Dengue virus sustains viability of infected cells by counteracting apoptosis-mediated DNA breakage

AbstractDengue is the most important arboviral disease inflicting mankind. This mosquito-borne Flavivirus causes mild to severe dengue fever which in some cases leads to life-threatening conditions namely, dengue haemorrhagic fever and dengue shock syndrome. Annual infection is estimated at 390 million globally with 96 million manifesting clinically. So, ≥80% infections are asymptomatic and self-limiting. Dengue virus (DV) non-structural protein 1 (NS1) is a proven virotoxin abundantly present in the victim’s blood. We found that DV-infected or only NS1-expressing cells both can induce Cleaved Caspase3, due to antiviral response of host cells. NS1-transfected cells also showed nuclear damage and significant levels of DNA breaks suggestive of ensuing apoptosis. So, it was established that NS1 alone is capable of causing apoptosis. Surprisingly, despite secreting similar amount of soluble NS1, the DV-infected cells showed intact nuclear morphology and background levels of DNA nicks. These observations suggested that DV downregulates apoptosis of infected cells, which is a viral strategy against host defence. Furthermore, DV-infected cells counteracted Camptothecin-induced apoptotic DNA break. DV-infection was also found to keep the infected cells metabolically more active than only NS1 expressing cells. So, DV bypasses cellular defence against virus i.e. apoptosis by counteracting cellular DNA break and keeps the infected cells metabolically active to support virus replication for longer period which eventually results in high virus titer in circulation. Our findings reveal another level of intricacy involving dengue virus-host interactions and perhaps explain why ≥80% DV infections are asymptomatic/self-limiting despite the presence of NS1 virotoxin in infected cells.Author SummaryNS1, a virotoxin, abundantly present in Dengue patients blood, is a major player behind disease patho-biogenesis including plasma leakage. Despite the presence of NS1 in blood, Dengue is asymptomatic and self-limiting in more than 80% dengue virus (DV) infected people. We investigated this observation and found that plasmid-mediated NS1 expression and secretion in cells are sufficient to cause programmed cell death (apoptosis) and associated cellular DNA breakage. However, cells infected with dengue virus and secreting equivalent amounts of NS1 didn’t exhibit apoptotic DNA breakage. Consequently, DV-infected cells showed better survival than cells in which only NS1 was transiently expressed by transfection with expression plasmid. We also found that DV can even prevent chemical induced apoptotic DNA damage in infected host cells. So, DV bypasses host antiviral defence i.e. apoptosis by counteracting cellular DNA breakages and keeps the infected cells metabolically active to prolong virus replication.

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A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Journal of Virology ◽

10.1128/jvi.00867-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7170-7186 ◽

Cited By ~ 26

Author(s):

Laurent Chatel-Chaix ◽

Wolfgang Fischl ◽

Pietro Scaturro ◽

Mirko Cortese ◽

Stephanie Kallis ◽

...

Keyword(s):

Amino Acid ◽

Dengue Virus ◽

Virus Replication ◽

Strong Evidence ◽

Drug Target ◽

Nonstructural Protein ◽

Rna Replication ◽

Replication Cycle ◽

Infected Cells ◽

Ns3 Protease

ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

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Dengue Virus Induces Increased Activity of the Complement Alternative Pathway in Infected Cells

Journal of Virology ◽

10.1128/jvi.00633-18 ◽

2018 ◽

Vol 92 (14) ◽

Cited By ~ 10

Author(s):

Sheila Cabezas ◽

Gustavo Bracho ◽

Amanda L. Aloia ◽

Penelope J. Adamson ◽

Claudine S. Bonder ◽

...

Keyword(s):

