Function and interaction of coronavirus ion channel proteins

Objective: COVID-19 has emerged as a significant global health crisis causing devastating effects on world population accounting for over 6 million deaths worldwide. Although acute RTI is the prevalent cause of morbidity, kidney outcomes centered on a spectrum of AKI have evolved over the course of the pandemic. Especially the emerging variants have posed a daunting challenge to the scientific communities, prompting an urging requirement for global contributions in understanding the viral dynamics. In addition to canonical genes, several subgroup- specific accessory genes are located between the S and E genes of coronaviruses regarding which little is known. Previous studies have shown that accessory proteins (aps) in viruses function as viroporins that regulate viral infection, propagation and egress [1]. In this study we attempted to characterize the function of aps of coronavirus variants as ion channels. Furthermore, we also probed the interaction of ap4 with the host system. Method(s): Serial passaging (selection pressure), growth kinetics, confocal imaging, genome sequence analysis and proteomics were performed in Huh-7, MRC5 cells and/or human monocyte derived macrophages. Potassium uptake assay was performed in a Saccharo myces cerevisiae strain, which lacks the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using Two Electrode Voltage Clamp (TEVC) method. Result(s): Serial passaging demonstrated the acquisition of several frameshift mutations in ORF4 resulting in C-terminally truncated protein versions (ap4 and ap4a) and indicate a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length ap4 was expressed compared to the C-terminally truncated protein ap4a. Confocal imaging showed that ap4 and ap4a are not exclusively located in a single cellular compartment. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a weak K+ selective ion channel. In addition, accessory proteins of other virus variants also elicited microampere range of currents. Conclusion(s): Our study provides the first evidence that ap4 and other accessory proteins of coronavirus variants act as viroporins. Future studies are aimed at demonstrating the role of ap4 during the viral life cycle by modulating ion homeostasis of host cell in vivo (interacting proteins obtained from proteomic studies) and thereby serve as a tool for potential drug target.

Fatal COVID-19 in an infant prompted an early diagnosis of ZNFX1 deficiency in a sibling

Background: NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. ZNFX1 deficiency in humans is very rare;to date, only fifteen cases have been reported by Vavassori S et al. (10.1016/j.jaci.2021.03.045). The disease presented in all cases as severe viral infections complicated by multisystem inflammation evolved to multiorgan failure with a high mortality rate. Pediatric Allergy and Immunology Section at Queen Rania Children's Hospital in Jordan had confirmed the diagnosis of ZNFX1 deficiency in an infant at his first presentation with severe viral illness based on the positive family history of one sibling death caused by complicated COVID-19 infection. Case presentation: A 12-month-old boy was born to consanguineous parents, full-term, with no NICU admission. He was doing well till the age of four months when he was admitted to the hospital with fever, hypoactivity, and maculopapular skin rash. On admission, he was ill, hypoactive, and febrile, and a physical exam showed hepatosplenomegaly and maculopapular skin rash. His lab showed thrombocytopenia, elevated transaminases, hyperferritinemia, and high CRP;he was treated with broad-spectrum antibiotics, but he continued to deteriorate, and his infectious workup was unrevealing, including COVID-19 PCR. His older sibling died at eight months in 2020 when she got a COVID-19 infection, deceased after rapid deterioration evolved to multiorgan failure. Unfortunately, she had no stored DNA, as she was treated at a peripheral hospital. Based on this presentation and the fatal COVID-19 infection, pediatric immunology service got consulted;we did an immunological workup, which showed normal lymphocyte subsets, Immunoglobulins, and bacterial antibodies. Whole exome sequencing showed a homozygous frameshift mutation in the ZNFX1 gene, protein change defect had detected;p.Tyr555MetfsTer6, and nucleotide change variant: c.1663_1665delTACinsAT. Family screening showed heterozygous for the same variant in both parents and a healthy sibling. The patient was diagnosed with the hemophagocytic lymphohistiocytosis-like disease and treated with steroids, intravenous immunoglobulin, and antimicrobials, he showed complete recovery, and we are going to do bone marrow transplantation as his brother is 8/8 HLA matched.Copyright © 2023 Elsevier Inc.

Alternative structures of RNA control gene expression and offer therapeutic strategies

RNA viruses are diverse and abundant pathogens responsible for numerous human ailments, from common colds to AIDS, SARS, Ebola, and other dangerous diseases. RNA viruses possess relatively compact genomes and have therefore evolved multiple mechanisms to maximize their coding capacities, often using overlapping reading frames. In this way, one RNA sequence can encode multiple proteins via mechanisms including alternative splicing and ribosomal frameshifting. Many such processes in gene expression involve the RNA folding into three-dimensional structures that can recruit ribosomes without initiation factors, hijack host proteins, cause ribosomes to frameshift, and expose or occlude regulatory protein binding motifs to ultimately control each key process in the viral life cycle. I will discuss the RNA structure of HIV-1 and SARS-CoV-2 and the importance of alternative conformations assumed by the same RNA sequence in controlling gene expression of viruses and bacteria.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Surfactant Protein B Reduces Respiratory Viral Infectivity in Human Lungs

