Wheat cell–free‐based protein arrays currently available in our research group.
Dengue, one of the most common mosquito‐borne viral infectious diseases in the world, is caused by the dengue virus (DENV). This enveloped RNA virus has immunologically distinct serotypes that increase the risk of life‐threatening diseases, such as dengue haemorrhagic fever. However, no effective antiviral therapy against DENV infection has yet been established. As seen in other RNA viruses, various cellular factors have been reported to participate in efficient DENV replication. On the other hand, increasing recent evidence demonstrates that host cells harbour inhibitory factors that limit the DENV replication. In particular, it is well known that the response of interferons (IFNs), the first line of a host defence system against invading pathogens, evokes the expression of a number of genes that negatively regulate various steps of virus replication. This set of inhibitory genes, called interferon‐stimulated genes (ISGs), is considered to be a central force in IFN‐mediated antiviral responses. In this chapter, we focus our attention on the cellular factors involved in DENV infection, particularly to those that modulate DENV replication through their association with viral RNA. In addition, we also summarize general experimental approaches for identifying the host factors of RNA viruses, including DENV.
- dengue virus
- cellular factors
- RNA untranslated regions
- interferon‐stimulated genes
- identification systems
Dengue virus (DENV) is an enveloped and positive‐strand RNA virus that belongs to the genus
DENV is a mosquito‐borne virus widely distributed in the tropical and subtropical areas of the world. This flavivirus infection is transmitted to humans via the bite of infected mosquitos. The primary vector of DENV infection is
2. Brief overview of DENV replication
DENV infection begins with its entry into a permissive cell via receptor‐mediated endocytosis (Figure 1). So far, various types of human cells, such as macrophages, lymphocytes, hepatocytes, and endothelial cells, are reportedly susceptible to DENV infection. Among them, monocyte lineage cells (i.e., dendritic cells [DCs] and macrophages) are thought to be the primary targets of DENV in humans. As the entry receptors of DENV, several cellular proteins, including C‐type lectin receptors (e.g., DC‐SIGN/CD209, mannose receptor/CD206) and phosphatidylserine receptors (e.g., TIM, TAM), have been demonstrated .
Upon entry into the cell, a membrane fusion between DENV envelope (E) glycoprotein and endosomal vesicle occurs, leading to the release of viral RNA into the cytoplasm. The DENV genome is a single‐stranded positive‐sense RNA and is approximately 10‐kb long. The viral RNA contains a single long open reading frame (ORF) flanked by highly structured 5′ and 3′ untranslated regions (UTRs), with a type 1 cap (m7GpppAmp) at the 5′ terminus and no poly(A) tract at the 3’ terminus . The single ORF is translated to a large polyprotein, which is subsequently cleaved co‐ and post‐translationally into three structural (capsid [C], pre‐membrane [prM], and E) and seven non‐structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by viral (NS3) and host proteases (Figure 1A). Of the NS proteins, NS5, the largest viral protein, functions as an RNA‐dependent RNA polymerase (RdRp), which synthesizes a complementary minus‐strand RNA template and, in turn, produces many copies of positive‐strand viral genomic RNA. Viral RNA replication takes place in the so‐called replication complexes (RCs) composed of viral RNA and proteins as well as hypothetical cellular proteins, which are formed on the endoplasmic reticulum (ER) membrane. This membrane structure, rearranged by DENV infection, is also the place of viral RNA translation, protein processing, and progeny virion assembly . The immature particles then bud into the ER lumen and are transported through the secretion pathway of the trans‐Golgi network, in which progeny virions undergo a maturation process via a conformational change of prM and E proteins on the virion surface. Finally, mature, infectious virions egress from infected cells by exocytosis (Figure 1B) [5, 8].
