Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses

Abstract

Double-stranded RNA (dsRNA) longer than 30 bp is a key activator of the innate immune response against viral infections. It is widely assumed that the generation of dsRNA during genome replication is a trait shared by all viruses. However, to our knowledge, no study exists in which the production of dsRNA by different viruses is systematically investigated. Here, we investigated the presence and localization of dsRNA in cells infected with a range of viruses, employing a dsRNA-specific antibody for immunofluorescence analysis. Our data revealed that, as predicted, significant amounts of dsRNA can be detected for viruses with a genome consisting of positive-strand RNA, dsRNA, or DNA. Surprisingly, however, no dsRNA signals were detected for negative-strand RNA viruses. Thus, dsRNA is indeed a general feature of most virus groups, but negative-strand RNA viruses appear to be an exception to that rule.

FIG. 1.

The monoclonal antibody J2 specifically recognizes dsRNA. Vero cells were transfected with poly(I:C) as indicated in Materials and Methods. After an incubation period of 6 h, the cells were fixed and analyzed for dsRNA using the mouse monoclonal antibody J2 (red). To visualize the cell nuclei, histone H4 was stained using a specific rabbit antiserum (green). Shown are untransfected cells (1), poly(I:C)-transfected cells (2), and poly(I:C)-transfected cells treated with either 2 U of RNase III (3) or 2 U of RNase A (4). Bar, 20 μm. All pictures were taken with the same magnification.

FIG. 2.

dsRNA in cells infected with positive-strand RNA and dsRNA viruses. Vero cells were infected at a multiplicity of infection of 5 with EMCV (2), SARS-CoV (3), or ReoV (4) or left uninfected (1). At 5 h (EMCV), 16 h (SARS-CoV), or 48 h (ReoV) postinfection, cells were fixed and analyzed by immunofluorescence as indicated in the legend to Fig. 1. Bar, 20 μm. All pictures were taken with the same magnification. The differences in size and morphology of the nuclei are most probably caused by initiation of apoptosis or by disturbances in nuclear-cytoplasmic transport, as has been described for several viruses (6, 21, 23, 27).

FIG. 3.

dsRNA in cells infected with DNA viruses. Vero cells were infected at a multiplicity of infection of 5 with AdV (2), HSV (3), or Vac (4) or left uninfected (1). At 7 h (AdV, HSV) or 5 h (Vac) postinfection, cells were fixed and analyzed by immunofluorescence as indicated in the legend to Fig. 1. Bar, 20 μm. All pictures were taken with the same magnification.

FIG. 4.

Negative-strand RNA viruses. Vero cells were infected at a multiplicity of infection of 5 with FLUAV (A, panels 2 and 4) or LACV (B, panels 2 and 4) or left uninfected (A and B, panels 1 and 3). At 5 h postinfection, cells were fixed and analyzed by immunofluorescence either for dsRNA as indicated in the legend to Fig. 1 (A and B, panels 1 and 2) or for viral antigens (A and B, panels 3 and 4). Bar, 20 μm. All pictures were taken with the same magnification.