleavage, C3b, binds to C3 convertase changing its substrate specificity, so that the enzyme becomes a C5 convertase. The fragment C5b, generated from the cleavage of C5, binds to the infected cell membrane, initiating the assembly of a complex formed by C6, C7, C8, and C9, which promotes cell lysis. (2) Viral dsRNAs produced during the replication of RNA viruses are recognized by PRRs. Binding of dsRNA leads the cytosolic PPRs to associate with the mitochondrial antiviral signaling protein (MAVS) through its caspase-recruitment domain (CARD), recruiting the TANK-protein kinase 1 (TBK1) and IκB kinase-ε (IKKε). These kinases phosphorylate IRF-3, which forms homodimers or heterodimers with IRF-7, which, in turn, translocate to the nucleus, inducing the expression of type I IFN and pro-inflammatory cytokines. This pathway also includes the participation of the adaptor protein STING, which acts in mitochondrial-associated membrane (MAM) to mediate RIG-I downstream signaling. (3) Type I IFN-mediated antiviral responses occurs via the expression of several IFN-stimulated genes (ISGs). IFN binding to its heterodimeric IFN-α receptor (IFNAR1/2) activates Janus kinase 1 (JAK1) or tyrosine kinase 2 (TYK2), leading to the phosphorylation of the signal transducer and activator of transcription (STAT) proteins, which dimerize and translocate to the nucleus. STAT1–STAT2 heterodimer binds to IFN regulatory factor 9 (IRF9) and migrates to the nucleus, inducing the expression of ISGs through its binding to the IFN-stimulated response elements (ISRE). Type I and type II IFNs can also induce dimerization of STAT3, which translocate to the nucleus, where it binds to gamma-activated sequences (GAS), stimulating the production of both pro- and anti-inflammatory cytokines; (B) Participation of DENV NSPs in the evasion of the host immune response. (1) DENV NS1 inhibits complement activation by interacting with different components of the complement system, including C1 proenzyme, C1s, C4, C4b, and MBL. The formation of the complex C4-NS1-C1s/C1 results in degradation of C4, impairing the formation of C3 convertase. NS1 binding to MBL protects DENV against MBL-mediated virus neutralization by the lectin pathway of complement activation. (2) DENV NSPs impair the innate immune response mediated by viral dsRNA recognition. NS4B interacts with the CARD domain of MAVS, impairing its binding to the cytoplasmic PRRs. Moreover, this protein, by inducing the formation of convoluted membranes (CM) and promoting mitochondrial elongation, inhibits the translocation of PRRs to MAMs. NS2B/NS3 interacts with IKKε and cleaves STING and NS2A together with NS4B, inhibiting the phosphorylation of TBK1 and its substrate IRF3. These steps impair the activation of transcription factors IRF-3 and IRF-7. (3) DENV NSPs inhibit INF-stimulated signaling in different points. NS4B interacts with STAT1, blocking its phosphorylation, and NS5 mediates STAT2 degradation, so both proteins inhibit the expression of ISGs by interfering in ISRE activation. Additionally, NS1 interacts with STAT3, inhibiting the formation of its homodimers, thus preventing GAS-induced gene expression. Red arrows represent the events induced by NSPs, while dashed red arrows represent those ones that are blocked by NSPs. ER, endoplasmic reticulum; MT, mitochondria.

Figure 3

DENV NSPs-induced host metabolic alterations. During infection, DENV NS4B induces the formation of the convoluted membrane (CM), the membranous structure where viral polyprotein is processed and the resulting proteins accumulate. NS3 recruits the enzyme fatty acid synthase (FASN) to the virus replication sites, besides stimulating its enzymatic activity, increasing de novo FA biosynthesis, which provides lipids for inducing the formation of CM, as well as for the assembly of the viral envelope. Fatty acids (FA) are mobilized from lipid droplets (LD) to undergo β-oxidation mitochondria, providing energy to the high-energy demanding virus replication process. NS4B is able to inhibit phosphorylation of cytoplasmic protein dynamin-related protein 1 (Drp1), preventing the mitochondrial fission process. Fused mitochondria accumulate in infected cells, increasing the efficiency of the oxidative metabolism. Red arrows represent the events induced by NSPs, while dashed red arrows represent those ones that are blocked by NSPs. MFN1, mitofusin 1; MFN2, mitofusin 2; Opa1, optic atrophy protein 1; ER, endoplasmic reticulum; MT, mitochondria.