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

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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Murine Norovirus Replication Induces G0/G1Cell Cycle Arrest in Asynchronously Growing Cells

Journal of Virology ◽

10.1128/jvi.03673-14 ◽

2015 ◽

Vol 89 (11) ◽

pp. 6057-6066 ◽

Cited By ~ 16

Author(s):

Colin Davies ◽

Chris M. Brown ◽

Dana Westphal ◽

Joanna M. Ward ◽

Vernon K. Ward

Keyword(s):

Cell Cycle ◽

Viral Replication ◽

Host Cell ◽

Virus Replication ◽

Cell Cycle Progression ◽

Cycle Phase ◽

Murine Norovirus ◽

Cycle Progression ◽

Infected Cells ◽

Favorable Conditions

ABSTRACTMany viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1phase and an accumulation of cells in the G0/G1phase. The accumulation in G0/G1phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1phase may be representative of other members of theCaliciviridae.IMPORTANCENoroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1phase. Furthermore, we show that MNV replication is enhanced in the G1phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1phase for RNA virus replication.

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Dengue Fever and Severe Dengue in Barbados, 2008–2016

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed5020068 ◽

2020 ◽

Vol 5 (2) ◽

pp. 68

Author(s):

Kirk Osmond Douglas ◽

Sudip Kumar Dutta ◽

Byron Martina ◽

Fatih Anfasa ◽

T. Alafia Samuels ◽

...

Keyword(s):

Dengue Virus ◽

Dengue Fever ◽

Small Sample Size ◽

Health Planning ◽

Case Fatality ◽

Small Sample ◽

Severe Dengue ◽

Medical Attention ◽

Enzyme Linked Immunosorbent Assay ◽

Virus Serotype

Analysis of the temporal, seasonal and demographic distribution of dengue virus (DENV) infections in Barbados was conducted using national surveillance data from a total of 3994 confirmed dengue cases. Diagnosis was confirmed either by DENV–specific real time reverse transcriptase polymerase chain reaction (rRT–PCR), or non–structural protein 1 (NS1) antigen or enzyme linked immunosorbent assay (ELISA) tests; a case fatality rate of 0.4% (10/3994) was observed. The dengue fever (DF) prevalence varied from 27.5 to 453.9 cases per 100,000 population among febrile patients who sought medical attention annually. DF cases occurred throughout the year with low level of transmission observed during the dry season (December to June), then increased transmission during rainy season (July to November) peaking in October. Three major dengue epidemics occurred in Barbados during 2010, 2013 and possibly 2016 with an emerging three–year interval. DF prevalence among febrile patients who sought medical attention overall was highest among the 10–19 years old age group. The highest DF hospitalisation prevalence was observed in 2013. Multiple serotypes circulated during the study period and Dengue virus serotype 2 (DENV–2) was the most prevalent serotype during 2010, whilst DENV–1 was the most prevalent serotype in 2013. Two DENV–1 strains from the 2013 DENV epidemic were genetically more closely related to South East Asian strains, than Caribbean or South American strains, and represent the first ever sequencing of DENV strains in Barbados. However, the small sample size (n = 2) limits any meaningful conclusions. DF prevalence was not significantly different between females and males. Public health planning should consider DENV inter–epidemic periodicity, the current COVID–19 pandemic and similar clinical symptomology between DF and COVID–19. The implementation of routine sequencing of DENV strains to obtain critical data can aid in battling DENV epidemics in Barbados.