Endothelial Cells ◽

Dengue Virus ◽

Vascular Permeability ◽

Alternative Pathway ◽

Denv Infection ◽

Factor H ◽

Severe Dengue ◽

Complement Alternative Pathway ◽

Viral Pathogen ◽

Infected Cells

ABSTRACTSevere dengue virus (DENV) infection is associated with overactivity of the complement alternative pathway (AP) in patient studies. Here, the molecular changes in components of the AP during DENV infectionin vitrowere investigated. mRNA for factor H (FH), a major negative regulator of the AP, was significantly increased in DENV-infected endothelial cells (EC) and macrophages, but, in contrast, production of extracellular FH protein was not. This discord was not seen for the AP activator factor B (FB), with DENV induction of both FB mRNA and protein, nor was it seen with Toll-like receptor 3 or 4 stimulation of EC and macrophages, which induces both FH and FB mRNA and protein. Surface-bound and intracellular FH protein was, however, induced by DENV, but only in DENV antigen-positive cells, while in two other DENV-susceptible immortalized cell lines (ARPE-19 and human retinal endothelial cells), FH protein was induced both intracellularly and extracellularly by DENV infection. Regardless of the cell type, there was an imbalance in AP components and an increase in markers of complement AP activity associated with DENV-infected cells, with lower FH relative to FB protein, an increased ability to promote AP-mediated lytic activity, and increased deposition of complement component C3b on the surface of DENV-infected cells. For EC in particular, these changes are predicted to result in higher complement activity in the local cellular microenvironment, with the potential to induce functional changes that may result in increased vascular permeability, a hallmark of dengue disease.IMPORTANCEDengue virus (DENV) is a significant human viral pathogen with a global medical and economic impact. DENV may cause serious and life-threatening disease, with increased vascular permeability and plasma leakage. The pathogenic mechanisms underlying these features remain unclear; however, overactivity of the complement alternative pathway has been suggested to play a role. In this study, we investigate the molecular events that may be responsible for this observed alternative pathway overactivity and provide novel findings of changes in the complement system in response to DENV infection in primary cell types that are a major target for DENV infection (macrophages) and pathogenesis (endothelial cells)in vivo. Our results suggest a new dimension of cellular events that may influence endothelial cell barrier function during DENV infection that could expand strategies for developing therapeutics to prevent or control DENV-mediated vascular disease.

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Mitochondrial Import of Dengue Virus NS3 Protease and Cleavage of GrpEL1, a Cochaperone of Mitochondrial Hsp70

Journal of Virology ◽

10.1128/jvi.01178-20 ◽

2020 ◽

Vol 94 (17) ◽

Author(s):

Chaitanya Gandikota ◽

Fareed Mohammed ◽

Lekha Gandhi ◽

Deepti Maisnam ◽

Ushodaya Mattam ◽

...

Keyword(s):

Virus Infection ◽

Dengue Virus ◽

Severe Dengue ◽

Clinical Samples ◽

Mitochondrial Matrix ◽

Virus Infections ◽

Mitochondrial Import ◽

Mitochondrial Transport ◽

Infected Cells ◽

Mitochondrial Hsp70

ABSTRACT Dengue virus infections, which have been reported in nearly 140 countries, pose a significant threat to human health. The genome of dengue virus encodes three structural and seven nonstructural (NS) proteins along with two untranslated regions, one each on both ends. Among them, dengue protease (NS3) plays a pivotal role in polyprotein processing and virus multiplication. NS3 is also known to regulate several host proteins to induce and maintain pathogenesis. Certain viral proteins are known to interact with mitochondrial membrane proteins and interfere with their functions, but the association of a virus-coded protein with the mitochondrial matrix is not known. In this report, by using in silico analysis, we show that NS3pro alone is capable of mitochondrial import; however, this is dependent on its innate mitochondrial transport signal (MTS). Transient-transfection and protein import studies confirm the import of NS3pro to the mitochondrial matrix. Similarly, NS3pro-helicase (amino acids 1 to 464 of NS3) also targets the mitochondria. Intriguingly, reduced levels of matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), were noticed in NS3pro-expressing, NS3pro-helicase-expressing, and virus-infected cells. Upon the use of purified components, GrpEL1 undergoes cleavage, and the cleavage sites have been mapped to KR81A and QR92S. Importantly, GrpEL1 levels are seriously compromised in severe dengue virus-infected clinical samples. Our studies provide novel insights into the import of NS3 into host mitochondria and identify a hitherto unknown factor, GrpEL1, as a cleavage target, thereby providing new avenues for dengue virus research and the design of potential therapeutics. IMPORTANCE Approximately 40% of the world’s population is at risk of dengue virus infection. There is currently no specific drug or potential vaccine for these infections. Lack of complete understanding of the pathogenesis of the virus is one of the hurdles that must be overcome in developing antivirals for this virus infection. In the present study, we observed that the dengue virus-coded protease imports to the mitochondrial matrix, and our report is the first ever of a virus-coded protein, either animal or human, importing to the mitochondrial matrix. Our analysis indicates that the observed mitochondrial import is due to an inherited mitochondrial transport signal. We also show that matrix-localized GrpE protein homolog 1 (GrpEL1), a cochaperone of mitochondrial Hsp70 (mtHsp70), is also the substrate of dengue virus protease, as observed in vitro and ex vivo in virus-infected cells and dengue virus-infected clinical samples. Hence, our studies reveal an essential aspect of the pathogenesis of dengue virus infections, which may aid in developing antidengue therapeutics.