3. Cellular factors involved in DENV replication: interaction with viral RNA untranslated regions
As seen in other RNA and DNA viruses, flaviviruses, including DENV, have been shown to utilize biological processes of the host to replicate efficiently in infected cells through the interaction between viral and cellular proteins. Although the biological relevance of many of the host interactors in
The DENV 5′ and 3′ UTRs are approximately 100 and 350–700 nucleotides long, respectively (Figure 1A). The primary sequences located within or adjacent to the UTRs have been shown to be essential for virus replication . For instance, complementary sequences, termed CS (cyclization sequence) elements, found in the ORF of capsid and the 3′ UTR and UAR (upstream of the AUG region) elements, found in the 5′ and 3′ UTRs, are reported to physically anneal to mediate DENV genome cyclization. This cyclization is likely to recruit the DENV RdRp at the 5′ end of the viral genome and then likely to facilitate viral RNA amplification . Additionally, DENV UTRs form stable secondary and tertiary structures, and these structural integrities are thought to be important for the regulation of viral RNA synthesis and translation process. Therefore, albeit the sequences are diverse, UTR secondary structures are highly conserved among flaviviruses . Furthermore, these regions have been demonstrated to contain several cellular proteins .
An early study that employed an
PTB is a ubiquitous RNA‐binding protein known to be involved in splicing, polyadenylation, stability, and translation of cellular mRNA . With regard to its role in virus infection, PTB has been shown to bind to the UTRs of picornaviruses [21, 22] and the hepatitis C virus (HCV) ; it functions as an internal ribosome entry site, (IRES)‐trans‐acting factor, to activate viral translation [24, 25]. On the other hand, several studies have raised questions about the involvement of PTB in the translation process of those RNA viruses [26–28]. Interestingly, both La and PTB were implicated in HCV replication . In the case of DENV infection, PTB is reportedly required for efficient replication. Furthermore, PTB interacted with a DENV protein, NS4, as well as viral RNA, suggesting that PTB associates with DENV RCs . Although it remains unclear whether PTB regulates DENV RNA simplification or the translation process (or another step of virus infection), PTB may function as a molecular chaperone to stabilize the structured viral RNA [30, 31]. In addition to the host factors mentioned above, recent studies using tobramycin RNA aptamer affinity chromatography identified DDX6 (DEAD‐box RNA helicase) and ERI3 (putative 3′–5′ RNA exonuclease) as DENV UTR‐binding cellular proteins that promote DENV replication [32, 33].
The genome of flaviviruses, including DENV, encodes enzymes required for viral RNA synthesis (i.e., RdRp and helicase) and viral protein processing (i.e., protease); however, the viral protein translation process must rely fully on the translational machinery of the host , except for the methyltransferase activity conferred by NS5, which adds a type 1 7‐methylguanosine cap to the 5′ terminus of viral RNA. It is, therefore, not surprising that cellular factors are associated with DENV RNA during viral translation [8, 35]. At the initiation step of eukaryotic mRNA translation, a 5′ cap structure is first recognized by a eukaryotic initiation factor, eIF4E, which, in turn, leads to the recruitment of eIF4G. eIF4G serves as a scaffold protein that binds the DEAD‐box RNA helicase eIF4A and also the poly(A)‐binding protein (PABP), resulting in the circularization of mRNA . This complex formation induces the association of the 43S ribosomal subunit through the binding of the eIF3 complex. Finally, the 60S ribosomal subunit joins the initiation complex that enables the elongation process of translation . In the case of DENV, its genomic RNA is 5′ capped but lacks 3′‐end poly(A) sequences . Nevertheless, PABP is shown to interact with DENV 3′ UTR
4. Experimental procedures for identifying the cellular factors
In the following sections, we summarize several general approaches to search for the cellular factors involved in virus infections.
