RNA Intermediates Research Articles

Human LINE-1 retrotransposons: impacts on the genome and regulation by host factors.

Genome sequencing revealed that nearly half of the human genome is comprised of transposable elements. Although most of these elements have been rendered inactive due to mutations, full-length intact long interspersed element-1 (LINE-1 or L1) copies retain the ability to mobilize through RNA intermediates by a so-called "copy-and-paste" mechanism, termed retrotransposition. L1 is the only known autonomous mobile genetic element in the genome, and its retrotransposition contributes to inter- or intra-individual genetic variation within the human population. However, L1 retrotransposition also poses a threat to genome integrity due to gene disruption and chromosomal instability. Moreover, recent studies suggest that aberrant L1 expression can impact human health by causing diseases such as cancer and chronic inflammation that might lead to autoimmune disorders. To counteract these adverse effects, the host cells have evolved multiple layers of defense mechanisms at the epigenetic, RNA and protein levels. Intriguingly, several host factors have also been reported to facilitate L1 retrotransposition, suggesting that there is competition between negative and positive regulation of L1 by host factors. Here, we summarize the known host proteins that regulate L1 activity at different stages of the replication cycle and discuss how these factors modulate disease-associated phenotypes caused by L1.

An extensive survey of phytoviral RNA 3' uridylation identifies extreme variations and virus-specific patterns.

Viral RNAs can be uridylated in eukaryotic hosts. However, our knowledge of uridylation patterns and roles remains rudimentary for phytoviruses. Here, we report global 3' terminal RNA uridylation profiles for representatives of the main families of positive single-stranded RNA phytoviruses. We detected uridylation in all 47 viral RNAs investigated here, revealing its prevalence. Yet, uridylation levels of viral RNAs varied from 0.2% to 90%. Unexpectedly, most poly(A) tails of grapevine fanleaf virus (GFLV) RNAs, including encapsidated tails, were strictly monouridylated, which corresponds to an unidentified type of viral genomic RNA extremity. This monouridylation appears beneficial for GFLV because it became dominant when plants were infected with nonuridylated GFLV transcripts. We found that GFLV RNA monouridylation is independent of the known terminal uridylyltransferases (TUTases) HEN1 SUPPRESSOR 1 (HESO1) and UTP:RNA URIDYLYLTRANSFERASE 1 (URT1) in Arabidopsis (Arabidopsis thaliana). By contrast, both TUTases can uridylate other viral RNAs like turnip crinkle virus (TCV) and turnip mosaic virus (TuMV) RNAs. Interestingly, TCV and TuMV degradation intermediates were differentially uridylated by HESO1 and URT1. Although the lack of both TUTases did not prevent viral infection, we detected degradation intermediates of TCV RNA at higher levels in an Arabidopsis heso1 urt1 mutant, suggesting that uridylation participates in clearing viral RNA. Collectively, our work unveils an extreme diversity of uridylation patterns across phytoviruses and constitutes a valuable resource to further decipher pro- and antiviral roles of uridylation.

Differential role of TLR7 signaling in SARS-CoV-2-induced antiviral and inflammatory responses

Abstract Since its emergence in 2019, SARS-CoV-2 has caused significant morbidity and mortality in humans. SARS-CoV-2-induced impaired antiviral and excessive inflammatory responses drive acute lung injury and severe pneumonia. However, the key pattern recognition receptors that elicit effective antiviral and pathogenic inflammatory responses are not well defined. Since CoVs are single-stranded RNA (ssRNA) viruses that make double-stranded RNA (dsRNA) intermediates during replication, we assessed the antiviral and pro-inflammatory roles of ssRNA and dsRNA mimics in mouse macrophages. We show that R837-TLR7 signaling induced excessive inflammation, while PolyIC-TLR3/MDA activation caused robust IFN production. Since R837-TLR7 induced high levels of inflammatory cytokines, using a novel mouse-adapted SARS-COV-2, we examined the role of TLR7 signaling in COVID-19 pathogenesis. Our results show that TLR7 KO mice were more susceptible to SARS-CoV-2 infection as compared to WT mice. Further investigation of SARS-CoV-2 infected lungs showed significantly reduced mRNA levels of antiviral interferons (IFNα/β/λ), ISGs (ISG15 and CXCL10), and several pro-inflammatory cytokine/chemokines in TLR7 KO lungs compared to WT. Interestingly, immunolabelling of cells isolated from infected WT and TLR7 KO lungs showed a high neutrophil count in TLR7 KO mice compared to WT mice. Furthermore, reduced lung IFN levels and increased mortality correlated with high lung viral titer in TLR7 KO mice. In summary, our findings conclusively establish that TLR7 signaling is protective during SARS-CoV-2 infection, and despite TLR7-mediated robust inflammatory cytokine production, TLR7-induced IFN and ISG responses play a key role in host protection.