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Extracellular Vesicles Mediate Receptor-Independent Transmission of Novel Tick-Borne Bunyavirus

Journal of Virology ◽

10.1128/jvi.02490-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 873-886 ◽

Cited By ~ 17

Author(s):

Jesus A. Silvas ◽

Vsevolod L. Popov ◽

Adriana Paulucci-Holthauzen ◽

Patricia V. Aguilar

Keyword(s):

Virus Infection ◽

Extracellular Vesicles ◽

Virus Replication ◽

Case Fatality ◽

Host Protein ◽

Host Cells ◽

Limited Information ◽

Recent Emergence ◽

Infected Cells ◽

Cytoplasmic Structures

ABSTRACTSevere fever with thrombocytopenia syndrome (SFTS) virus is a newly recognized member of the genusPhlebovirusin the familyBunyaviridae. The virus was isolated from patients presenting with hemorrhagic manifestations and an initial case fatality rate of 12 to 30% was reported. Due to the recent emergence of this pathogen, there is limited knowledge on the molecular virology of SFTS virus. Recently, we reported that the SFTS virus NSs protein inhibited the activation of the beta interferon (IFN-β) promoter. Furthermore, we also found that SFTS virus NSs relocalizes key components of the IFN response into NSs-induced cytoplasmic structures. Due to the important role these structures play during SFTS virus replication, we conducted live cell imaging studies to gain further insight into the role and trafficking of these cytoplasmic structures during virus infection. We found that some of the SFTS virus NSs-positive cytoplasmic structures were secreted to the extracellular space and endocytosed by neighboring cells. We also found that these secreted structures isolated from NSs-expressing cells and SFTS virus-infected cells were positive for the viral protein NSs and the host protein CD63, a protein associated with extracellular vesicles. Electron microscopy studies also revealed that the isolated CD63-immunoprecipitated extracellular vesicles produced during SFTS virus infection contained virions. The virions harbored within these structures were efficiently delivered to uninfected cells and were able to sustain SFTS virus replication. Altogether, these results suggest that SFTS virus exploits extracellular vesicles to mediate virus receptor-independent transmission to host cells and open the avenue for novel therapeutic strategies against SFTS virus and related pathogens.IMPORTANCESFTS virus is novel bunyavirus associated with hemorrhagic fever illness. Currently, limited information is available about SFTS virus. In the present study, we demonstrated that extracellular vesicles produced by SFTS virus-infected cells harbor infectious virions. We sought to determine whether these “infectious” extracellular vesicles can mediate transmission of the virus and confirmed that the SFTS virions were efficiently transported by these secreted structures into uninfected cells and were able to sustain efficient replication of SFTS virus. These results have significant impact on our understanding of how the novel tick-borne phleboviruses hijack cellular machineries to establish infection and point toward a novel mechanism for virus replication among arthropod-borne viruses.

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Wolbachia wStri Blocks Zika Virus Growth at Two Independent Stages of Viral Replication

mBio ◽

10.1128/mbio.00738-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 17

Author(s):

M. J. Schultz ◽

A. L. Tan ◽

C. N. Gray ◽

S. Isern ◽

S. F. Michael ◽

...

Keyword(s):

Dengue Virus ◽

Viral Replication ◽

Yellow Fever ◽

Virus Replication ◽

Zika Virus ◽

Chikungunya Virus ◽

Yellow Fever Virus ◽

Rna Viruses ◽

Content Type ◽

Infected Cells

ABSTRACTMosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacteriumWolbachia pipientisfrom supergroup A is a recent strategy employed to reduce the capacity for major vectors in theAedesmosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup BWolbachia wStri, isolated fromLaodelphax striatellus, was shown to inhibit multiple lineages of ZIKV inAedes albopictuscells. Here, we show thatwStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%.wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited bywStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry intowStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate inWolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis inwStri-infected cells. This study’s findings increase the potential for application ofwStri to block additional arboviruses and also identify specific blocks in viral infection caused byWolbachiacoinfection.IMPORTANCEDengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont,Wolbachia wStri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use ofWolbachia wStri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy.