4.1. Conventional methods using living cells
4.1.1. Gene expression analysis and proteomic analysis
Studies using a microarray system, quantitative RT‐PCR analysis, and GeneChip analysis have revealed that the expression levels of many mRNAs in host cells are dramatically altered upon DENV infection [40–42]. It is highly possible that some of these DENV‐responsible genes and their products function as crucial positive or negative regulators of DENV replication in cells. Indeed, tumour necrosis factor–related apoptosis‐inducing ligand (TRAIL), whose mRNA expression level significantly increased with DENV infection, was found to be a negative regulator of DENV replication . In addition to the analysis of the gene expression profile, the global host protein expression profile upon DENV infection was also investigated by comprehensive proteomic analysis. Conventionally, the total host proteins extracted from infected cells and control cells were separated by two‐dimensional PAGE (2‐D PAGE), and the proteins whose expression levels were altered by the viral infection were picked up, and their amino acid sequences were determined with mass spectrometry (MS) analysis. Previous studies have identified many host factors that respond to DENV infection [43, 44]. More recently, a proteomic analysis based on stable isotope labelling by amino acids in cell culture (SILAC), which overcame the limitations of sensitivity and resolution of 2‐D PAGE, was developed and applied to DENV research [45, 46]. The technologies of high‐throughput gene expression analysis and proteomic analysis are thought to be powerful tools for understanding the global cellular expression profile of host cells upon DENV infection, both at the gene and at the protein levels. From the results of these assays alone, however, it is difficult to distinguish whether DENV‐responsible host factors that are identified are indeed involved in the regulation of viral replication. To understand the roles and functions of these hit factors requires further functional analysis.
4.1.2. RNAi screening
RNA interference (RNAi) is a well‐known approach for identifying novel host factors in virus‐infected cells. When the knockdown of a host gene by RNAi alters the efficiency of viral replication, it is highly possible that this gene works as a host factor of the virus. Currently, commercial siRNA pools that cover most human genes are available, and many genome‐wide comprehensive screenings for many kinds of viruses have been performed thus far [47–49]. In studies of flaviviruses, hundreds of host factor proteins involved with the early steps of WNV infection have been identified by silencing more than 20,000 human genes from a small interfering RNA (siRNA) pool . An additional bioinformatic study, followed by the siRNA screening, revealed that many of these hits were involved in the ubiquitin‐proteasome pathway and the ER‐associated degradation (ERAD) pathway, both of which are essential for many steps of viral replication. In addition to host factors in human cells, one study aimed to identify host factors from insect cells, another host of DENV . In that study of DENV‐adapted cells from
4.1.3. Yeast two‐hybrid analysis
For researchers attempting to identify host factors that directly interact with viral proteins, yeast two‐hybrid (Y2H) analysis is a common and effective way; numerous host factors of many viruses, including DENV, have been identified using this system [52–58]. Taking advantage of good throughput, several large screenings were performed to build DENV host interactomes using human cDNA libraries. One study reported that a Y2H analysis using either partial or full‐length DNA fragments encoding each DENV protein and the human liver cDNA library identified 105 viral‐host interactions; further knockdown experiments using siRNAs revealed that six proteins were essential for the efficient viral replication of DENV . Another study also carried out a Y2H assay using NS3 and NS5 proteins from DENV and other flaviviruses as bait, and 108 human proteins were identified as interacting with NS3 or NS5 or both . In addition, Y2H assays were performed to identify DENV proteins interacting with host factors from human and mosquito cDNA libraries. They identified several common host factors conserved in both humans and mosquitos . These studies identified many host proteins that interact with DENV proteins; however, few of the hits overlap in independent studies. Although this might be caused mainly by the fact that the assay in each study was performed in different conditions and with different cDNA libraries, it is also possible that each study includes many false positives and false negatives.
4.2. Wheat germ–based protein array system
4.2.1. Overview of the protein array technology
As described above, the living cell‐based methods are powerful tools for identifying viral‐host interactions since the assays could be carried out under physiological conditions, at least partially. However, these methods have several disadvantages. First, proteins whose expression levels are quite low or that show cytotoxicity are hard to analyse. Second, immunoprecipitation assays are commonly used to detect the interaction between a protein and a protein; however, the number of interactions that can be detected at one time is limited. Therefore, it is highly possible that many researchers have potentially overlooked important but difficult‐to‐detect interactions in their first screenings using living cells. To solve these problems, we recently developed a novel biochemical screening method based on a wheat germ cell–free protein synthesis system (wheat cell‐free system) and high‐throughput binding assay. The wheat cell‐free system enables the synthesis of various kinds of eukaryotic proteins in a 96‐well format . So far, proteins having several transmembrane domains, relatively large molecular weights, and cytotoxic activity were successfully synthesized by this system [63, 64]. This robust protein synthesis system allows us to establish a “protein array”, from which tens to thousands of recombinant proteins sorted by their functions were arrayed into each well of a 96‐well plate . The protein arrays currently available in our research group are shown in Table 1 [65–70]. Based on these protein arrays, we have established a high‐throughput binding assay for identifying proteins directly bound to target proteins from the array. To perform hundreds to tens of thousands of binding reactions, a luminescent‐based binding assay, called AlphaScreen, was employed [65, 66, 71]. This assay is able to use crude translation products of wheat cell‐free synthesis to detect binding reactions by mixing these crude proteins in a 384‐well plate, followed by adding two beads and the antibody for detection. Because of its flexibility, AlphaScreen can be used not only as a binding assay but also as a protein cleavage assay for a viral protease. The principle of the assays is shown in Figure 2.