Detection of double-stranded RNA intermediates of four major SARS-CoV-2 variants in formalin-fixed paraffin-embedded lung tissue of Syrian golden hamsters using monoclonal antibodies

Detection of double-stranded RNA intermediates of four major SARS-CoV-2 variants in formalin-fixed paraffin-embedded lung tissue of Syrian golden hamsters using monoclonal antibodies

Topical application of coat protein specific dsRNA molecules confers resistance against cognate Potato virus Y (PVY) infecting potato of North-East India

Sustainable agriculture relies on techniques and technology that combine efficiency with the smallest possible ecological and environmental impact. RNA interference (RNAi), a post-transcriptional eukaryotic pathway in eukaryotes can be co-opted for the control of plant pathogens in the topical application system. RNAi is the biological mechanism of mRNA degradation triggered by endogenous or exogenous double-stranded small interfering RNAs (dsRNA/siRNA) and regulates the target gene expression. Topically applied double-stranded RNA (dsRNA) molecules on plants imitate the viral RNA intermediate which is involved in plant virus replication and thereby triggers post-transcriptional gene silencing via., RNAi pathway and reducing viral replication. In this study, a non-transgenic-based RNAi approach was adopted to induce resistance against PVY in potato crop. dsRNA was successfully produced through in vitro transcription and hybridization. To test the effect of dsRNA molecules, in vitro synthesized dsRNA were applied on potato leaves along with PVY. dsRNA-treated plants showed higher resistance to viral disease and exhibited normal growth compared with PVY-treated plants. Therefore, the topical application of dsRNA molecules from the PVY CP gene could serve as a promising method for suppressing PVY infection and ultimately enhancing potato yield.

A structural understanding of influenza virus genome replication.

Influenza virus contains a single-stranded negative-sense RNA genome. Replication of the genome is carried out by the viral RNA-dependent RNA polymerase in the context of the viral ribonucleoprotein (RNP) complex, through a positive-sense complementary RNA intermediate. Genome replication is tightly controlled through interactions with accessory viral and host factors. Propelled by developments in recombinant protein expression, and technical improvements in X-ray crystallography and cryo-electron microscopy, snapshots of the replication process have been captured. Here, we review how recent structural data shed light on the molecular mechanisms of influenza virus genome replication, in particular, encapsidation of nascent RNA, de novo RNP assembly, and regulation of replication initiation through interactions with host and viral cues.

Antagonism of ALAS1 by the Measles Virus V protein contributes to degradation of the mitochondrial network and promotes interferon response.