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Targeting cap-dependent translation to inhibit Chikungunya virus replication: selectivity of p38 MAPK inhibitors to virus-infected cells due to autophagy-mediated down regulation of phospho-ERK

Journal of General Virology ◽

10.1099/jgv.0.001629 ◽

2021 ◽

Vol 102 (7) ◽

Author(s):

Prashant Mudaliar ◽

Parvanendhu Pradeep ◽

Rachy Abraham ◽

Easwaran Sreekumar

Keyword(s):

Protein Expression ◽

P38 Mapk ◽

Virus Replication ◽

Chikungunya Virus ◽

Host Cells ◽

Hek293 Cells ◽

Viral Titre ◽

Mtor Inhibition ◽

Infected Cells ◽

Eif2 Alpha

The 5′ capped, message-sense RNA genome of Chikungunya virus (CHIKV) utilizes the host cell machinery for translation. Translation is regulated by eIF2 alpha at the initiation phase and by eIF4F at cap recognition. Translational suppression by eIF2 alpha phosphorylation occurs as an early event in many alphavirus infections. We observe that in CHIKV-infected HEK293 cells, this occurs as a late event, by which time the viral replication has reached an exponential phase, implying its minimal role in virus restriction. The regulation by eIF4F is mediated through the PI3K-Akt-mTOR, p38 MAPK and RAS-RAF-MEK-ERK pathways. A kinetic analysis revealed that CHIKV infection did not modulate AKT phosphorylation, but caused a significant reduction in p38 MAPK phosphorylation. It caused degradation of phospho-ERK 1/2 by increased autophagy, leaving the PI3K-Akt-mTOR and p38 MAPK pathways for pharmacological targeting. mTOR inhibition resulted in moderate reduction in viral titre, but had no effect on CHIKV E2 protein expression, indicating a minimal role of the mTOR complex in virus replication. Inhibition of p38 MAPK using SB202190 caused a significant reduction in viral titre and CHIKV E2 and nsP3 protein expression. Furthermore, inhibiting the two pathways together did not offer any synergism, indicating that inhibiting the p38 MAPK pathway alone is sufficient to cause restriction of CHIKV replication. Meanwhile, in uninfected cells the fully functional RAS-RAF-MEK-ERK pathway can circumvent the effect of p38 MAPK inhibition on cap-dependent translation. Thus, our results show that host-directed antiviral strategies targeting cellular p38 MAPK are worth exploring against Chikungunya as they could be selective against CHIKV-infected cells with minimal effects on uninfected host cells.

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Loss of IKK subunits limits NF-κB signaling in reovirus infected cells

10.1101/843680 ◽

2019 ◽

Author(s):

Andrew J. McNamara ◽

Pranav Danthi

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Virus Replication ◽

Nuclear Translocation ◽

Innate Immune ◽

Host Cells ◽

Reovirus Infection ◽

Cellular Environment ◽

Ikk Complex ◽

Infected Cells

ABSTRACTViruses commonly antagonize innate immune pathways that are primarily driven by Nuclear Factor-κB (NF-κB), Interferon Regulatory Factor (IRF) and Signal Transducer and Activator of Transcription (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased RNA-seq based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that while vgRNA and reovirus infection both induce a similar IRF dependent gene expression program, gene expression driven by the NF-κB family of transcription factors is lower in infected cells. Potent agonists of NF-κB, such as Tumor Necrosis Factor alpha (TNFα) and vgRNA, failed to induce NF-κB dependent gene expression in infected cells. We demonstrate that NF-κB signaling is blocked due to loss of critical members of the Inhibitor of KappaB Kinase (IKK) complex, NF-κB Essential MOdifier (NEMO) and IKKβ. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-κB, thereby preventing gene expression. Our studies demonstrate that reovirus infection selectively blocks NF-κB, likely to counteract its antiviral effects and promote efficient viral replication.IMPORTANCEHost cells mount a response to curb virus replication in infected cells and prevent infection of neighboring, as yet uninfected cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-κB is inactive. Further, we demonstrate that NF-κB is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-κB function. Based on previous evidence that active NF-κB limits reovirus infection, we conclude that inactivating NF-κB is a viral strategy to produce a cellular environment that is favorable for virus replication.