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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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Virus recovery rates for wild-type and live-attenuated vaccine strains of African horse sickness virus serotype 7 in orally infected South African Culicoides species

Medical and Veterinary Entomology ◽

10.1111/j.1365-2915.2007.00706.x ◽

2007 ◽

Vol 21 (4) ◽

pp. 377-383 ◽

Cited By ~ 23

Author(s):

G. J. VENTER ◽

J. T. PAWESKA

Keyword(s):

South African ◽

African Horse Sickness ◽

Culicoides Species ◽

Wild Type ◽

African Horse Sickness Virus ◽

Recovery Rates ◽

Live Attenuated Vaccine ◽

Attenuated Vaccine ◽

Virus Serotype ◽

Virus Recovery

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The antiviral activity of sulfated polysaccharides against dengue virus is dependent on virus serotype and host cell

Antiviral Research ◽

10.1016/j.antiviral.2005.02.001 ◽

2005 ◽

Vol 66 (2-3) ◽

pp. 103-110 ◽

Cited By ~ 170

Author(s):

L TALARICO ◽

C PUJOL ◽

R ZIBETTI ◽

P FARIA ◽

M NOSEDA ◽

...

Keyword(s):

Antiviral Activity ◽

Dengue Virus ◽

Host Cell ◽

Sulfated Polysaccharides ◽

Virus Serotype

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Emergence of genotype I of dengue virus serotype 3 during severe dengue epidemic in Sri Lanka, 2017

Japanese Journal of Infectious Diseases ◽

10.7883/yoken.jjid.2020.854 ◽

2021 ◽

Author(s):

Mya Myat Ngwe Tun ◽

Rohitha Muthugala ◽

Lakmali Rajamanthri ◽

Takeshi Nabeshima ◽

Corazon C. Buerano ◽

...

Keyword(s):

Sri Lanka ◽

Dengue Virus ◽

Severe Dengue ◽

Genotype I ◽

Virus Serotype ◽

Dengue Epidemic

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

10.1101/2020.06.19.162479 ◽

2020 ◽

Author(s):

Himadri Nath ◽

Keya Basu ◽

Abhishek De ◽

Subhajit Biswas

Keyword(s):

Dengue Virus ◽

Virus Replication ◽

Host Cells ◽

Severe Dengue ◽

Dna Breaks ◽

Dna Breakage ◽

Major Player ◽

Infected Cells ◽

Dna Break ◽

Cellular Dna

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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Dengue Virus Serotype 4 Is Responsible for the Outbreak of Dengue in East Java City of Jember, Indonesia

Viruses ◽

10.3390/v12090913 ◽

2020 ◽

Vol 12 (9) ◽

pp. 913

Author(s):

Aryati Aryati ◽

Billy J. Wrahatnala ◽

Benediktus Yohan ◽

May Fanny ◽

Faradila K. N. Hakim ◽

...

Keyword(s):

Dengue Virus ◽

Clinical Symptoms ◽

Secondary Infection ◽

Herd Immunity ◽

Antibody Detection ◽

Severe Dengue ◽

Envelope Gene ◽

Genotype I ◽

Denv Serotypes ◽

Virus Serotype

Outbreaks of dengue virus (DENV) in Indonesia have been mainly caused by the DENV serotype-1; -2; or -3. The DENV-4 was the least-reported serotype in Indonesia during the last five decades. We recently conducted a molecular epidemiology study of dengue in the Jember regency, East Java province, Indonesia. Dengue is endemic in the region and outbreaks occur annually. We investigated the clinical characteristics and etiology of dengue-like febrile illness in this regency to understand the disease dynamics. A total of 191 patients with clinical symptoms similar to dengue were recruited during an 11-month study in 2019–2020. Children accounted for the majority of cases and dengue burden was estimated in 41.4% of the cases based on NS1 antigen, viral RNA, and IgG/IgM antibody detection with the majority (73.4%) being primary infections. Secondary infection was significantly associated with a higher risk of severe dengue manifestation. All four DENV serotypes were detected in Jember. Strikingly, we observed the predominance of DENV-4, followed by DENV-3, DENV-1, and DENV-2. Genotype determination using Envelope gene sequence revealed the classification into Genotype I, Cosmopolitan Genotype, Genotype I, and Genotype II for DENV-1, -2, -3, and -4, respectively. The predominance of DENV-4 in Jember may be associated with a new wave of DENV infections and spread in a non-immune population lacking a herd-immunity to this particular serotype.

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