Transcriptional Regulatory Sequences Research Articles

M6A methylation of transcription leader sequence impacts discontinuous transcription of subgenomic mRNAs.

The SARS-CoV-2 genome has been shown to be m6A methylated at several positions in vivo. Strikingly, a DRACH motif, the recognition motif for adenosine methylation, resides in the core of the transcriptional regulatory leader sequence (TRS-L) at position A74, which is highly conserved and essential for viral discontinuous transcription. Methylation at position A74 correlates with viral pathogenicity. Discontinuous transcription produces a set of subgenomic mRNAs that function as templates for translation of all structural and accessory proteins. A74 is base-paired in the short stem-loop structure 5'SL3 that opens during discontinuous transcription to form long-range RNA-RNA interactions with nascent (-)-strand transcripts at complementary TRS-body sequences. A74 can be methylated by human METTL3/METTL14 complex in vitro. Here, we investigate its impact on the structural stability of 5'SL3 and the long-range TRS-leader:TRS-body duplex formation necessary for synthesis of subgenomic mRNAs of all four viral structural proteins. Methylation uniformly destabilizes 5'SL3 and long-range duplexes and alters their relative populations in equilibrium, suggesting that m6A74 modification acts as a regulator for the abundance of viral structural proteins due to this destabilization.

Comparing the molecular evolution and recombination patterns of predominant PRRSV-2 lineages co-circulating in China.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses widespread epidemics in swine herds, yet the drivers underlying lineage replacements/fitness dynamics remain unclear. To delineate the evolutionary trajectories of PRRSV-2 lineages prevalent in China, we performed a comprehensive longitudinal phylodynamic analysis of 822 viral sequences spanning 1991-2022. The objectives encompassed evaluating lineage dynamics, genetic diversity, recombination patterns and glycosylation profiles. A significant shift in the dominance of PRRSV-2 sub-lineages has been observed over the past 3 decades, transitioning from sub-lineage 8.7 to sub-lineage 1.8, followed by extensive diversification. The analysis revealed discordant recombination patterns between the two dominant viral sub-lineages 1.8 and 8.7, underscoring that modular genetic exchanges contribute significantly to their evolutionary shaping. Additionally, a strong association was found between recombination breakpoint locations and transcriptional regulatory sequences (TRSs). Glycosylation patterns also demonstrated considerable variability across sub-lineages and temporally, providing evidence for immune-driven viral evolution. Furthermore, we quantified different evolutionary rates across sub-lineages, with sub-lineage 1.8 uniquely displaying the highest nucleotide substitution rates. Taken together, these findings provide refined insight into the evolutionary mechanisms underpinning cyclic shifts in dominance among regionally circulating PRRSV sub-lineages.

Endogenous retroviral solo-LTRs in human genome.

Human endogenous retroviruses (HERVs) are derived from the infection and integration of exogenetic retroviruses. HERVs account for 8% of human genome, and the majority of HERVs are solitary LTRs (solo-LTRs) due to homologous recombination. Multiple findings have showed that solo-LTRs could provide an enormous reservoir of transcriptional regulatory sequences involved in diverse biological processes, especially carcinogenesis and cancer development. The link between solo-LTRs and human diseases still remains poorly understood. This review focuses on the regulatory modules of solo-LTRs, which contribute greatly to the diversification and evolution of human genes. More importantly, although inactivating mutations, insertions and deletions have been identified in solo-LTRs, the inherited regulatory elements of solo-LTRs initiate the expression of chimeric lncRNA transcripts, which have been reported to play crucial roles in human health and disease. These findings provide valuable insights into the evolutionary and functional mechanisms underlying the presence of HERVs in human genome. Taken together, in this review, we will present evidences showing the regulatory and encoding capacity of solo-LTRs as well as the significant impact on various aspects of human biology.

A recombination-resistant genome for live attenuated and stable PEDV vaccines by engineering the transcriptional regulatory sequences.

Coronaviruses are important pathogens of humans and animals, and vaccine developments against them are imperative. Due to the ability to induce broad and prolonged protective immunity and the convenient administration routes, live attenuated vaccines (LAVs) are promising arms for controlling the deadly coronavirus infections. However, potential recombination events between vaccine and field strains raise a safety concern for LAVs. The porcine epidemic diarrhea virus (PEDV) remodeled TRS (RMT) mutant generated in this study replicated efficiently in both cell culture and in pigs and retained protective immunogenicity against PEDV challenge in pigs. Furthermore, the RMT PEDV was resistant to recombination and genetically stable. Therefore, RMT PEDV can be further optimized as a backbone for the development of safe LAVs.

Identification of subgenomic mRNAs derived from the coronavirus 1a/1b protein gene: Implications for coronavirus transcription

Synthesis of coronavirus subgenomic mRNA (sgmRNA) is guided by the transcription regulatory sequence (TRS). sgmRNA derived from the body TRS (TRS-B) located at the 1a/1b protein gene is designated 1ab/sgmRNA. In the current study, we comprehensively identified the 1ab/sgmRNAs synthesized from TRS-Bs located at the 1a/1b protein genes of different coronavirus genera both in vitro and in vivo by RT‒PCR and sequencing. The results suggested that the degree of sequence homology between the leader TRS (TRS-L) and TRS-B may not be a decisive factor for 1ab/sgmRNA synthesis. This observation led us to revisit the coronavirus transcription mechanism and to propose that the disassociation of coronavirus polymerase from the viral genome may be a prerequisite for sgmRNA synthesis. Once the polymerase can disassociate at TRS-B, the sequence homology between TRS-L and TRS-B is important for sgmRNA synthesis. The study therefore extends our understanding of transcription mechanisms.