Viruses have evolved countless mechanisms to subvert and impair the host innate immune response. Measles virus (MeV), an enveloped, non-segmented, negative-strand RNA virus, alters the interferon response through different mechanisms, yet no viral protein has been described as directly targeting mitochondria. Among the crucial mitochondrial enzymes, 5'-aminolevulinate synthase (ALAS) is an enzyme that catalyzes the first step in heme biosynthesis, generating 5'-aminolevulinate from glycine and succinyl-CoA. In this work, we demonstrate that MeV impairs the mitochondrial network through the V protein, which antagonizes the mitochondrial enzyme ALAS1 and sequesters it to the cytosol. This re-localization of ALAS1 leads to a decrease in mitochondrial volume and impairment of its metabolic potential, a phenomenon not observed in MeV deficient for the V gene. This perturbation of the mitochondrial dynamics demonstrated both in culture and in infected IFNAR-/- hCD46 transgenic mice, causes the release of mitochondrial double-stranded DNA (mtDNA) in the cytosol. By performing subcellular fractionation post infection, we demonstrate that the most significant source of DNA in the cytosol is of mitochondrial origin. Released mtDNA is then recognized and transcribed by the DNA-dependent RNA polymerase III. The resulting double-stranded RNA intermediates will be captured by RIG-I, ultimately initiating type I interferon production. Deep sequencing analysis of cytosolic mtDNA editing divulged an APOBEC3A signature, primarily analyzed in the 5'TpCpG context. Finally, in a negative feedback loop, APOBEC3A an interferon inducible enzyme will orchestrate the catabolism of mitochondrial DNA, decrease cellular inflammation, and dampen the innate immune response.

LINE-1 retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5' single-cell RNA-Seq.

LINE-1 retrotransposons are sequences capable of copying themselves to new genomic loci via an RNA intermediate. New studies implicate LINE-1 in a range of diseases, especially in the context of aging, but without an accurate understanding of where and when LINE-1 is expressed, a full accounting of its role in health and disease is not possible. We therefore developed a method-5' scL1seq-that makes use of a widely available library preparation method (10x Genomics 5' single cell RNA-seq) to measure LINE-1 expression in tens of thousands of single cells. We recapitulated the known pattern of LINE-1 expression in tumors-present in cancer cells, absent from immune cells-and identified hitherto undescribed LINE-1 expression in human epithelial cells and mouse hippocampal neurons. In both cases, we saw a modest increase with age, supporting recent research connecting LINE-1 to age related diseases.

Activation of the NLRP1 inflammasome in human keratinocytes by the dsDNA mimetic poly(dA:dT).

The accrual of cytosolic DNA leads to transcription of type I IFNs, proteolytic maturation of the IL-1 family of cytokines, and pyroptotic cell death. Caspase-1 cleaves pro-IL1β to generate mature bioactive cytokine and gasdermin D which facilitates IL-1 release and pyroptotic cell death. Absent in melanoma-2 (AIM2) is a sensor of dsDNA leading to caspase-1 activation, although in human monocytes, cGAS-STING acting upstream of NLRP3 mediates the dsDNA-activated inflammasome response. In healthy human keratinocytes, AIM2 is not expressed yet caspase-1 is activated by the synthetic dsDNA mimetic poly(dA:dT). Here, we show that this response is not mediated by either AIM2 or the cGAS-STING-NLRP3 pathway and is instead dependent on NLRP1. Poly(dA:dT) is unique in its ability to activate NLRP1, as conventional linear dsDNAs fail to elicit NLRP1 activation. DsRNA was recently shown to activate NLRP1 and prior work has shown that poly(dA:dT) is transcribed into an RNA intermediate that stimulates the RNA sensor RIG-I. However, poly(dA:dT)-dependent RNA intermediates are insufficient to activate NLRP1. Instead, poly(dA:dT) results in oxidative nucleic acid damage and cellular stress, events which activate MAP3 kinases including ZAKα that converge on p38 to activate NLRP1. Collectively, this work defines a new activator of NLRP1, broadening our understanding of sensors that recognize poly(dA:dT) and advances the understanding of the immunostimulatory potential of this potent adjuvant.

Transposons Acting as Competitive Endogenous RNAs: In-Silico Evidence from Datasets Characterised by L1 Overexpression.

