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. 2015 Sep;89(18):9383-92.
doi: 10.1128/JVI.01299-15. Epub 2015 Jul 1.

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

Kyung-No Son  1 , Zhiguo Liang  1 , Howard L Lipton  2
Affiliations

Double-Stranded RNA Is Detected by Immunofluorescence Analysis in RNA and DNA Virus Infections, Including Those by Negative-Stranded RNA Viruses

Kyung-No Son et al. J Virol. 2015 Sep.

Abstract

Early biochemical studies of viral replication suggested that most viruses produce double-stranded RNA (dsRNA), which is essential for the induction of the host immune response. However, it was reported in 2006 that dsRNA could be detected by immunofluorescence antibody staining in double-stranded DNA and positive-strand RNA virus infections but not in negative-strand RNA virus infections. Other reports in the literature seemed to support these observations. This suggested that negative-strand RNA viruses produce little, if any, dsRNA or that more efficient viral countermeasures to mask dsRNA are mounted. Because of our interest in the use of dsRNA antibodies for virus discovery, particularly in pathological specimens, we wanted to determine how universal immunostaining for dsRNA might be in animal virus infections. We have detected the in situ formation of dsRNA in cells infected with vesicular stomatitis virus, measles virus, influenza A virus, and Nyamanini virus, which represent viruses from different negative-strand RNA virus families. dsRNA was also detected in cells infected with lymphocytic choriomeningitis virus, an ambisense RNA virus, and minute virus of mice (MVM), a single-stranded DNA (ssDNA) parvovirus, but not hepatitis B virus. Although dsRNA staining was primarily observed in the cytoplasm, it was also seen in the nucleus of cells infected with influenza A virus, Nyamanini virus, and MVM. Thus, it is likely that most animal virus infections produce dsRNA species that can be detected by immunofluorescence staining. The apoptosis induced in several uninfected cell lines failed to upregulate dsRNA formation.

Importance: An effective antiviral host immune response depends on recognition of viral invasion and an intact innate immune system as a first line of defense. Double-stranded RNA (dsRNA) is a viral product essential for the induction of innate immunity, leading to the production of type I interferons (IFNs) and the activation of hundreds of IFN-stimulated genes. The present study demonstrates that infections, including those by ssDNA viruses and positive- and negative-strand RNA viruses, produce dsRNAs detectable by standard immunofluorescence staining. While dsRNA staining was primarily observed in the cytoplasm, nuclear staining was also present in some RNA and DNA virus infections. The nucleus is unlikely to have pathogen-associated molecular pattern (PAMP) receptors for dsRNA because of the presence of host dsRNA molecules. Thus, it is likely that most animal virus infections produce dsRNA species detectable by immunofluorescence staining, which may prove useful in viral discovery as well.

