Jumper enables discontinuous transcript assembly in coronaviruses

Genes in SARS-CoV-2 and other viruses in the order of Nidovirales are expressed by a process of discontinuous transcription which is distinct from alternative splicing in eukaryotes and is mediated by the viral RNA-dependent RNA polymerase. Here, we introduce the DISCONTINUOUS TRANSCRIPT ASSEMBLYproblem of finding transcripts and their abundances given an alignment of paired-end short reads under a maximum likelihood model that accounts for varying transcript lengths. We show, using simulations, that our method, JUMPER, outperforms existing methods for classical transcript assembly. On short-read data of SARS-CoV-1, SARS-CoV-2 and MERS-CoV samples, we find that JUMPER not only identifies canonical transcripts that are part of the reference transcriptome, but also predicts expression of non-canonical transcripts that are supported by subsequent orthogonal analyses. Moreover, application of JUMPER on samples with and without treatment reveals viral drug response at the transcript level. As such, JUMPER enables detailed analyses of Nidovirales transcriptomes under varying conditions.

CORSID enables de novo identification of transcription regulatory sequences and genes in coronaviruses

Genes in coronaviruses are preceded by transcription regulatory sequences (TRSs), which play a critical role in gene expression mediated by the viral RNA-dependent RNA-polymerase via the process of discontinuous transcription. In addition to being crucial for our understanding of the regulation and expression of coronavirus genes, we demonstrate for the first time how TRSs can be leveraged to identify gene locations in the coronavirus genome. To that end, we formulate the TRS AND GENE IDENTIFICATION (TRS-GENE-ID) problem of simultaneously identifying TRS sites and gene locations in unannotated coronavirus genomes. We introduce CORSID (CORe Sequence IDentifier), a computational tool to solve this problem. We also present CORSID-A, which solves a constrained version of the TRS-GENE-ID problem, the TRS IDENTIFICATION (TRS-ID) problem, identifying TRS sites in a coronavirus genome with specified gene annotations. We show that CORSID-A outperforms existing motif-based methods in identifying TRS sites in coronaviruses and that CORSID outperforms state-of-the-art gene finding methods in finding genes in coronavirus genomes. We demonstrate that CORSID enables de novo identification of TRS sites and genes in previously unannotated coronaviruses. CORSID is the first method to perform accurate and simultaneous identification of TRS sites and genes in coronavirus genomes without the use of any prior information.

Evidence for a long-r ange RNA-RNA interaction between ORF8 and the downstream region of the Spike polybasic insertion of SARS-CoV-2

SARS-CoV-2 has affected people all over the world as the causative agent of COVID-19. The virus is related to the highly lethal SARS-CoV responsible for the 2002-2003 SARS outbreak in Asia. Intense research is ongoing to understand why both viruses have different spreading capacities and mortality rates. Similar to other betacoronaviruses, long-range RNA-RNA interactions occur between different parts of the viral genomic RNA, resulting in discontinuous transcription and production of various sub-genomic RNAs. These sub-genomic RNAs are then translated into different viral proteins. An important difference between both viruses is a polybasic insertion in the Spike region of SARS-CoV-2, absent in SARS-CoV. Here we show that a 26-base-pair long-range RNA-RNA interaction occurs between the genomic region downstream of the Spike insertion and ORF8 in SARS-CoV-2. Predictions suggest that the corresponding ORF8 region forms the most energetically favorable interaction with that of Spike region from amongst all possible candidate regions within SARS-CoV-2 genomic RNA. We also found signs of sequence covariation in the predicted interaction using a large dataset with 27,592 full-length SARS-CoV-2 genomes. In particular, a synonymous mutation in ORF8 accommodated for base pairing with Spike [G23675 C28045U], and a non-synonymous mutation in Spike accommodated for base pairing with ORF8 [C23679U G28042] both of which were in close proximity of one another. The predicted interactions can potentially be related to regulation of sub-genomic RNA production rates.

Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.

In vivo structure and dynamics of the SARS-CoV-2 RNA genome

The dynamics of SARS-CoV-2 RNA structure and their functional relevance are largely unknown. Here we develop a simplified SPLASH assay and comprehensively map the in vivo RNA-RNA interactome of SARS-CoV-2 genome across viral life cycle. We report canonical and alternative structures including 5′-UTR and 3′-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in both cells and virions. We provide direct evidence of interactions between Transcription Regulating Sequences, which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE. More importantly, we find that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome domains remain stable but with strengthened demarcation of local domains and weakened global cyclization. Taken together, our analysis reveals the structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, the alternative conformations and the maintenance of global genome organization during the whole life cycle of SARS-CoV-2, which we anticipate will help develop better antiviral strategies.

Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned a droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNAs from anonymized COVID-19 positive subjects and we found a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Further, we observed that expression profiles of sgmRNAs analyzed in a subset of 110 samples subjected to meta-transcriptomics sequencing were highly correlated with those obtained by ddPCR. Our results, providing a comprehensive and dynamic snapshot of SARS-CoV-2 sgmRNAs expression and replication, may contribute to provide a better understanding of SARS-CoV-2 transcription and expression mechanisms, and support the development of more accurate molecular diagnostic tools and for the stratification of COVID-19 patients.

In vivo structure and dynamics of the RNA genome of SARS-Cov-2

The SARS-CoV-2 coronavirus, which causes the COVID-19 pandemic, is one of the largest positive strand RNA viruses. Here we developed a simplified SPLASH assay and comprehensively mapped the in vivo RNA-RNA interactome of SARS-CoV-2 RNA during the viral life cycle. We observed canonical and alternative structures including 3’-UTR and 5’-UTR, frameshifting element (FSE) pseudoknot and genome cyclization in cells and in virions. We provide direct evidence of interactions between Transcription Regulating Sequences (TRS-L and TRS-Bs), which facilitate discontinuous transcription. In addition, we reveal alternative short and long distance arches around FSE, forming a “high-order pseudoknot” embedding FSE, which might help ribosome stalling at frameshift sites. More importantly, we found that within virions, while SARS-CoV-2 genome RNA undergoes intensive compaction, genome cyclization is weakened and genome domains remain stable. Our data provides a structural basis for the regulation of replication, discontinuous transcription and translational frameshifting, describes dynamics of RNA structures during life cycle of SARS-CoV-2, and will help to develop antiviral strategies.

Novel RT-ddPCR Assays for determining the transcriptional profile of SARS-CoV-2

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription.Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.HighlightsWe developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2Our panel targets nongenic and genic regions in genomic and subgenomic RNAsAll assays detect 1-10 copies and are linear over 3-4 orders of magnitudeAll assays correlated with the clinical Abbott SARS-CoV-2 Viral Load AssayClinical samples showed higher copy numbers for targets at the 3’ end of the genome

SARS-CoV-2 highly conserved s2m element dimerizes via a kissing complex and interacts with host miRNA-1307-3p

ABSTRACTThe ongoing COVID-19 pandemic highlights the necessity for a more fundamental understanding of the coronavirus life cycle. The causative agent of the disease, SARS-CoV-2, is being studied extensively from a structural standpoint in order to gain insight into key molecular mechanisms required for its survival. Contained within the untranslated regions of the SARS-CoV-2 genome are various conserved stem-loop elements that are believed to function in RNA replication, viral protein translation, and discontinuous transcription. While the majority of these regions are variable in sequence, a 41-nucleotide s2m element within the 3’ UTR is highly conserved among coronaviruses and three other viral families. In this study, we demonstrate that the s2m element of SARS-CoV-2 dimerizes by forming an intermediate homodimeric kissing complex structure that is subsequently converted to a thermodynamically stable duplex conformation. This process is aided by the viral nucleocapsid protein, potentially indicating a role in mediating genome dimerization. Furthermore, we demonstrate that the s2m element interacts with multiple copies of host cellular miRNA-1307-3p. Taken together, our results highlight the potential significance of the dimer structures formed by the s2m element in key biological processes and implicate the motif as a possible therapeutic drug target for COVID-19 and other coronavirus-related diseases.

Extensive High-Order Complexes within SARS-CoV-2 Proteome Revealed by Compartmentalization-Aided Interaction Screening

SummaryBearing the largest single-stranded RNA genome in nature, SARS-CoV-2 utilizes sophisticated replication/transcription complexes (RTCs), mainly composed of a network of nonstructural proteins and nucleocapsid protein, to establish efficient infection. Here, we developed an innovative interaction screening strategy based on phase separation in cellulo, namely compartmentalization of protein-protein interactions in cells (CoPIC). Utilizing CoPIC screening, we mapped the interaction network among RTC-related viral proteins. We identified a total of 47 binary interactions among 14 proteins governing replication, discontinuous transcription, and translation of coronaviruses. Further exploration via CoPIC led to the discovery of extensive ternary complexes composed of these components, which infer potential higher-order complexes. Taken together, our results present an efficient, and robust interaction screening strategy, and indicate the existence of a complex interaction network among RTC-related factors, thus opening up new opportunities to understand SARS-CoV-2 biology and develop therapeutic interventions for COVID-19.