Purpose of Study: COVID pneumonia caused by SARS-CoV-2 can result in a depletion of surfactant & lung injury, which resembles neonatal respiratory distress syndrome. Exogenous surfactant has shown promise as a therapeutic option in intubated hospitalized patients. Our preliminary data in human lung organoids (LOs) with a deficiency of surfactant protein B (SP-B) showed an increased viral load compared to normal LOs. Single cell RNA sequencing (scRNAseq) revealed that SP-B-deficient cells showed increased viral entry genes (ACE2 receptor) & dysregulated inflammatory markers emanating from the lung cells themselves. Our objective was to determine: (1) cell-specific transcriptional differences between normal & SP-B deficient human lung cells after infection with SARS-CoV-2 and (2) a therapeutic role of SP-B protein & surfactant in COVID-19 pneumonia. Methods Used: We used normal and SP-B mutant (homozygous, frameshift, loss of function mutation p.Pro133GlnfsTer95, previously known as 121ins2) human induced pluripotent stem cells (hiPSC) and differentiated them into 3D proximal lung organoids. The organoids were infected with the delta variant of SARS-CoV-2 for 24 hours at an MOI of 1. Infected and uninfected organoids were fixed in trizol in triplicate and underwent processing for bulk RNA sequencing. We tested for differentially expressed genes using the program DEseq. We also plated normal iPSC derived lung organoids as a monolayer and pre-treated them with 1mg/ml of Poractant alfa or 5 uM of recombinant SP-B protein. The delta strain of SARS-CoV-2 was added to the 96 wells at an MOI of 0.1 for one hour with shaking, then an overlay with DMEM/CMC/FBS was added and left on for 23 hours. The plate was fixed and stained for nucleocapsid (NC) protein. Summary of Results: Bioinformatic analysis of the bulk RNA sequencing data showed an increase in the multiple cytokines and chemokines in the SP-B mutant LOs compared to control. We also saw differential gene expression patterns in the SP-B mutant LOs including a reduction in SFTPC, FOXA2, and NKX2-1 and an increase in IL1A, VEGFA, PPARG and SMAD3. In the exogenous surfactant experiments, there was a decrease in total expression of viral NC in the Poractant alfa & rSP-B-treated cells compared to SARS-CoV-2 infection alone (p<0.001). Conclusion(s): Surfactant modulates the viral load of SARS-CoV-2 infection in the human lung. Deficiency in SP-B results in the dysregulation of the lung epithelial inflammatory signaling pathways resulting in worsening infections.

Molnupiravir Exhibits a High Barrier to the Development of SARS-CoV-2 Resistance in vitro

Background. Molnupiravir is an orally available prodrug of the antiviral nucleoside analog N-Hydroxycytidine (NHC). In preclinical studies NHC has shown broad-spectrum antiviral activity against multiple RNA viruses including SARS-CoV-2. Incorporation of NHC by viral polymerases impairs replication by introducing errors into the viral genome. NHC has been shown to have a high barrier to the development of resistance in vitro with RSV, Influenza and Venezualen Equine Encephalitis viruses. In these studies, we have explored the potential for SARS-CoV-2 to develop resistance to NHC in cell culture. Methods. Vero E6 cells were infected with SARS-CoV-2 (WA-1) in triplicate in the presence of NHC or a C3L-protease inhibitor (MRK-A). Culture supernatants from wells with the highest drug concentration exhibiting a cytopathic effect (CPE) score of>=2+ were repassaged and at each passage, IC50 values were estimated based on CPE scoring. At each passage, full genome next generation sequencing (NGS) was performed on the viral RNA Results. No change in susceptibility to NHC (EC50 fold change <= 1.1) was noted in 2 of 3 cultures and a 2-fold change was observed in one culture after 30 passages. In contrast, a 3- to 4-fold decreases in susceptibility to the 3CL protease inhibitor were seen by passage by 12, with increasing resistance of 4.6- to 15.7-fold observed by passage 30. NHC passaged viruses exhibited 53 to 99 amino acid changes, including substitutions and deletions (both in-frame and frameshift), across 25 different viral proteins as compared with 10 to 13 changes in 13 proteins in the MRK-A cultures. With NHC, 3 to 4 changes were observed in the viral polymerase;however, these were randomly distributed, and none were observed more than once. In contrast, the 3CL protease passaged virus had a nsp5 T21I substitution detected in all 3 cultures. Conclusion. No evidence of SARS-CoV-2 phenotypic or genotypic resistance was observed following 30 passages with NHC. A random pattern of amino acid changes were observed across multiple proteins consistent with the mechanism of action of NHC. In the same study, resistance was readily selected to a control 3CL protease inhibitor. Together these data support previous reports demonstrating the high barrier to resistance of NHC.

