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RNA interference modulates replication of dengue virus in Drosophila melanogaster cells.

Mukherjee S, Hanley KA - BMC Microbiol. (2010)

Bottom Line: The four serotypes did not differ in mean titer.While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown.Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003, USA.

ABSTRACT

Background: Mosquito-borne dengue virus (DENV, genus Flavivirus) has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi) induced by exogenous small interfering RNAs (siRNAs) has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi.

Results: Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI) 0.1 and MOI 10; each strain achieved titers > 4.0 log10pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2), resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

Conclusions: These findings indicate that DENV can replicate in Drosophila S2 cells and that the RNAi pathway plays a role in modulating DENV replication in these cells. S2 cells offer a useful cell culture model for evaluation of the interaction between DENV and the RNAi response.

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Cartoon representing the major enzymes involved in the overlapping branches of the siRNA and the miRNA pathways in Drosophila melanogaster. While this cartoon was designed to emphasize the differences between the two pathways, it is important to stress that there is also extensive interaction and overlap between the two branches (some of which are represented by dotted arrows). This latter point is discussed in more detail in the text. [siRISC: RNA Induced Silencing Complex associated with siRNA; miRISC: miRNA associated RISC; miRNP: miRNA associated Ribo-Nucleo Protein complex; Ago: Argonaute (exhibits slicer activity); Dcr: Dicer; Spn-E: Spindle-E protein (involved in assembly of RISC); PIWI (co-purifies with Dcr-1 in Drosophila germline cells); R2D2 (bridges initiator and effector steps of siRNA pathway); ATP: adenosine triphosphate] [11,12,46,51-57].
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Figure 1: Cartoon representing the major enzymes involved in the overlapping branches of the siRNA and the miRNA pathways in Drosophila melanogaster. While this cartoon was designed to emphasize the differences between the two pathways, it is important to stress that there is also extensive interaction and overlap between the two branches (some of which are represented by dotted arrows). This latter point is discussed in more detail in the text. [siRISC: RNA Induced Silencing Complex associated with siRNA; miRISC: miRNA associated RISC; miRNP: miRNA associated Ribo-Nucleo Protein complex; Ago: Argonaute (exhibits slicer activity); Dcr: Dicer; Spn-E: Spindle-E protein (involved in assembly of RISC); PIWI (co-purifies with Dcr-1 in Drosophila germline cells); R2D2 (bridges initiator and effector steps of siRNA pathway); ATP: adenosine triphosphate] [11,12,46,51-57].

Mentions: In response to this exigency, investigators have pursued novel methods to prevent and treat dengue disease. In particular, there is considerable excitement about the potential to utilize RNA interference (RNAi) (Figure 1) to treat flavivirus infection in the host and control flavivirus transmission by the vector [7]. The RNAi pathway is composed of two major branches (Figure 1). The small interfering RNA (siRNA) branch is triggered by perfectly or nearly-perfectly base-paired exogenous dsRNA and results in RNA degradation, while the cellular microRNA branch (miRNA) is triggered by imperfectly base-paired dsRNA and results in translation repression [8-10]. Although siRNAs and miRNAs are processed via discrete pathways, specific enzymes may participate in both pathways. For example, recent evidence from Drosophila indicates that Dicer (Dcr)-1 is critical for both RNA degradation and translation repression, while Dcr-2 is required only for RNA degradation [11,12], and that Argonaute (Ago)-1 and Ago-2 proteins overlap in their functions [13].


RNA interference modulates replication of dengue virus in Drosophila melanogaster cells.

Mukherjee S, Hanley KA - BMC Microbiol. (2010)

Cartoon representing the major enzymes involved in the overlapping branches of the siRNA and the miRNA pathways in Drosophila melanogaster. While this cartoon was designed to emphasize the differences between the two pathways, it is important to stress that there is also extensive interaction and overlap between the two branches (some of which are represented by dotted arrows). This latter point is discussed in more detail in the text. [siRISC: RNA Induced Silencing Complex associated with siRNA; miRISC: miRNA associated RISC; miRNP: miRNA associated Ribo-Nucleo Protein complex; Ago: Argonaute (exhibits slicer activity); Dcr: Dicer; Spn-E: Spindle-E protein (involved in assembly of RISC); PIWI (co-purifies with Dcr-1 in Drosophila germline cells); R2D2 (bridges initiator and effector steps of siRNA pathway); ATP: adenosine triphosphate] [11,12,46,51-57].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874549&req=5