LINE L1 are transposable elements that can replicate within the genome by passing through RNA intermediates. The vast majority of these element copies in the human genome are inactive and just between 100 and 150 copies are still able to mobilize. During evolution, they could have been positively selected for beneficial cellular functions. Nonetheless, L1 deregulation can be detrimental to the cell, causing diseases such as cancer. The activity of miRNAs represents a fundamental mechanism for controlling transcript levels in somatic cells. These are a class of small non-coding RNAs that cause degradation or translational inhibition of their target transcripts. Beyond this, competitive endogenous RNAs (ceRNAs), mostly made by circular and non-coding RNAs, have been seen to compete for the binding of the same set of miRNAs targeting protein coding genes. In this study, we have investigated whether autonomously transcribed L1s may act as ceRNAs by analyzing public dataset in-silico. We observed that genes sharing miRNA target sites with L1 have a tendency to be upregulated when L1 are overexpressed, suggesting the possibility that L1 might act as ceRNAs. This finding will help in the interpretation of transcriptomic responses in contexts characterized by the specific activation of transposons.

An update on post-transcriptional regulation of retrotransposons.

Retrotransposons, including LINE-1, Alu, SVA, and endogenous retroviruses, are one of the major constituents of human genomic repetitive sequences. Through the process of retrotransposition, some of them occasionally insert into new genomic locations by a copy-paste mechanism involving RNA intermediates. Irrespective of de novo genomic insertions, retrotransposon expression can lead to DNA double-strand breaks and stimulate cellular innate immunity through endogenous patterns. As a result, retrotransposons are tightly regulated by multi-layered regulatory processes to prevent the dangerous effects of their expression. In recent years, significant progress was made in revealing how retrotransposon biology intertwines with general post-transcriptional RNA metabolism. Here, I summarize current knowledge on the involvement of post-transcriptional factors in the biology of retrotransposons, focusing on LINE-1. I emphasize general RNA metabolisms such as methylation of adenine (m6 A), RNA 3'-end polyadenylation and uridylation, RNA decay and translation regulation. I discuss the effects of retrotransposon RNP sequestration in cytoplasmic bodies and autophagy. Finally, I summarize how innate immunity restricts retrotransposons and how retrotransposons make use of cellular enzymes, including the DNA repair machinery, to complete their replication cycles.

MTR4 adaptor PICT1 functions in two distinct steps during pre-rRNA processing

Ribosome biogenesis proceeds with the successive cleavage and trimming of the large 47S rRNA precursor, where the RNA exosome plays major roles in concert with the Ski2-like RNA helicase, MTR4. The recent finding of a consensus amino acid sequence, the arch-interacting motif (AIM), for binding to the arch domain in MTR4 suggests that recruitment of the RNA processing machinery to the maturing pre-rRNA at appropriate places and timings is mediated by several adaptor proteins possessing the AIM sequence. In yeast Saccharomyces cerevisiae, Nop53 plays such a role in the maturation of the 3′-end of 5.8S rRNA. Here, we investigated the functions of PICT1 (also known as GLTSCR2 or NOP53), a mammalian ortholog of Nop53, during ribosome biogenesis in human cells. PICT1 interacted with MTR4 and exosome in an AIM-dependent manner. Overexpression of PICT1 mutants defecting AIM sequence and siRNA-mediated depletion of PICT1 showed that PICT1 is involved in two distinct pre-rRNA processing steps during the generation of 60S ribosomes; first step is the early cleavage of 32S intermediate RNA, while the second step is the late maturation of 12S precursor into 5.8S rRNA. The recruitment of MTR4 and RNA exosome via the AIM sequence was required only during the late processing step. Although, the depletion of MTR4 and PICT1 induced stabilization of the tumor suppressor p53 protein in cancer cell lines, the depletion of the exosome catalytic subunits, RRP6 and DIS3, did not exert such an effect. These results suggest that recruitment of the RNA processing machinery to the 3′-end of pre-5.8S rRNA may be involved in the induction of the nucleolar stress response, but the pre-rRNA processing capabilities themselves were not involved in this process.

Viral replication site distribution for rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization.

We made 2 Z-based in situ hybridization (ISH) probes for the detection of rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) nucleic acid in formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that had died during an outbreak of RHD in Washington, USA. One probe system was made for detection of negative-sense RNA (i.e., the replicative intermediate RNA for the virus), and the other probe system was constructed for detection of genomic and mRNA of the virus (viral mRNA). Tissue sets were tested separately, and the viral mRNA probe system highlighted much broader tissue distribution than that of the replicative intermediate RNA probe system. The latter was limited to liver, lung, kidney, spleen, myocardium, and occasional endothelial staining, whereas signal for the viral mRNA was seen in many more tissues. The difference in distribution suggests that innate phagocytic activity of various cell types may cause overestimation of viral replication sites when utilizing ISH of single-stranded, positive-sense viruses.