|Protein array||Origin||Number of proteins||References|
|Protein kinase||Human and mouse||400||Masaki et al. , Miyakawa et al. , Kudoh et al. |
|E3 ligase||Human and mouse||250||Takahashi et al. , Tan et al. |
|Single transmembrane protein||Human and mouse||730||Unpublished|
|Auto‐antigen protein||Human and mouse||2100||Matsuoka et al. |
4.2.2. Practical applications for viral research
We and other research groups have done several assays using this technology to identify host proteins that interacted with viral proteins. In researching the HCV (a virus related to DENV), several protein kinases were found to bind directly with the HCV non‐structural protein NS5A using the protein kinase array; by phosphorylating NS5A through additional functional analysis using a cultured cell system, one protein kinase, Casein kinase I‐α, was found to regulate viral replication . The protein kinase array was used to identify other host factors that functionally interacted with human immunodeficiency virus (HIV) proteins [67, 68]. In the case of the protease assay, one research article demonstrated that a protease from xenotropic murine leukaemia virus‐related virus (XMRV) and 24 cellular proteins that were target candidates of the viral protease were synthesized;
4.2.3. Wheat cell–free protein array for DENV research
Thus far, NS3 and NS5 proteins of DENV have been considered difficult to synthesize as full‐length active recombinant proteins using a conventional protein expression system, such as for
5. Restriction of DENV infection by cellular inhibitors
As previously mentioned, DENV hijacks the host's biological process for its efficient replication. Meanwhile, it has become apparent that DENV infection can be limited by cellular factors. In this sense, the innate immune response induced by IFN is considered to be the first line of defence against DENV 
Generally, RNA viruses that infect target cells are sensed by the pattern recognition receptors (PRRs), which specifically recognize a component of invading viruses. As for DENV infection, the membrane‐bound Toll‐like receptors (TLR3, TLR7, TLR8) and the cytosolic receptors (retinoic acid‐inducible gene I [RIG‐I], melanoma differentiation‐associated gene 5 [MDA5]) are reported to be the PRRs for viral RNA [74–77]. These recognitions in turn activate adaptor molecules of the PRR, leading to the activation of a downstream phosphorylation cascade and the subsequent production of IFN and pro‐inflammatory cytokines (Figure 4) . Among the IFNs produced, type I IFNs, including IFNα, IFN‐β, and IFN‐ω, play an important role in antiviral immunity . The type I IFN then binds to its receptors (IFNAR) on neighbouring cells and signals to induce the phosphorylation of signal transducers and the activators of transcriptions 1 and 2 (STAT1 and 2) in cytoplasm. This phosphorylation of STAT1/2 triggers the formation of IFN‐stimulated gene factor 3 (ISGF3) with IFN‐regulatory factor 9 (IRF9). Finally, the ISGF3 complex translocates to the nucleus and acts as a transcription activator for the expression of a number of genes by binding to the IFN‐stimulated response elements (ISREs) on chromosomes (Figure 4) .