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Figures

FIG 1
FIG 1
Reactivity of the 9D5 MAb with dsRNA duplexes of various sizes and TMEV and influenza A virus RF RNAs. (A) (Left) Electrophoresis on a 1.7% agarose gel stained with ethidium bromide. The gel contained large molecular size ssRNA markers, a 47-mer RNA duplex, small-molecular-size (100- to 1,000-bp) poly(I·C), and large-molecular-size (1 to 8 kbp) poly(I·C) in the four lanes from left to right, respectively. (Right) Northwestern blot showing the reactivity of MAb 9D5 with 100-bp to 8-kbp poly(I·C). (B) (Left) Electrophoresis on a 1% agarose gel of in vitro-transcribed TMEV RNA and total RNA from TMEV-infected BHK-21 cells (6 h p.i.) stained with ethidium bromide. Lanes: 1, high-range ssRNA markers; 2, in vitro-transcribed TMEV genomic RNA; 3, total RNA from infected cells showing the TMEV positive-strand genome and dsRNA RF along with cellular RNAs. (Right) Northwestern blot of the membrane on the left showing the reactivity of RF and RI RNAs with 9D5 antibodies to dsRNA. (C) (Left) Electrophoresis on a 1% agarose gel of total RNA from uninfected and IAV-infected MDCK cells stained with ethidium bromide. Lanes: 1, ssRNA markers; 2, uninfected MDCK cells; 3, infected MDCK cells. (Middle) Northwestern blot of lane 2 from the panel on the left with no reactivity. (Left) Blot of the membrane of lane 3 from the panel on the left showing the reactivity of IAV genome RF segments. (D) Sensitivity of the 9D5 MAb for the detection of TMEV RF RNA from infected BHK-21 cells. (Top) A 1% agarose gel stained with ethidium bromide showing barely detectable TMEV RF RNA; (bottom) Northwestern blot showing the reactivity of TMEV RF RNA with as little as 50 ng of antibody. (E) Titration of MAb 9D5 (3.27 μg/μl) and MAb J2 (1.2 μg/μl) showing their relative reactivity with TMEV-infected M1-D cells by immunofluorescence intensity.
FIG 2
FIG 2
Immunofluorescence analysis of cell monolayers infected with positive-strand RNA viruses. Uninfected (A, D, G) and infected (B, E, H) cell monolayers were stained with a 1:2,000 dilution of either MAb 9D5 or polyclonal antibody 170A to dsRNA, and infected cells were stained with specific antiviral antibodies (C, F, I). The fields in the center (B, E, H) and right (C, F, I) panels are identical. (A to C) Uninfected and TMEV-infected M1-D macrophages. (A) Uninfected cells. (B) TMEV-infected cells showing strong punctate cytoplasmic staining (green). (Insets) A loss of staining after preincubation with RNase III (left) but retention of staining after preincubation with RNase A (right). (C) TMEV-infected cells stained with a 1:3,000 dilution of rabbit polyclonal antibodies to TMEV virions showing cytoplasmic staining (red). (Inset) Colocalization of dsRNA and virus antigen(s). (D to F) Uninfected and EV71-infected Vero B6 cells. Strong punctate cytoplasmic dsRNA staining (green) was shown in uninfected cells (D) but not in infected cells (E). (F) EV71-infected cells exhibited cytoplasmic staining with a 1:2,000 dilution of mouse MAb to EV71 (red; virus protein specificity is not known). (Insets) Colocalization of both antigens. (G to I) Uninfected and MHV-infected DBT astrocytoma cells. Strong punctate cytoplasmic staining (green) with the 170A polyclonal antibody was shown in infected cells (H) but not in uninfected cells (G), while MHV-infected cells exhibited cytoplasmic staining with a 1:500 dilution of mouse MAb to MHV M protein (red) (I). The inset in panel I shows the colocalization of both antigens. The results of incubations of RNases with EV71 and MHV are the same as those for incubations of RNases with TMEV (not shown). Bars, 10 μm. All insets have the same magnification as the full panels.
FIG 3
FIG 3