Impact of Genetic Testing in Nephrology: A Family's Experience with Dent Disease 2

Diagnosis of a genetic kidney disease can enhance treatment/management, allow patient/family counseling, and enable patient referral to specialists or clinical trials. Here we present a case study describing the use of a >380 gene panel associated with kidney diseases (The RenasightTM test, Natera, Inc.) to diagnose Dent disease 2 in a patient and their family members. A 41-year-old male was referred to Nephrology for evaluation of elevated SCr (4.6 mg/dL) and proteinuria. The patient’s medical history was unremarkable except for glaucoma in infancy. A renal biopsy identified glomerulomegaly. Genetic testing identified a likely pathogenic, hemizygous, frame-shift variant (c.311del;p.Cys104Phefs*2) in exon 5 of OCRL, an X-linked gene, which is associated with Dent disease 2. This genetic diagnosis prompted changes to the patient’s treatment plan, including patient counseling and preparation for renal replacement therapy (RRT). The patient’s 46-year-old brother was hospitalized due to COVID-19 symptoms with a SCr of 19.1 mg/dL. Due to limited medical history, it was unclear if he was presenting with acute kidney injury or chronic kidney disease. Although there was no evidence of nephrolithiasis or renal tubular acidosis typically associated with Dent disease 2, the family history prompted genetic testing that confirmed the presence of the familial variant in this patient. These genetic findings prevented delay in treatment, namely, initiation of RRT. Given the X-linked inheritance of Dent disease 2, the patients’ mother is an obligate carrier of the p.Cys104Phefs*2 variant in OCRL. Therefore, the third brother is an appropriate candidate for genetic testing due to his 50% chance of inheriting the familial variant. In this family, identification of an OCRL variant via broad panel renal genetic testing impacted patient counseling, management, and family testing. Notably, without genetic testing for the proband, his brother’s condition may have gone undiagnosed due to the atypical presentation, demonstrating the variability of OCRL-related conditions. Genetic testing can enable accurate disease diagnosis in individuals with an atypical presentation, syndromic kidney disease and/or a family history.

WHOLE EXOME SEQUENCING IDENTIFIES A RARE NONSENSE MUTATION IN NACAD AS A POSSIBLE CAUSE OF COVID ORCHITIS IN BROTHERS

OBJECTIVE: This project sought to uncover genetic explanations as to why certain men face increased susceptibility to developing COVID orchitis. Our goal was to identify genetic variants associated with COVID orchitis in a group of patients, aided by whole-exome sequencing and protein phenotyping of affected patients. MATERIALS AND METHODS:We identified and examined six COVID- 19 patients who all were confirmed with polymerase chain reaction (PCR), including three COVID-19 (+) men without orchitis (controls) and three COVID (+) men with orchitis (bilateral testicular pain for at least 5 days around the time of testing PCR positive). Of note, among the three men with COVID-19 who had orchitis, two of them were siblings.DNA extraction and whole exome sequencing were performed on blood using the QIAmp blood maxi kit on five of the six patients. Variants were prioritized by being shared between the three patients affected with orchitis, absent in controls, and introducing nonsense, frameshift, splicing or non-synonymous amino acid changes and less than 10% in population prevalence. Based on WES findings, DuoSet® Human ACE2 reagent kit 2 (catalog number: DY933- 05) was purchased from R&D Systems, USA, and used to measure the level of soluble ACE2 in the plasma samples. RESULTS: The average age of the men in the study was 25 years old. The average duration of COVID symptoms (fever, sore throat, cough, body aches) were 7 days. Among the men who developed bilateral testis pain, the symptoms lasted for an average of 22 days. The median sperm concentration and sperm motility was 19 million/cc and 60% around 3 months after original infection. A list of 16 variants was generated that found to be shared between the two siblings with COVID orchitis along with the unrelated subject with COVID orchitis, and not present in the two controls. Among the 16 variants, a nonsynonymous non-frameshit deletion in NACAD variant on chromosome 7 with a frequency of 3.9% prevalence in ExAC was prioritized based on known involvement in the ACE2 pathway, read depth, and genotype quality. Phenotypically, we found that circulating levels of solubleACE2 was 3.72 ng/ml among men who had COVID orchitis and was lower than men who developed COVID without orchitis. CONCLUSIONS: We observed a stop mutation in NACAD in 2 brothers and 1 unrelated man who developed COVID orchitis. Interestingly, we found lower circulating ACE2 serum levels in both brothers with orchitis and the one nonrelated orchitis subject but normal serum levels in all controls. NACAD when involved with cellular ability to shuttle out ACE2 becomes critical for COVID symptomatology. With decreased transcellular and extracellular transport of ACE2 being possible in subjects with the gene mutation, it can be postulated more ACE2 will be found intracellularly leading to increased cellular entry of SARS CoV-2 and possibility of orchitis sequelae. IMPACT STATEMENT: These findings provide an explanation as to why genetic variations can lead to some patients developing comorbidities such as orchitis from COVID-19.