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Aedes aegypti uses RNA interference in defense against Sindbis virus infection.

Campbell CL, Keene KM, Brackney DE, Olson KE, Blair CD, Wilusz J, Foy BD - BMC Microbiol. (2008)

Bottom Line: We show virus-dependent effects on RNAi component transcript and protein levels during infection.We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication.These data define important features of RNAi anti-viral defense in Ae. aegypti.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthropod-borne Infectious Diseases Laboratory; Microbiology, Immunology, and Pathology Department, Colorado State University, Fort Collins, USA. corey.campbell@colostate.edu

ABSTRACT

Background: RNA interference (RNAi) is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus).

Results: SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.

Conclusion: We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.

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Ae aegypti Ago2 protein depletion and accumulation varied in a virus-dependent manner. Protein was extracted from pools of ten midguts from unfed, bloodfed or virus/bloodfed adult female Ae aegypti at the time points indicated. Equivalent protein amounts were separated by 4–15% gradient PAGE prior to blotting. Anti-Ae aegypti Ago2 antibody recognizes the carboxy-terminal peptide sequence YERMQIRTEIQDGHPMFFV. The loading control is a 19 kDa Ago2 cross-reacting band. "U", unfed female, "B", non-infectious bloodfed female, "M", MRE16-eGFP-infected, "T", TR339-eGFP-infected. Blots are representative of two independent experiments.
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Figure 5: Ae aegypti Ago2 protein depletion and accumulation varied in a virus-dependent manner. Protein was extracted from pools of ten midguts from unfed, bloodfed or virus/bloodfed adult female Ae aegypti at the time points indicated. Equivalent protein amounts were separated by 4–15% gradient PAGE prior to blotting. Anti-Ae aegypti Ago2 antibody recognizes the carboxy-terminal peptide sequence YERMQIRTEIQDGHPMFFV. The loading control is a 19 kDa Ago2 cross-reacting band. "U", unfed female, "B", non-infectious bloodfed female, "M", MRE16-eGFP-infected, "T", TR339-eGFP-infected. Blots are representative of two independent experiments.

Mentions: The lack of statistically significant increases in Ago2 transcript levels during SINV infection led us to ask whether protein levels fluctuated. Figure 5 shows Ago2 protein profiles in MRE16-eGFP and TR339-eGFP infected midguts compared to unfed and un-infected blood-fed controls. Bloodfeeding alone reduces Ago2 protein levels compared to midguts from unfed mosquitoes, and these proteins re-accumulated over a time period concomitant with the expected time course of bloodmeal digestion, vitellogenesis, and a return to the unfed state.


Aedes aegypti uses RNA interference in defense against Sindbis virus infection.

Campbell CL, Keene KM, Brackney DE, Olson KE, Blair CD, Wilusz J, Foy BD - BMC Microbiol. (2008)

Ae aegypti Ago2 protein depletion and accumulation varied in a virus-dependent manner. Protein was extracted from pools of ten midguts from unfed, bloodfed or virus/bloodfed adult female Ae aegypti at the time points indicated. Equivalent protein amounts were separated by 4–15% gradient PAGE prior to blotting. Anti-Ae aegypti Ago2 antibody recognizes the carboxy-terminal peptide sequence YERMQIRTEIQDGHPMFFV. The loading control is a 19 kDa Ago2 cross-reacting band. "U", unfed female, "B", non-infectious bloodfed female, "M", MRE16-eGFP-infected, "T", TR339-eGFP-infected. Blots are representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2278134&req=5

Figure 5: Ae aegypti Ago2 protein depletion and accumulation varied in a virus-dependent manner. Protein was extracted from pools of ten midguts from unfed, bloodfed or virus/bloodfed adult female Ae aegypti at the time points indicated. Equivalent protein amounts were separated by 4–15% gradient PAGE prior to blotting. Anti-Ae aegypti Ago2 antibody recognizes the carboxy-terminal peptide sequence YERMQIRTEIQDGHPMFFV. The loading control is a 19 kDa Ago2 cross-reacting band. "U", unfed female, "B", non-infectious bloodfed female, "M", MRE16-eGFP-infected, "T", TR339-eGFP-infected. Blots are representative of two independent experiments.
Mentions: The lack of statistically significant increases in Ago2 transcript levels during SINV infection led us to ask whether protein levels fluctuated. Figure 5 shows Ago2 protein profiles in MRE16-eGFP and TR339-eGFP infected midguts compared to unfed and un-infected blood-fed controls. Bloodfeeding alone reduces Ago2 protein levels compared to midguts from unfed mosquitoes, and these proteins re-accumulated over a time period concomitant with the expected time course of bloodmeal digestion, vitellogenesis, and a return to the unfed state.

Bottom Line: We show virus-dependent effects on RNAi component transcript and protein levels during infection.We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication.These data define important features of RNAi anti-viral defense in Ae. aegypti.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthropod-borne Infectious Diseases Laboratory; Microbiology, Immunology, and Pathology Department, Colorado State University, Fort Collins, USA. corey.campbell@colostate.edu

ABSTRACT

Background: RNA interference (RNAi) is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquito's anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus).

Results: SINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.

Conclusion: We show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.

Show MeSH
Related in: MedlinePlus