Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Summary

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Abstract

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Introduction

The initial step of the induction of the innate immune response is host recognition of double-stranded (ds) RNA by the pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5)¹. For positive-sense RNA viruses, dsRNA can usually be readily detected using the J2 or K1 anti-dsRNA monoclonal antibodies (Mab)². Interaction between dsRNA and PRRs in positive-strand RNA viruses, such as picornavirus, has been characterized using confocal microscopy³. However, for negative-sense RNA virus, visualization and....

Protocol

1. Preparation of A549 cells and JUNV infection

1. Seed 2 x 10⁵ human lung epithelial A549 cells onto poly-D-lysine (PDL) coated glass coverslips in 12-well plates at 24 hours prior to infection.
2. Prepare aliquots of 150 μL of JUNV¹³ at a multiplication of infection (MOI) of 1.0 plaque forming unit per cell diluted in Dulbecco’s Modified Eagle’s Medium (DMEM) media supplemented with 2% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P/S).
3. Remove media from cell culture. Add virus inoculum on each well containing coverslip and incubate for 1.5 h at 37 °C. Shake the plates ....

Results

This protocol was applied to study the distribution and colocalization between the RLRs (RIG-I and MDA-5) and dsRNA in JUNV-infected cells. As shown in Figure 1 and Figure 2, the accumulation of dsRNA increases over time as viral infection progresses. Concentrated MDA-5 (Figure 1) and RIG-I (Figure 2) signals were found colocalized with the punctate structures of the NP .......

Discussion

For positive-sense RNA viruses and dsDNA viruses, dsRNA is readily detected with the widely-used J2 anti-dsRNA antibody. However, negative-sense RNA viruses are believed to produce dsRNA at a low or below the detection level using the same antibody². Thus, many aspects of viral RNA and PRR interaction are largely unclear for negative-sense RNA viruses. We attempted to stain for dsRNA in arenavirus infection using the J2 antibody but the fluorescence signals were not differentiable compared to the .......

Materials

Name	Company	Catalog Number	Comments
APEX Alexa Fluor 647 antibody labeling kit	Invitrogen	A10475
BSA	Sigma Aldrich	A4503
DAPI	Cell Signaling	4083	1:1,000 dilution
Donkey-anti rabbit Alexa Fluor 594	Invitrogen	A-11058	1:2,000 dilution; Lot #: 1454437
Dulbecco's modified Eagle's medium	Corning	10-013-CV
Fetal Bovine Serum	Atlanta Bio	S11150
Glass microscope slides	Fisher	12-550-15
Goat-anti mouse Alexa Fluor 488	Invitrogen	A-11029	1:2000 dilution; Lot #: 1874804
Human lung epithelial A549 cells	ATCC	CCL-185
Methanol	Fisher	A412	Stored at -20 °C
Mouse MAb anti-JUNV NP	BEI	NA05-AG12	Conjugated to Alexa-647 at 1:1,000 dilution
Mouse MAb pan-Enterovirus 9D5 Reagent	Millipore Sigma	3361	ready for use; diluted to 1:2; Lot #: 3067445
PBS supplemented with Ca and Mg	Corning	21-030-CV
PDL Coated coverslips	Neuvitro	H-12-1.5-pdl
ProLong Gold antifade	Invitrogen	P10144
Rabbit MAb MDA-5	Abcam	ab126630	1:250 dilution; Lot #: GR97758-7
recombiant Candid#1 strain of JUNV	Lab generated	Lab generated	As previously described in reference 13.
RIG-I mouse MAb conjugated to Alexa-594	Santa Cruz	sc-376845	1:1000 dilution; Lot #: AO218
Triton X-100	Sigma Aldrich	T8787