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. 2002 Dec;3(12):1175-80.
doi: 10.1093/embo-reports/kvf234. Epub 2002 Nov 21.

Rotavirus gene silencing by small interfering RNAs

Miguel Angel Déctor  1 , Pedro Romero, Susana López, Carlos F Arias
Affiliations

Rotavirus gene silencing by small interfering RNAs

Miguel Angel Déctor et al. EMBO Rep. 2002 Dec.

Abstract

RNA interference is an evolutionarily conserved double-stranded RNA-triggered mechanism for suppressing gene expression. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, from which the spike protein VP4 projects. Here, we show that a small interfering RNA corresponding to the VP4 gene efficiently inhibits the synthesis of this protein in virus-infected cells. A large proportion of infected cells had no detectable VP4 and the yield of viral progeny was reduced. Most of the virus particles purified from these cells were triple-layered, but lacked VP4, and were poorly infectious. We also show that VP4 might not be required for the last step of virus morphogenesis. The VP4 gene silencing was specific, since the synthesis of VP4 from rotavirus strains that differ in the target sequence was not affected. These findings offer the possibility of carrying out reverse genetics in rotaviruses.

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Figures

Figure 1
Figure 1
Rotavirus gene silencing. (A) Rotavirus RRV viral progeny produced in cells transfected with siRNAs to VP4 and lamin A/C, expressed as a percentage of the virus yield obtained in mock-transfected cells. The viral progeny titer in mock-transfected cells was 2.1 × 106 ffu/ml. (B) Immunoblot analysis of VP4 synthesized in cells transfected with the indicated siRNA as compared with the synthesis of viral protein VP2, at various times p.i. (C) Immunofluorescence detection of VP4 and the non-structural protein NSP5 in rotavirus RRV- or RF-infected cells in the presence of siRNAVP4 or the unrelated siRNALam A/C.
Figure 2
Figure 2
Virus particles synthesized in the presence of siRNAVP4. (A) Isopicnic CsCl gradients of viral particles assembled in the presence of either siRNAVP4 or siRNALamA/C. (B) Gel electrophoresis analysis of the viral particles present in the bands detected in the isopicnic gradients shown in (A). The lane numbers correspond to the number of the band in the gradient. The migration of the viral structural proteins in the gel is indicated. RI refers to the relative infectivity of TLPs and spikeless TLPs as compared with that of DLPs. The infectivities of the particles obtained from siRNALamA/C-treated cells were (ffu/ml) DLPs, 6.7 × 106; TLPs, 4.8 × 109. The infectivities of the particles obtained from siRNAVP4-transfected cells were (ffu/ml) DLPs, 1 × 107; spikeless TLPs, 4.8 × 107; and TLPs 9.9 × 109. (C) EM analysis of DLPs, TLPs and spikeless TLPs purified from siRNAVP4-treated cells, corresponding to bands 3, 4 and 5 of the gradient shown in (A). Magnification ×85 000. The inserts show viral particles amplified 1.5-fold. Scale bars, 90 nm.
Figure 3
Figure 3
Morphogenesis of RRV rotavirus particles in the presence of mock-transfected or siRNAVP4-transfected MA104 cells. Dense viroplasmic inclusions (V) are abundant in the cytoplasm of rotavirus-infected cells, adjacent to the ER. From these structures DLPs bud (arrowheads) into the lumen of the ER (ERL), resulting in membrane-enveloped particles (small arrows), which later lose the membrane to produce mature triple-layered virions (large arrows). The pictures shown are representative of at least 20 different virus-infected cells. Magnification ×14 000. Scale bars, 400 nm.
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