Arthropod-Borne Virus Surveillance as a Tool to Study the Australian Mosquito Virome

Mosquitoes (n = 4381 in 198 pools) were collected in March and April 2018 to survey the presence of West Nile virus Kunjin strain in mosquito populations around crocodile farms in the Darwin region of the Northern Territory (NT) of Australia. While no Kunjin virus was detected in these mosquitoes, we applied our viral replicative intermediates screening system termed monoclonal antibodies to viral RNA intermediates in cells or MAVRIC to this set of samples. This resulted in the detection of 28 pools with virus replicating in C6/36 mosquito cells and the identification of three insect viruses from three distinct virus classes. We demonstrate the persistence of the insect-specific flavivirus Palm Creek virus in Coquillettidia xanthogaster mosquitoes from Darwin over almost a decade, with limited genetic drift. We also detected a novel Hubei macula-like virus 3 strain in samples from two mosquito genera, suggesting the virus, for which the sequence was originally detected in spiders and soybean thrips, might be involved in a horizontal transmission cycle between arthropods and plants. Overall, these data demonstrate the strength of the optimized MAVRIC system and contribute to our general knowledge of the mosquito virome and insect viruses.

Flipped over U: structural basis for dsRNA cleavage by the SARS-CoV-2 endoribonuclease.

Coronaviruses generate double-stranded (ds) RNA intermediates during viral replication that can activate host immune sensors. To evade activation of the host pattern recognition receptor MDA5, coronaviruses employ Nsp15, which is auridine-specific endoribonuclease. Nsp15 is proposed to associate with the coronavirus replication-transcription complex within double-membrane vesicles to cleave these dsRNA intermediates. How Nsp15 recognizes and processes dsRNA is poorly understood because previous structural studies of Nsp15 have been limited to small single-stranded (ss) RNA substrates. Here we present cryo-EM structures of SARS-CoV-2 Nsp15 bound to a 52nt dsRNA. We observed that the Nsp15 hexamer forms a platform for engaging dsRNA across multiple protomers. The structures, along with site-directed mutagenesis and RNA cleavage assays revealed critical insight into dsRNA recognition and processing. To process dsRNA Nsp15 utilizes a base-flipping mechanism to properly orient the uridine within the active site for cleavage. Our findings show that Nsp15 is a distinctive endoribonuclease that can cleave both ss- and dsRNA effectively.

Involvement of retroelements in the autoimmune response in humans

Retroelements are mobile genomic components requiring an RNA intermediate which is reverse-transcribed into complementary DNA for transposition. Human genome contains a vast amount of retroelements including retrotransposons and endogenous retroviruses. These elements are categorized according to presence or absence of long terminal repeats, LTRs or non-LTRs, as well as autonomous and non-autonomous according to involvement of reverse transcriptase. The retroelements have been accumulated in mammalian genomes over all evolutionary times through vertical transmission, and many of them were inactivated through accumulation of mutations. However, the retroelements entered into genome within the last 200,000 years are mostly functional. Some of the active retroelements are associated with varying autoimmune diseases because anti-retroelement antibodies might cross-react with other proteins in the human body. For instance, autoimmunity and inflammation could be stimulated by increased expression of long interspersed element 1 (LINE-1 or L1) or decreased L1 degradation. Different regulation of L1 expression might be related to the genetic and sex-related variations or environmental factors. Activation of retroelements is also controlled by epigenetic silencing mechanisms such as histone modification. Elevated levels of L1 retroelements could trigger the production of type I interferon, a crucial innate defense mechanism in mammals against viruses, and systemic autoimmune response is induced. Loss-of-function in some deoxyribonucleases (DNases) such as three prime repair exonuclease 1 that degrades reverse-transcribed DNA is also related to autoimmune diseases. Additionally, human endogenous retroviruses also play a role in autoimmune diseases. Involvement of retroelements in autoimmune disorders is exemplified with three diseases, i.e. systemic lupus erythematosus, Aicardi–Goutières syndrome, and multiple sclerosis.