Figure 1: Cartoon representing the major enzymes involved in the overlapping branches of the siRNA and the miRNA pathways in Drosophila melanogaster. While this cartoon was designed to emphasize the differences between the two pathways, it is important to stress that there is also extensive interaction and overlap between the two branches (some of which are represented by dotted arrows). This latter point is discussed in more detail in the text. [siRISC: RNA Induced Silencing Complex associated with siRNA; miRISC: miRNA associated RISC; miRNP: miRNA associated Ribo-Nucleo Protein complex; Ago: Argonaute (exhibits slicer activity); Dcr: Dicer; Spn-E: Spindle-E protein (involved in assembly of RISC); PIWI (co-purifies with Dcr-1 in Drosophila germline cells); R2D2 (bridges initiator and effector steps of siRNA pathway); ATP: adenosine triphosphate] [11,12,46,51-57].
Mentions: In response to this exigency, investigators have pursued novel methods to prevent and treat dengue disease. In particular, there is considerable excitement about the potential to utilize RNA interference (RNAi) (Figure 1) to treat flavivirus infection in the host and control flavivirus transmission by the vector [7]. The RNAi pathway is composed of two major branches (Figure 1). The small interfering RNA (siRNA) branch is triggered by perfectly or nearly-perfectly base-paired exogenous dsRNA and results in RNA degradation, while the cellular microRNA branch (miRNA) is triggered by imperfectly base-paired dsRNA and results in translation repression [8-10]. Although siRNAs and miRNAs are processed via discrete pathways, specific enzymes may participate in both pathways. For example, recent evidence from Drosophila indicates that Dicer (Dcr)-1 is critical for both RNA degradation and translation repression, while Dcr-2 is required only for RNA degradation [11,12], and that Argonaute (Ago)-1 and Ago-2 proteins overlap in their functions [13].

Bottom Line: The four serotypes did not differ in mean titer.While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown.Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003, USA.

ABSTRACT

Background: Mosquito-borne dengue virus (DENV, genus Flavivirus) has emerged as a major threat to global human health in recent decades, and novel strategies to contain the escalating dengue fever pandemic are urgently needed. RNA interference (RNAi) induced by exogenous small interfering RNAs (siRNAs) has shown promise for treatment of flavivirus infections in hosts and prevention of transmission by vectors. However, the impact of RNAi triggered by authentic virus infection on replication of DENV, or any flavivirus, has received little study. The objectives of the current study were threefold: first, to assess the utility of Drosophila melanogaster S2 cells for the study of DENV, second to investigate the impact of multiple enzymes in the RNAi pathway on DENV replication; and third to test for variation in the response of the four serotypes of DENV to modulation of RNAi.

Results: Three strains from each of the four DENV serotypes showed replication in S2 cells following infection at multiplicity of infection (MOI) 0.1 and MOI 10; each strain achieved titers > 4.0 log10pfu/ml five days after infection at MOI 10. The four serotypes did not differ in mean titer. S2 cells infected with DENV-1, 2, 3 or 4 produced siRNAs, indicating that infection triggered an RNAi response. Knockdown of one of the major enzymes in the RNAi pathway, Dicer-2 (Dcr-2), resulted in a 10 to 100-fold enhancement of replication of all twelve strains of DENV in S2 cells. While serotypes did not differ in their average response to Dcr-2 knockdown, strains within serotypes showed significant differences in their sensitivity to Dcr-2 knockdown. Moreover, knockdown of three additional components of the RNAi pathway, Argonaute 2 (Ago-2), Dcr-1 and Ago-1, also resulted in a significant increase in replication of the two DENV strains tested, and the magnitude of this increase was similar to that resulting from Dcr-2 knockdown.

Conclusions: These findings indicate that DENV can replicate in Drosophila S2 cells and that the RNAi pathway plays a role in modulating DENV replication in these cells. S2 cells offer a useful cell culture model for evaluation of the interaction between DENV and the RNAi response.

Show MeSH
Related in: MedlinePlus