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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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Replication kinetics of DENV-2 Tonga in S2 cells depleted of specified components of the RNAi pathway.
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Figure 7: Replication kinetics of DENV-2 Tonga in S2 cells depleted of specified components of the RNAi pathway.

Mentions: Subsequent analyses focused on two DENV strains that had shown the smallest (DENV-2 Tonga) and an intermediate (DENV-4 Taiwan) response to Dcr-2 knockdown (Figure 6). A multistep growth curve revealed that knockdown of Dcr-2 resulted in enhancement of replication of both strains within 48 hrs pi, and by 72 hrs pi both strains had achieved a titer 10 - 100 - fold higher in Dcr-2 depleted cells than control cells (Figures 7 and 8). A similar pattern was observed following knockdown of Dcr-1, Ago-1 and Ago-2 (Figures 7 and 8); titers of both DENV strains were significantly higher in cells depleted of each enzyme than control cells 96 hrs pi (unpaired t-tests; df = 4, P < 0.02 for all comparisons).


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

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

Replication kinetics of DENV-2 Tonga in S2 cells depleted of specified components of the RNAi pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Replication kinetics of DENV-2 Tonga in S2 cells depleted of specified components of the RNAi pathway.
Mentions: Subsequent analyses focused on two DENV strains that had shown the smallest (DENV-2 Tonga) and an intermediate (DENV-4 Taiwan) response to Dcr-2 knockdown (Figure 6). A multistep growth curve revealed that knockdown of Dcr-2 resulted in enhancement of replication of both strains within 48 hrs pi, and by 72 hrs pi both strains had achieved a titer 10 - 100 - fold higher in Dcr-2 depleted cells than control cells (Figures 7 and 8). A similar pattern was observed following knockdown of Dcr-1, Ago-1 and Ago-2 (Figures 7 and 8); titers of both DENV strains were significantly higher in cells depleted of each enzyme than control cells 96 hrs pi (unpaired t-tests; df = 4, P < 0.02 for all comparisons).

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