Crowning Touches in Positive-Strand RNA Virus Genome Replication Complex Structure and Function.

Positive-strand RNA viruses, the largest genetic class of eukaryotic viruses, include coronaviruses and many other established and emerging pathogens. A major target for understanding and controlling these viruses is their genome replication, which occurs in virus-induced membrane vesicles that organize replication steps and protect double-stranded RNA intermediates from innate immune recognition. The structure of these complexes has been greatly illuminated by recent cryo-electron microscope tomography studies with several viruses. One key finding in diverse systems is the organization of crucial viral RNA replication factors in multimeric rings or crowns that among other functions serve as exit channels gating release of progeny genomes to the cytosol for translation and encapsidation. Emerging results suggest that these crowns serve additional important purposes in replication complex assembly, function, and interaction with downstream processes such as encapsidation. The findings provide insights into viral function and evolution and new bases for understanding, controlling, and engineering positive-strand RNA viruses.

Diverse and mobile: eccDNA-based identification of carrot low-copy-number LTR retrotransposons active in callus cultures.

SUMMARYLong terminal repeat retrotransposons (LTR‐RTs) are mobilized via an RNA intermediate using a ‘copy and paste’ mechanism, and account for the majority of repetitive DNA in plant genomes. As a side effect of mobilization, the formation of LTR‐RT‐derived extrachromosomal circular DNAs (eccDNAs) occurs. Thus, high‐throughput sequencing of eccDNA can be used to identify active LTR‐RTs in plant genomes. Despite the release of a reference genome assembly, carrot LTR‐RTs have not yet been thoroughly characterized. LTR‐RTs are abundant and diverse in the carrot genome. We identified 5976 carrot LTR‐RTs, 2053 and 1660 of which were attributed to Copia and Gypsy superfamilies, respectively. They were further classified into lineages, families and subfamilies. More diverse LTR‐RT lineages, i.e. lineages comprising many low‐copy‐number subfamilies, were more frequently associated with genic regions. Certain LTR‐RT lineages have been recently active in Daucus carota. In particular, low‐copy‐number LTR‐RT subfamilies, e.g. those belonging to the DcAle lineage, have significantly contributed to carrot genome diversity as a result of continuing activity. We utilized eccDNA sequencing to identify and characterize two DcAle subfamilies, Alex1 and Alex3, active in carrot callus. We documented 14 and 32 de novo insertions of Alex1 and Alex3, respectively, which were positioned in non‐repetitive regions.

Small and intermediate size structural RNAs in the unicellular parasite Cryptosporidium parvum as revealed by sRNA-seq and comparative genomics.

Small and intermediate-size noncoding RNAs (sRNAs and is-ncRNAs) have been shown to play important regulatory roles in the development of several eukaryotic organisms. However, they have not been thoroughly explored in Cryptosporidium parvum, an obligate zoonotic protist parasite responsible for the diarrhoeal disease cryptosporidiosis. Using Illumina sequencing of a small RNA library, a systematic identification of novel small and is-ncRNAs was performed in C. parvum excysted sporozoites. A total of 79 novel is-ncRNA candidates, including antisense, intergenic and intronic is-ncRNAs, were identified, including 7 new small nucleolar RNAs (snoRNAs). Expression of select novel is-ncRNAs was confirmed by RT-PCR. Phylogenetic conservation was analysed using covariance models (CMs) in related Cryptosporidium and apicomplexan parasite genome sequences. A potential new type of small ncRNA derived from tRNA fragments was observed. Overall, a deep profiling analysis of novel is-ncRNAs in C. parvum and related species revealed structural features and conservation of these novel is-ncRNAs. Covariance models can be used to detect is-ncRNA genes in other closely related parasites. These findings provide important new sequences for additional functional characterization of novel is-ncRNAs in the protist pathogen C. parvum.

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.