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Inhibition of Tulane virus replication in vitro with RNA interference.

ABSTRACT: RNA interference (RNAi), a conserved mechanism triggered by small interfering RNA (siRNA), has been used for suppressing gene expression through RNA degradation. The replication of caliciviruses (CVs) with RNAi was studied using the Tulane virus (TV) as a model. Five siRNAs targeting the non-structural, the major (VP1) and minor (VP2) structural genes of the TV were developed and the viruses were quantified using quantitative real time PCR (qPCR) and tissue culture infective dose (TCID(50) ) assay. Treatment of the cells with siRNA 4 hr before viral inoculation significantly reduced viral titer by up to 2.6 logs and dramatically decreased viral RNA copy numbers and viral titers 48 hr post infection in four of the five siRNAs studied. The results were confirmed by Western blot, in which the major structural protein VP1 was markedly reduced in both the cells and the culture medium. Two small protein bands of the shell (S) and protruding (P) domains of the viral capsid protein were also detected in the cell lysates, although their role in viral replication remains unknown. Since the TV shares many biological properties with human noroviruses (NoVs), the successful demonstration of RNAi in TV replication would provide valuable information in control of acute gastroenteritis caused by human NoVs.

SUBMITTER: Fan Q

PROVIDER: S-EPMC3508507 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Inhibition of Tulane virus replication in vitro with RNA interference.

Fan Qiang Q Wei Chao C Xia Ming M Jiang Xi X

Journal of medical virology 20130101 1

RNA interference (RNAi), a conserved mechanism triggered by small interfering RNA (siRNA), has been used for suppressing gene expression through RNA degradation. The replication of caliciviruses (CVs) with RNAi was studied using the Tulane virus (TV) as a model. Five siRNAs targeting the non-structural, the major (VP1) and minor (VP2) structural genes of the TV were developed and the viruses were quantified using quantitative real time PCR (qPCR) and tissue culture infective dose (TCID(50) ) ass ...[more]

PMID: 23154881

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Project description: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.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.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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Inhibition of Tulane virus replication in vitro with RNA interference.

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Inhibition of Tulane virus replication in vitro with RNA interference.

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