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.