Molecular Mechanisms of SARS-CoV-2 Infection and Therapeutic Targets

National Institute Of Allergy And Infectious Diseases

Investigators

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Abstract

The single-stranded RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proven to be easily transmissible in humans. Infected individuals present with a range of COVID-19 disease-associated characteristics: on the low end of the spectrum patients are asymptomatic or display mild symptoms and on the high end of the spectrum patients require intensive medical treatment and may die of respiratory failure. Many factors can contribute to the course of the disease, one of the most important being the host's ability to mount an appropriate immune response. Before last year, few details were known about the immunological response to SARS-CoV-2. We are interested in investigating disease susceptibility of the host based on genetic defects and discovering possible targets for drug development. In collaboration with various sites in Italy and other smaller domestic and international sites, we are participating in a project led by Dr. Helen Su to sequence roughly 2,000 genomes of COVID-19 positive patients. We found that nearly nine percent of patients have a mutation associated with pathways recognizing viral RNA, specifically gene IFIH1 and its encoded protein MDA5 or gene DDX58 and its encoded protein RIG-I. Once MDA5 or RIG-I bind viral RNA, MAVS protein is activated, inducing a host interferon (IFN) response. Half of the IFIH1 variants in our cohort led to loss-of-function (LOF), inhibiting the host's ability to recognize viral pathogens. In vitro studies showed that SARS-CoV-2 has increased viral titers in MDA5- or MAVS-deficient cells. Interestingly, RIG-I-deficient cells did not increase viral replication, leading us to conclude that MDA5 is the primary SARS-CoV-2 RNA viral sensor in fibroblast cells. Co-infection experiments with SARS-CoV-2 or other common RNA viruses like influenza and rhinovirus led us to conclude that RIG-I and MDA5 sense different viral stimuli. Upon stimulation, cells from patients with MDA5 mutations have a significant decrease in IFN-beta production compared to controls. We hypothesize that MDA5 and MAVS limits viral replication and are currently investigating this mechanism. Our genetic analysis efforts also led to the discovery of an enrichment in rare variants predicted to be LOF at the 13 human loci known to govern Toll-like receptor 3 (TLR3) and interferon regulatory factor 7 (IRF7) dependent type I IFN immunity to influenza virus in 659 patients with life-threatening COVID-19 pneumonia relative to 534 subjects with asymptomatic or benign infection. By testing these and other rare variants at these 13 loci, we experimentally defined LOF variants underlying autosomal-recessive or autosomal-dominant deficiencies in 23 patients (3.5 percent), 17 to 77 years of age. We showed that human fibroblasts with mutations affecting this circuit are vulnerable to SARS-CoV-2. Inborn errors of TLR3- and IRF7-dependent type I IFN immunity can underlie life-threatening COVID-19 pneumonia in patients with no prior severe infection. There are at least 2-3 dozen other potentially pathogenic variants to be studied in detail for roles in anti-viral immunity. SARS-CoV, a coronavirus similar to SARS-CoV-2, was first reported in 2003 and is comprised of eight accessory proteins called open reading frames (ORFs). Using a 3a-deficient strain of SARS-CoV, we observed host cell death was dramatically reduced compared to normal virus. SARS-CoV-2 also encodes the ORF 3a protein, and preliminary experiments have shown it induces cell death. However, the exact mechanism is unknown. Using bioID mass spectrometry, we identified proteins that interact with ORF 3a. We screened roughly 8,000 FDA approved drug compounds for activity based upon inhibition of cell death induced by SARS-CoV-2. The screen came back with a few compounds. However, after further cytopathogenic assay analysis, there was no significant cell death blocking compared to controls. At this time, there are no further compound candidates to analyze.