As seen above, IFN is considered to be an inducer of the antiviral state, and it has been well demonstrated that actual antiviral effector molecules in the IFN response are a subset of the genes upregulated by IFN, which are called IFN‐stimulated genes (ISGs). Thus far, hundreds of genes have been classified as ISGs, and many of them are reported to have inhibitory effects on divergent families of viruses, including flaviviruses [80–83]. Importantly, several ISGs have also been shown to restrict DENV infection, and their suppressive effects are likely to be exerted at the multiple steps of virus replication, including virus entry (IFITMs [84, 85], ADAP2 ), viral RNA/protein synthesis (ISG20 , viperin [88, 89]), and infectious virion production (tetherin , ISG15 [91, 92]). In addition, a comprehensive study using an overexpression of a cDNA library derived from known ISGs demonstrates the involvement of many more ISGs in the restriction of DENV . However, the precise mechanisms of these ISG‐mediated anti‐DENV activities remain unclear, and we will have to wait for future studies for detailed analysis.
6. Identification of RyDEN/C19orf66 as novel anti‐DENV ISG
It has been well demonstrated that a gain‐of‐function (i.e., overexpression) screen of cDNA is a powerful approach to identifying antiviral ISGs [82, 83, 93, 94]. In a recent study, we employed the gain‐of‐function strategy using a pool of cDNA library derived from type 1 IFN‐treated cells to search for ISGs suppressing DENV replication in human cells . In this approach, a human cervical carcinoma cell line, HeLa, was treated with type I IFN (a mixture of human IFN‐α and ‐ω) for 24 hours at a concentration that had been reported to inhibit DENV infection
The ORF of RyDEN (
The anti‐DENV activity of RyDEN was confirmed by creating stable cell lines that expressed epitope tag‐fused human hepatoma cell lines, and all DENV serotypes (i.e., DENV‐1–4) were found to be inhibited by RyDEN expressions. In line with the fact that this gene was first identified by the gain‐of‐function approach using an IFN‐derived cDNA library, expressions of RyDEN in various human cell lines were upregulated by IFN treatment to a greater or lesser extent, indicating that RyDEN is a bona fide anti‐DENV ISG. More importantly, when the endogenous expression of RyDEN mRNA was knocked down by RNAi, the suppressive activity of type I IFNs against DENV became less effective, suggesting that RyDEN was a major contributor of the IFN‐mediated anti‐DENV response .
One question to ponder: what is the molecular mechanism by which RyDEN suppresses DENV replication? Affinity purification‐mass spectrometry analysis with affinity tag‐fused RyDEN found that RyDEN interacted with two other cellular proteins, poly(A)‐binding protein cytoplasmic 1 (PABPC1) and La motif‐related protein 1 (LARP1). PABPC1 is a member of the PABP family; as described above, this protein bridges the 5′ and 3′ ends of mRNA by binding both the eIF4G and the poly(A) tail, which stimulates the initiation of translation . LARP1 is also an RNA‐binding protein and one of the LARPS, which shares the signature motif with the La autoantigen, called the La motif (LM). Therefore, Larp and La proteins are categorized as being in the same family . Intriguingly, LARP1 is shown to interact with PABP to stimulate the mRNA translation process [99, 100]. Considering the positive effect of PABP on translation , the association of PABPC1 and LARP1 with RyDEN suggests that RyDEN might interfere with the translation of DENV RNA by inhibiting PABPC1 and LARP1 functions. Indeed, this speculation was supported by the following findings: (i) an RNAi‐mediated knockdown of PABPC1 and LARP1 significantly reduced the level of DENV replication, (ii) PABPC1 interacted with DENV RNA in infected cells, (iii) the expression of RyDEN suppressed the expression of the reporter protein from a DENV‐based sub‐genomic RNA replicon that lacked structural (C, prM, E) genes, and (iv) the recruitment of RyDEN to the DENV RNA 3’ UTR was enhanced by the presence of PABPC1
7. Concluding remarks
This chapter highlighted the molecular interactions between DENV and host factors, particularly focused on the cellular regulation of DENV replication. As is well known, IFN response is one of the host controls of DENV infection and pathogenesis [96, 103, 104]. Although the effector molecules in the IFN response that actually interfere with virus replication remain fully clarified, profound efforts have been made to identify the IFN‐inducible cellular factors restricting RNA virus replication, including DENV . It should be noted that antagonistic effects of DENV infection on IFN signalling and production, which are mediated by several means using viral factors, are observed [105–107]. However, the characterization of anti‐DENV ISGs is to illuminate the “heel of Achilles” of DENV, which will provide the underpinnings for the development of antivirals against dengue.