Comparison of Dengue Virus Type 2-Specific Small RNAs from RNA Interference-Competent and –Incompetent Mosquito Cells

ABSTRACT Dengue virus is a positive-strand RNA virus and a member of the genus Flavivirus, which includes West Nile, yellow fever, and tick-borne encephalitis viruses. Flavivirus genomes are translated as a single polyprotein that is subsequently cleaved into 10 proteins, the first of which is the viral capsid (C) protein. Dengue virus type 2 (DENV2) and other mosquito-borne flaviviruses initiate translation of C from a start codon in a suboptimal context and have multiple in-frame AUGs downstream. Here, we show that an RNA hairpin structure in the capsid coding region (cHP) directs translation start site selection in human and mosquito cells. The ability of the cHP to direct initiation from the first start codon is proportional to its thermodynamic stability, is position dependent, and is sequence independent, consistent with a mechanism in which the scanning initiation complex stalls momentarily over the first AUG as it begins to unwind the cHP. The cHP of tick-borne flaviviruses is not maintained in a position to influence start codon selection, which suggests that this coding region cis element may serve another function in the flavivirus life cycle. Here, we demonstrate that the DENV2 cHP and both the first and second AUGs of C are necessary for efficient viral replication in human and mosquito cells. While numerous regulatory elements have been identified in the untranslated regions of RNA viral genomes, we show that the cHP is a coding-region RNA element that directs start codon selection and is required for viral replication.

Download Full-text

RNA Silencing of Dengue Virus Type 2 Replication in Transformed C6/36 Mosquito Cells Transcribing an Inverted-Repeat RNA Derived from the Virus Genome

Journal of Virology ◽

10.1128/jvi.76.24.12925-12933.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12925-12933 ◽

Cited By ~ 107

Author(s):

Zach N. Adelman ◽

Irma Sanchez-Vargas ◽

Emily A. Travanty ◽

Jon O. Carlson ◽

Barry J. Beaty ◽

...

Keyword(s):

Dengue Virus ◽

Cell Lines ◽

Virus Type ◽

Inverted Repeat ◽

Rna Silencing ◽

Defense Mechanism ◽

Northern Blot Hybridization ◽

Mosquito Cells ◽

Dengue Virus Type 2

ABSTRACT Double-stranded RNA (dsRNA) initiates cellular posttranscriptional responses that are collectively called RNA silencing in a number of different organisms, including plants, nematodes, and fruit flies. In plants, RNA silencing has been associated with protection from virus infection. In this study, we demonstrate that dsRNA-mediated interference also can act as a viral defense mechanism in mosquito cells. C6/36 (Aedes albopictus) cells were stably transformed with a plasmid designed to transcribe an inverted-repeat RNA (irRNA) derived from the genome of dengue virus type 2 (DEN-2) capable of forming dsRNA. Clonal cell lines were selected with an antibiotic resistance marker and challenged with DEN-2. The cell lines were classified as either susceptible or resistant to virus replication, based on the percentage of cells expressing DEN-2 envelope (E) antigen 7 days after challenge. Eight out of 18 (44%) cell lines designed to express irRNA were resistant to DEN-2 challenge, with more than 95% of the cells showing no DEN-2 antigen accumulation. One of the DEN-2-resistant cell lines, FB 9.1, was further characterized. DEN-2 genome RNA failed to accumulate in FB 9.1 cells after challenge. Northern blot hybridization detected transcripts containing transgene sequences of both sense and antisense polarity, suggesting that DEN-2-specific dsRNA was present in the cells. In addition, a class of small RNAs 21 to 25 nucleotides in length was detected that specifically hybridized to labeled sense or antisense DEN-2 RNA derived from the target region of the genome. These observations were consistent with RNA silencing as the mechanism of resistance to DEN-2 in transformed mosquito cells.

Download Full-text

Review for "Glycogen synthetase kinase 3 inhibition drives MIC‐A/B to promote cytokine production by human natural killer cells in Dengue virus type 2 infection"

10.1002/eji.201948284/v1/review2 ◽

2019 ◽

Keyword(s):

Natural Killer Cells ◽

Dengue Virus ◽

Natural Killer ◽

Virus Type ◽

Cytokine Production ◽

Killer Cells ◽

Glycogen Synthetase ◽

Human Natural Killer Cells ◽

Dengue Virus Type 2

Download Full-text

Efﬁcacy of Catharanthus roseus Extract against Dengue Virus Type 2 Infection in Vitro

Indian Journal of Public Health Research & Development ◽

10.37506/v11/i2/2020/ijphrd/195051 ◽

2020 ◽

Vol 11 (2) ◽

pp. 1583

Author(s):

Noor Zarina Abd Wahab ◽

NorefrinaShafinaz Md. Nor ◽

Nazlina Ibrahim

Keyword(s):

Dengue Virus ◽

Virus Type ◽

Catharanthus Roseus ◽

Dengue Virus Type 2

Download Full-text

Reduced sphingosine kinase 1 activity in dengue virus type-2 infected cells can be mediated by the 3′ untranslated region of dengue virus type-2 RNA

Journal of General Virology ◽

10.1099/vir.0.055616-0 ◽

2013 ◽

Vol 94 (11) ◽

pp. 2437-2448 ◽

Cited By ~ 19

Author(s):

J. M. Carr ◽

T. Kua ◽

J. N. Clarke ◽

J. K Calvert ◽

J. R. Zebol ◽

...

Keyword(s):

Cell Death ◽

Dengue Virus ◽

Virus Type ◽

Elongation Factor ◽

Sphingosine Kinase ◽

Sphingosine Kinase 1 ◽

Diarrhea Virus ◽

Infected Cells ◽

Dengue Virus Type 2

Sphingosine kinase 1 (SphK1) is a lipid kinase with important roles including regulation of cell survival. We have previously shown reduced SphK1 activity in cells with an established dengue virus type-2 (DENV-2) infection. In this study, we examined the effect of alterations in SphK1 activity on DENV-2 replication and cell death and determined the mechanisms of the reduction in SphK1 activity. Chemical inhibition or overexpression of SphK1 after established DENV-2 infection had no effect on infectious DENV-2 production, although inhibition of SphK1 resulted in enhanced DENV-2-induced cell death. Reduced SphK1 activity was observed in multiple cell types, regardless of the ability of DENV-2 infection to be cytopathic, and was mediated by a post-translational mechanism. Unlike bovine viral diarrhea virus, where SphK1 activity is decreased by the NS3 protein, SphK1 activity was not affected by DENV-2 NS3 but, instead, was reduced by expression of the terminal 396 bases of the 3′ UTR of DENV-2 RNA. We have previously shown that eukaryotic elongation factor 1A (eEF1A) is a direct activator of SphK1 and here DENV-2 RNA co-localized and co-precipitated with eEF1A from infected cells. We propose that the reduction in SphK1 activity late in DENV-2-infected cells is a consequence of DENV-2 out-competing SphK1 for eEF1A binding and hijacking cellular eEF1A for its own replication strategy, rather than a specific host or virus-induced change in SphK1 to modulate viral replication. Nonetheless, reduced SphK1 activity may have important consequences for survival or death of the infected cell.

Download Full-text