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Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

Scott JC, Brackney DE, Campbell CL, Bondu-Hawkins V, Hjelle B, Ebel GD, Olson KE, Blair CD - PLoS Negl Trop Dis (2010)

Bottom Line: Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings.An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA.Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

View Article: PubMed Central - PubMed

Affiliation: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

ABSTRACT
The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

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viRNA genome coverage distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and Aedes aegypti mosquitoes.viRNA coverage across DENV genome for each library. Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, A. aegypti (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.
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pntd-0000848-g003: viRNA genome coverage distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and Aedes aegypti mosquitoes.viRNA coverage across DENV genome for each library. Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, A. aegypti (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.

Mentions: The sequences of the DENV2-specific small RNAs in the Aag2 cell library (5 dpi) were distributed evenly across the entire DENV2 genome, with the exception of a higher proportion of reads from one site around 10,000 nt (Fig. 3), further indicating that dsRNA replicative intermediates were the target of Dcr2 cleavage. The distribution along the viral genome of the DENV2 viRNAs from infected mosquitoes was somewhat different from that seen in the DENV2-infected Aag2 cells (Fig. 3), with several ‘hot spots’ for origins of either positive-sense or negative-sense viRNA, suggesting that ssRNA secondary structures in the DENV2 genome or its complement may also have been targeted by Dcr2, and to a greater extent in the mosquito than in cell culture. Analysis of potential secondary structures in the DENV2 RNA genome using mFold (www.bioinfo.rpi.edu/applications/mfold) predicts optimal-energy RNA configurations with extensive intrastrand base-pairing throughout the genome; however, most of these dsRNA structures lack perfect base-pairing over regions >21 bp so it is difficult to correlate them with siRNA “hot-spots”. The DENV2-specific small RNAs in C6/36 cells were not equally distributed, but instead were derived from a few specific regions of the genome, which might represent intrastrand secondary structure in the positive-strand virus genome.


Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells.

Scott JC, Brackney DE, Campbell CL, Bondu-Hawkins V, Hjelle B, Ebel GD, Olson KE, Blair CD - PLoS Negl Trop Dis (2010)

viRNA genome coverage distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and Aedes aegypti mosquitoes.viRNA coverage across DENV genome for each library. Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, A. aegypti (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2964303&req=5

pntd-0000848-g003: viRNA genome coverage distribution varies among DENV2-infected Aag2 and C6/36 cell cultures and Aedes aegypti mosquitoes.viRNA coverage across DENV genome for each library. Shown are Aag2 (5 dpi) library, C6/36 (5 dpi) library, A. aegypti (9 dpi) library. Red bars, negative-sense viRNAs; blue bars, positive-sense viRNAs. Note differences in Y-axes among graphs.
Mentions: The sequences of the DENV2-specific small RNAs in the Aag2 cell library (5 dpi) were distributed evenly across the entire DENV2 genome, with the exception of a higher proportion of reads from one site around 10,000 nt (Fig. 3), further indicating that dsRNA replicative intermediates were the target of Dcr2 cleavage. The distribution along the viral genome of the DENV2 viRNAs from infected mosquitoes was somewhat different from that seen in the DENV2-infected Aag2 cells (Fig. 3), with several ‘hot spots’ for origins of either positive-sense or negative-sense viRNA, suggesting that ssRNA secondary structures in the DENV2 genome or its complement may also have been targeted by Dcr2, and to a greater extent in the mosquito than in cell culture. Analysis of potential secondary structures in the DENV2 RNA genome using mFold (www.bioinfo.rpi.edu/applications/mfold) predicts optimal-energy RNA configurations with extensive intrastrand base-pairing throughout the genome; however, most of these dsRNA structures lack perfect base-pairing over regions >21 bp so it is difficult to correlate them with siRNA “hot-spots”. The DENV2-specific small RNAs in C6/36 cells were not equally distributed, but instead were derived from a few specific regions of the genome, which might represent intrastrand secondary structure in the positive-strand virus genome.

Bottom Line: Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings.An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA.Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

View Article: PubMed Central - PubMed

Affiliation: Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

ABSTRACT
The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.

Show MeSH
Related in: MedlinePlus