Host pattern recognition receptors TLR3, and the DExD/H-box helicases, RIG-I and MDA5 sense viral RNA
and activate IRF and NF-kB transcription factors culminating in generation of host anti-viral responses. Infection
of dendritic cells (DC) or mf with SARS-CoV-2 results in an abortive infection without viral replication. In contrast,
infection of normal human bronchial epithelial cells (NHBE) with SARS-CoV-2 results in robust viral replication.
Infection of both cell lineages with SARS-CoV-2 generates similar robust host anti-viral responses as measured
by induction of type 1 interferons (IFN1), interferon-stimulated genes (ISGs), TNF-a, IL-1, IL-6, IL-8, other
cytokines, chemokines and other pro-inflammatory mediators.
Alu elements make up ~10% of the human genome. Alu RNAs are abundant in human cells and, because
of their repetitive nature, can form double-stranded RNAs (dsRNA) and stimulate above-cited pattern recognition
receptors and a strong anti-viral response in the absence of viral infection. To prevent this, Alu RNAs are rapidly
A-to-I edited by adenosine deaminase specific for dsRNA, ADAR. Our preliminary studies show that severe
COVID-19 disease (COV-S) is associated with marked loss of A-to-I editing of endogenous Alu RNAs in both
blood and lung, while mild COVID-19 disease (COV-M) is associated with a partial loss of A-to-I editing. Infection
of DC as well as NHBE causes a marked loss of A-to-I editing of endogenous Alu RNAs. Our preliminary studies
show that unedited Alu RNAs activate host dsRNA sensors and stimulate transcriptional response leading to
induction of ISGs, IL-6, and IL-8. In contrast, the same Alu RNAs, if edited, as is seen in healthy controls or
mock-infected cells, fail to activate these gene expression programs.
Taken together, these results suggest the following hypothesis we propose to address. First, unedited Alu
RNAs are continuously synthesized and exist at high levels in cells. If unedited, Alu RNAs form dsRNAs that
stimulate potentially pathogenic anti-viral responses. However, Alu RNAs are continuously A-to-I edited so they
cannot form dsRNAs. In response to viral infection, this continuous cycle is rapidly disrupted by loss of A-to-I
editing by ADAR allowing accumulation of unedited Alu dsRNAs and stimulation of downstream anti-viral host
responses. It is tempting to speculate that the value to the host of this unique continuous cycle is to rapidly
stimulate anti-viral and pro-inflammatory host responses by Alu dsRNAs in response to viral infection to prevent
accumulation and spread of pathogenic viral particles.
To explore this hypothesis, we propose to infect mf, DC, and NHBE with SARS-CoV-2 and follow kinetics of
loss of A-to-I editing of endogenous Alu RNAs and host responses using RNA-seq and our computational
pipelines. We will also determine if RNAs that stimulate host responses are of viral origin or are Alu dsRNAs.
In aim II, we will investigate ability of unedited and edited Alu RNAs to stimulate anti-viral responses and employ
siRNA-mediated knockdown of Alu RNAs to demonstrate a direct role of Alu RNAs in the host anti-viral response.