Immunofluorescence analysis of dsRNA and virus proteins in negative-strand RNA virus infections. Uninfected (A, E, I, M) and infected (B, F, J, N) cell monolayers were stained with a 1:2,000 dilution of MAb 9D5 or polyclonal antibody 170A, and the other infected monolayers were stained with the specific antiviral antibodies (C, G, K, O). (A to D) Vero B6 cells infected or not infected with VSV. (A) Uninfected cells. (B) VSV-infected Vero B6 cells with punctate cytoplasmic staining (green) with MAb 9D5 (B). (C) VSV-infected cells stained with a 1:500 dilution of rabbit polyclonal antibodies to VSV G protein showing copious cytoplasmic staining (red). (D) Merged image of panels B and C. (E to H) Vero B6 cells infected or not infected with MeV. (E) Uninfected cells. (F) MeV-infected Vero B6 cells with punctate cytoplasmic staining with MAb 9D5. Note the large multinucleated giant cell. (G) MeV-infected cells were stained with human recombinant antibody to MeV NC protein and show diffuse cytoplasmic reactivity. (H) Merged image of panels F and G. (I to L) MDCK cells infected or not infected with IAV. (I) Uninfected cells. (J) IAV-infected MDCK cells revealing cytoplasmic staining (green) with MAb 9D5. (K) Cytoplasmic reactivity is seen with a 1:500 dilution of rabbit polyclonal antibody to IAV NC protein. (L) Merged image of panels J and K. (M to P) Vero B6 cells infected or not infected with NyaV. (M) Uninfected cells. (N) NyaV-infected Vero B6 cells revealing punctate areas of staining in the nucleus of infected cells. (O) Cytoplasmic reactivity with a 1:2,000 dilution of mouse antiserum to NyaV proteins is seen. (P) Merged image of panels N and O, with only a few of the stained nuclear dots in two cells showing colocalization (arrows). Bar, 10 μm (the magnification in panel A applies to all panels).
FIG 4
FIG 4
Immunofluorescence analysis of uninfected (A) and IAV-infected (B) Vero B6 cells stained with a 1:500 dilution of the J2 MAb showing that J2 also detects dsRNA in negative-strand RNA virus infections; images of the other negative-strand RNA viruses not shown.
FIG 5
FIG 5
Reactivity of dsRNA and viral proteins in cells infected with an ambisense RNA virus (LCMV) and an ssDNA virus (MVM) by immunofluorescence analysis. Uninfected (A, E) and infected (B, F) cell monolayers were stained with a 1:2,000 dilution of MAb 9D5 to dsRNA, and infected monolayers were stained with the specific antiviral antibodies (C, G). (A to D) LCMV-infected and uninfected Vero B6 cells. (A) Uninfected cells. (B) LCMV-infected Vero B6 cells revealing punctate cytoplasmic staining (green) with MAb 9D5. (C) LCMV-infected cells stained with a 1:100 dilution of MAb to the LCMV NP protein showing cytoplasmic staining (red). (D) Merged image of panels B and C. (E to G) MVM-infected and uninfected mouse A9 cells. (E) Uninfected cells. (F) MVM-infected mouse A9 cells showing punctate areas of various diameters with staining with MAb 9D5 in the nucleus (green). (G) MVM-infected monolayers showed cytoplasmic staining with a 1:200 dilution of polyclonal antibody to MVM NS1/2 (no DAPI counterstaining) (red). The images in panels F and G were not from the same field. Bars, 10 μm (the magnification in the panels with bars applies to all photomicrographs).
FIG 6
FIG 6
Comparison of nuclear images of cells infected with IAV, NyaV, and MVM stained for dsRNA. (A, B) IAV-infected MDCK cells at 16 and 8 h p.i., respectively, showing cytoplasmic staining (A) and nuclear staining (B); nuclear staining was seen only after the cell monolayers were first incubated with proteinase K. Infected cells with nuclear staining at 8 h did not reveal cytoplasmic staining; however, at 16 h, staining in the nucleus and cytoplasm was seen (not shown). (C) NyaV-infected Vero B6 cells revealed dot-like sites of staining in the nucleus. (D) MVM-infected A9 mouse fibroblasts showing increased numbers of smaller dot-like areas of staining in the nucleus compared with the numbers seen in IAV- and NyaV-infected cells.
FIG 7
FIG 7
Lack of induction of dsRNA reactivity with CXH and CXH plus TNF-α, which results in apoptosis, in uninfected M1-D macrophages. (A) Uninfected cells were incubated with 500 μM CXH for 6 h before staining with MAb 9D5 and showed no dsRNA reactivity. (B) TMEV-infected cells showing cytoplasmic dsRNA staining as a positive control. (C) DAPI-stained fragmented and blebbing nuclei, indicative of apoptosis, in uninfected cells. (D) Immunoblot analysis showing PARP and caspase-3 (Casp-3) cleavages, indicative of apoptosis, when cells were incubated with CHX at concentrations of ≥50 μM and slightly more robust cleavages when cells were incubated with both CHX at a concentration of 50 or 100 μM and TNF-α at a concentration of 5 or 10 μg.
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