Comparative study on retinoic acid-inducible gene-I of humans and bats

By Pooja Toshniwal PahariaReviewed by Danielle Ellis, B.Sc.Feb 15 2023

In a recent study posted to the bioRxiv* preprint server, researchers cloned and functionally characterized the virus ribonucleic acid (RNA) sensor, retinoic acid-inducible gene-I (RIG-I), from the microbat Myotis daubentonii (Yangochiroptera suborder) and the megabat Rousettus aegyptiacus (Yinpterochiroptera suborder), and compared them to the human ortholog.

Study: Functional comparisons of the virus sensor RIG-I from humans, the microbat Myotis daubentonii, and the megabat Rousettus aegyptiacus, and their response to SARS-CoV-2 infection. Image Credit: D. Kucharski K. Kucharska / Shutterstock

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Background

Chiroptera order bats are considered the main reservoirs for emerging zoonotic viral organisms. The tolerance of bats toward viruses highly pathogenic to humans has led to the hypothesis that bats might comprise a very active antiviral interferon (IFN)-inducing system. However, data on the functional characterization of the main constituents of the bat interferon system are limited.

The RIG-I gene can sense viral ribonucleic acid signatures that activate the antiviral signaling pathways to express interferon. The present study’s authors previously reported that cells derived from R. aegyptiacus  (Ro6E-J) and M. daubentoniid (MyDauNi) can respond to exogenously produced IFN.

About the study

In the present study, researchers extended their previous analysis by investigating whether MyDauNi cells could produce endogenous antiviral interferon in response to viral infections. They also functionally characterized RIG-I genes of suborders Yinpterochiroptera and Yangochiroptera.

Angiotensin-converting enzyme 2 (ACE2)-human embryonic kidney 293 (HEK293) ΔRIG-I cells were generated for cell culture experiments and treated with small interfering ribonucleic acid (siRNA). In addition, A549 cells, MyDauNi cells, and Ro6E-J cells were used. The cells were infected with stocks of Rift Valley fever virus (RVFV) clone 13, la crosse encephalitis virus (LACV), vesicular stomatitis virus (VSV), grown on baby hamster kidney (BHK) cells, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), propagated on VeroE6 cells.

Genomic RNA was isolated from VSV and RVFV clone 13, and quantified using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). For detecting human transcripts, assays using 18S ribosomal RNA IFN-β, RIG-I, C-X-C motif ligand-10 (CXCL-10), myxovirus resistance protein 1 (MxA), 2'-5'-oligoadenylate synthetase-1 (OAS-1) and melanoma differentiation-associated protein-5 (MDA-5) were used.

For detecting entire-length transcript sequences for M. daubentonii, the team used a de novo transcriptome assembly based on bulk RNA sequencing (RNA-Seq) data from their previous study. For R. aegyptiacus, the team used publicly available genome and annotation data to obtain transcript sequences. Virus titration experiments, complementary deoxyribonucleic acid (cDNA) cloning and bioinformatics analyses were performed.

The DNA sequences were translated in silico, and the resulting amino acids were aligned with the genetic sequences. Domains were manually assigned and confirmed using the simple modular architecture research tool (SMART) database. RIG-I trans-complementation assays were performed, and the RNA dependence of RIG-I was evaluated. Immunoblot and polyinosinic: polycytidylic acid (poly I:C) pull-down analyses were performed.

Results

Domain and sequence organization were highly conserved, with 93% to 95% sequence similarity between different species. All 3.0 RIG-I ortholog genes could regulate interferon induction to a comparable extent on being stimulated by viral ribonucleic acid at 37.0°C and 39.0°C. The effects could not be observed at 30.0°C. The findings indicated that bat and human RIG-I genes were functional at normal body temperature or higher but not at 30.0°C.

Similar to human retinoic acid-inducible gene-I, bat ortholog genes were activated optimally using double-stranded ribonucleic acid terminating with 5’-triphosphate and needed mitochondrial antiviral-signaling (MAVS) protein to demonstrate the antiviral effects. Human and R. aegyptiacus RIG-Is could sense SARS-CoV-2 infections through innate immunological mechanisms.

The bat RIG-I orthologs showed two differences from human RIG-I, a deletion of 2.0 amino acids at positions 236 and 237 and a 5.0 amino acid insertion at positions 491 and 492. M. daubentonii RIG-I comprised a deletion of one amino acid at position 195. The team detected two N-terminal caspase recruitment domains (CARDs), a helicase domain, a C-terminal regulatory domain, and a DExD-like helicase domain as being conserved among all the three RIG-I orthologs. RIG-I of bats and humans could rescue RIG-I gene deficiency, regardless of the background species.

RIG-I orthologs cloned from megabat and microbat cells could be stimulated by viral RNA and initiate antiviral signaling resulting in virus-responsive promoter transactivation. RIG-I orthologs of humans and R. aegyptiacus could induce IFN-β messenger RNA (mRNA) synthesis. However, for human and R. aegyptiacus RIG-I orthologs, the levels of cytokine messenger RNA induction by SARS-CoV-2 were 5.0-fold to >10.0-fold lower than those for RVFV mutant clone 13. MDA5 did not seem to contribute to the RIG-I-regulated interferon induction by SARS-CoV-2.

Overall, the study findings showed that RIG-I, expressed by megabats and microbats, is similar to the human counterpart. Bat RIG-I genes showed conserved domains and sequences. They showed comparable actions as the human ortholog in regulating interferon-activated and virus-activated genetic expression, temperature dependency, antiviral signaling, and ribonucleic acid ligand recognition. In addition, RIG-Is of humans and R. aegyptiacus (Yinpterochiroptera suborder) identified SARS-CoV-2 infections. The findings indicated that the zoonotic virus-harboring capability of bats seemed to arise from other characteristics.

*Important notice: bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.