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. 2013 Aug;87(16):9365-72.
doi: 10.1128/JVI.00567-13. Epub 2013 Jun 12.

Differential RNA sequence requirement for dengue virus replication in mosquito and mammalian cells

Sergio M Villordo  1 , Andrea V Gamarnik
Affiliations

Differential RNA sequence requirement for dengue virus replication in mosquito and mammalian cells

Sergio M Villordo et al. J Virol. 2013 Aug.

Abstract

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3' untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.

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Figures

Fig 1
Fig 1
Differential roles of the sHP in viral replication in mosquito and mammalian cells. (A) Schematic representation of the DENV genome showing relevant cis-acting elements: the promoter stem-loop A (SLA), the capsid region hairpin (cHP), the cyclization sequences (CS) 5′ upstream AUG region (5′UAR) and 3′UAR and 5′- and 3′CS, and the 3′ stem-loop (3′SL). The nucleotide sequence corresponding to the sHP structure is indicated on the right. (B) Replication of DENVs carrying substitutions within the sHP. Replication of viruses carrying the indicated mutations in BHK cells was monitored by immunofluorescence assay at different times posttransfection. Supernatants from mammalian BHK cells were used to infect mosquito C6/36 cells. The nucleotide sequence of the revertant virus Rev3 obtained from BHK cells transfected with Mut S3 is indicated; the arrows show the spontaneous changes generated in cell culture. (C) Replication of sHP mutants in the context of a DENV carrying luciferase as reporter. Luciferase activity was measured as a function of time after transfection of RNAs corresponding to the WT, a replication negative control with a mutation in the catalytic site of the NS5 polymerase (NS5mut), the sHP mutant Mut L, and the revertant virus Rev3. Error bars show standard deviations.
Fig 2
Fig 2
The loop sequence of the sHP is essential for DENV replication in mosquito cells. (A) Schematic representation of linear and circular conformations of the DENV genome showing the sequence corresponding to the sHP and the internal loop, respectively. Substitutions in each position of the loop and the internal loop are indicated as L1 to L6. Internal loop mutations IL1 and IL2 at the 5′ end of the genome are also indicated. (B and C) Replication of DENV mutants in the luciferase reporter system in mammalian and mosquito cells. Luciferase activity was measured as a function of time after transfection of the RNAs corresponding to the controls and mutants Mut L1 to L6, IL1, and IL2. Error bars show standard deviations.
Fig 3
Fig 3
Stability of the sHP structure is not the cause of the high vulnerability to nucleotide changes for DENV replication in mosquito cells. (A) Alignment showing high conservation of the sHP sequence in DENV types 1 to 4. The cyclization sequences 3′CS and 3′UAR are also indicated. (B) Schematic representation of mutants within the sHP that retain high stability. (C and D) Replication of DENV mutants in the luciferase reporter system in mammalian and mosquito cells as indicated in each case. Luciferase activity was measured as a function of time after transfection of viral RNAs corresponding to the controls and GAGA, UUCG, GAAAAA, and GAAAUA mutants. Error bars show standard deviations.
Fig 4
Fig 4
Substitutions within the stem of the sHP impair DENV replication in mosquito cells. (A and B) Replication of sHP mutants that change the sequence but maintain the stem structure in the context of the DENV luciferase reporter system. Luciferase activity in BHK and C6/36 cells was measured as a function of time after transfection of RNAs corresponding to WT and NS5Mut controls and mutants Rec 1, Rec 2, and Rec 3. Error bars show standard deviations. (C) Replication of DENV RNAs carrying substitutions, as indicated on the left, in mammalian BHK and mosquito C6/36 cells. Viral replication was monitored by immunofluorescence assay at different times posttransfection using specific anti-DENV antibodies.
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References

    1. Groat-Carmona AM, Orozco S, Friebe P, Payne A, Kramer L, Harris E. 2012. A novel coding-region RNA element modulates infectious dengue virus particle production in both mammalian and mosquito cells and regulates viral replication in Aedes aegypti mosquitoes. Virology 432:511–526 - PMC - PubMed
    1. Hanley KA, Manlucu LR, Gilmore LE, Blaney JE, Jr, Hanson CT, Murphy BR, Whitehead SS. 2003. A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4. Virology 312:222–232 - PubMed
    1. Helt AM, Harris E. 2005. S-phase-dependent enhancement of dengue virus 2 replication in mosquito cells, but not in human cells. J. Virol. 79:13218–13230 - PMC - PubMed
    1. Mondotte JA, Lozach PY, Amara A, Gamarnik AV. 2007. Essential role of dengue virus envelope protein N glycosylation at asparagine-67 during viral propagation. J. Virol. 81:7136–7148 - PMC - PubMed
    1. Samsa MM, Mondotte JA, Caramelo JJ, Gamarnik AV. 2012. Uncoupling cis-acting RNA elements from coding sequences revealed a requirement of the N-terminal region of dengue virus capsid protein in virus particle formation. J. Virol. 86:1046–1058 - PMC - PubMed

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