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Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

mBio ◽

10.1128/mbio.01865-15 ◽

2016 ◽

Vol 7 (1) ◽

Cited By ~ 29

Author(s):

Stacia L. Phillips ◽

Erik J. Soderblom ◽

Shelton S. Bradrick ◽

Mariano A. Garcia-Blanco

Keyword(s):

Mass Spectrometry ◽

Dengue Virus ◽

Virus Replication ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Viral Rna ◽

Host Proteins ◽

Infected Cells

ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. IMPORTANCE Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Viral RNA molecules physically interact with cellular RNA-binding proteins (RBPs) throughout the course of infection; the identification of such interactions will lead to the elucidation of the molecular mechanisms of virus replication. Until now, the identification of host proteins bound to dengue viral RNA has been accomplished using in vitro strategies. Here, we used a method for the specific purification of dengue viral ribonucleoprotein (RNP) complexes from infected cells and subsequently identified the associated proteins by mass spectrometry. We then validated a functional role for the majority of these proteins in mediating efficient virus replication. This approach has broad relevance to virology and RNA biology, as it could theoretically be used to purify any viral RNP complex of interest.

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Dengue Virus Replicative Intermediate RNA Detection by Reverse Transcription-PCR

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.1.198-200.2002 ◽

2002 ◽

Vol 9 (1) ◽

pp. 198-200 ◽

Cited By ~ 1

Author(s):

Gilberto Vaughan ◽

Hiram Olivera ◽

Leopoldo Santos-Argumedo ◽

Abraham Landa ◽

Baltasar Briseño ◽

...

Keyword(s):

Dengue Virus ◽

Virus Replication ◽

Reverse Transcription ◽

Cell Tropism ◽

Rna Detection ◽

Replicative Intermediate ◽

Reverse Transcription Pcr ◽

Infected Cells

ABSTRACT Dengue virus replication involves synthesis of a replicative intermediate RNA (RI-RNA), whose presence reveals an actual infection. We report on a simple and rapid reverse transcription-PCR for the detection of viral RI-RNA in infected cells. The product is demonstrated at 20 min postinfection. This method is useful for the study of virus-cell tropism.

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Specific Interaction of DDX6 with an RNA Hairpin in the 3´-UTR of the Dengue Genome Mediates G1 Phase Arrest

Journal of Virology ◽

10.1128/jvi.00510-21 ◽

2021 ◽

Author(s):

Opas Choksupmanee ◽

Worapol Tangkijthavorn ◽

Kenneth Hodge ◽

Krittanai Trisakulwattana ◽

Worawich Phornsiricharoenphant ◽

...

Keyword(s):

Cell Cycle ◽

Dengue Virus ◽

Host Cell ◽

Severe Dengue ◽

G1 Phase ◽

Live Attenuated Vaccine ◽

Attenuated Vaccine ◽

Virus Serotype ◽

Infected Cells ◽

Rna Hairpin

The extent to which viral genomic RNAs interact with host factors and contribute to host response and disease pathogenesis is not well known. Here, we report that the human RNA helicase DDX6 specifically binds to the viral most conserved RNA hairpin in the A3 element in the dengue 3´-UTR, with nanomolar affinities. DDX6 CLIP confirmed the interaction in HuH-7 cells infected by dengue virus serotype 2. This interaction requires three conserved residues, Lys 307 , Lys 367 , and Arg 369 , as well as the unstructured extension in the C-terminal domain of DDX6. Interestingly, alanine substitution of these three basic residues resulted in RNA-independent ATPase activity, suggesting a mechanism by which RNA-binding and ATPase activities are coupled in DEAD-box helicases. Furthermore, we applied a cross-omics gene enrichment approach to suggest that DDX6 is functionally related to cell cycle regulation and viral pathogenicity. Indeed, infected cells exhibited cell cycle arrest in G1 phase and a decrease in the early S phase. Exogeneous expression of intact DDX6, but not A3-binding-deficient mutants, alleviated these effects by rescue of the DNA pre-initiation complex expression. Disruption of the DDX6-binding site was found in dengue and Zika live-attenuated vaccine strains. Our results suggested that dengue virus has evolved an RNA aptamer against DDX6 to alter host cell states, and defined DDX6 as a new regulator of G1/S transition. Importance Dengue virus (DENV) is transmitted by mosquitoes to humans, infecting 390 million individuals per year globally. About 20% of infected patients shows a spectrum of clinical manifestation, ranging from a mild flu-like syndrome, dengue fever (DF), to life-threatening severe dengue (SD) diseases including dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). There is currently no specific treatment for dengue diseases and the molecular mechanism underlying dengue pathogenesis remains poorly understood. In this study, we combined biochemical, bioinformatics, high-content analysis, and RNA sequencing approaches to characterize a highly conserved interface of the RNA genome of DENV with a human factor named DDX6 in infected cells. The significance of our research is in identifying the mechanism for a viral strategy to alter host cell fates, which conceivably allows us to generate a model for live-attenuated vaccine and the design of new therapeutic reagent for dengue diseases.

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