The Ingenious Multiple Efficacies of the Integrated Gene Fragment of Torque Teno Mini Virus in Orchestrating Promyelocytic Leukemogenesis Via Hijacking Rara

Introduction Torque teno mini virus (TTMV) is a ubiquitous virus which symbiotic with humans, but it has recently been reported to cause acute promyelocytic leukemia (APL) through a unique form of genome fragment integration and forming the TTMV::RARA fusion gene, with 4 cases reported till now. However, the leukemogenesis mechanism of TTMV::RARA-APL has not been explored. Methods We recruited transcriptome sequencing (RNA-seq) data of 7 TTMV::RARA-APL cases, 3 of them also with whole genome sequencing (WGS) data, to analyze the properties of the TTMV integrated sequences. Clinical information was analyzed. In vitro cell experiments were performed to verify the functions of the conserved regulatory sequences and the fusion protein. Results These cases were collected from 4 centers, 5 males and 2 females, aged 3 to 35 (median 9) years, with 6 pediatric cases. Of which 3 pediatric cases were identified from a continuous acute myeloid leukemia (AML) cohort. In this cohort, 3 TTMV::RARA-APL were identified in 205 AML cases under 14 years, but no cases in another 930 AML cases, showing a strong tendency towards pediatrics (p=0.00022). Moreover, we totally identified 85 PML::RARA-APL and 3 TTMV::RARA-APL, and no other X::RAR cases in this cohort, indicating TTMV::RARA is the most common X::RAR fusion besides PML::RARA. All cases manifested typical APL morphological features, and RNA-seq expression analysis showed clustering with PML::RARA-APL cases. Initial effect and remission can be achieved with chemotherapy regimens containing all-trans retinoic acid (ATRA), but early recurrence is prone to occur. RARA-I387T mutation was observed in case #TR4, who relapsed after treatment with ATRA, suggesting that the leukemic cells were responsive to ATRA. However, primary resistance to ATRA and extensive resistance to various agents were observed in case #TR5, who was initially diagnosed with myeloid sarcoma and transformed into APL at relapse. The TTMV integrated sequences of 3 cases with WGS data were 1164bp, 2113bp, and 2859bp (Fig. 1A), respectively, and the common feature was the involvement of the transcription regulatory region and the open reading frame 2 (ORF2). Alignment of TTMV transcription regulatory sequences (Fig. 1B) revealed a highly conserved basal transcription factor region, including the canonical TATA box and GC box. While in the proximal promoter region showing sequence diversity, all 3 cases carried 4 RUNX1 binding elements and 1 octamer motif. In vitro experiments (Fig. 1C) showed that TTMV basal transcription element can initiate basal expression in 293T cells, while RUNX1 can significantly enhance the expression in response to the RUNX1 binding elements. RUNX1 is an important transcription factor protein in the differentiation of myeloid cells, and the 4 RUNX1 binding elements might explain the myelotropic of TTMV and confer an important basis in promyelocytic leukemogenesis. The TTMV-ORF2 coding sequences of all 7 cases were analyzed, with the common region being the N-terminus 58 amino acids, of which 20 were highly conserved (Fig. 1D, 1E). Co-Immunoprecipitation analysis confirmed that the TTMV::RARA protein forms homopolymers and heteropolymers with RXRA (Fig.1F), which is the property shared by all X::RARA proteins. Serine/Threonine protein phosphatase (S/T PPase) assays showed that the fusion protein exhibits strong S/T PPase activity (Fig. 1G). UAS/GAL4 reporter experiment showed that TTMV::RARA protein exhibited a constrained but concentration-dependent response to ATRA compared to RARA (Fig. 1H). However, fusion proteins with ORF2-C32A/C34A mutation and impaired phosphatase activity were significantly more responsive to ATRA (Fig. 1H). This suggests that the S/T PPase activity of the ORF2 coding region plays a role in retarded ATRA response. Conclusions This is the first systematic report on this novelty disease. Data suggests that TTMV::RARA-APL may be more common in children, which may be related to the ubiquity of TTMV and its acquisition early in life. The clinical manifestations and treatment responses are diverse, with overall dismal prognosis. Molecular investigation indicated the multifunctional TTMV integrated molecular machinery in orchestrating RUNX1 enforced myelotropic, aberrant priming expression, homodimer formation, and S/T PPase activity, and confer promyelocytic leukemogenesis via hijacking RARA.

Unveiling the role of PUS7-mediated pseudouridylation in host protein interactions specific for the SARS-CoV-2 RNA genome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive single-stranded RNA virus, engages in complex interactions with host cell proteins throughout its life cycle. While these interactions enable the host to recognize and inhibit viral replication, they also facilitate essential viral processes such as transcription, translation, and replication. Many aspects of these virus-host interactions remain poorly understood. Here, we employed the catRAPID algorithm and utilized the RNA-protein interaction detection coupled with mass spectrometry technology to predict and validate the host proteins that specifically bind to the highly structured 5' and 3' terminal regions of the SARS-CoV-2 RNA. Among the interactions identified, we prioritized pseudouridine synthase PUS7, which binds to both ends of the viral RNA. Using nanopore direct RNA sequencing, we discovered that the viral RNA undergoes extensive post-transcriptional modifications. Modified consensus regions for PUS7 were identified at both terminal regions of the SARS-CoV-2 RNA, including one in the viral transcription regulatory sequence leader. Collectively, our findings offer insights into host protein interactions with the SARS-CoV-2UTRs and highlight the likely significance of pseudouridine synthases and other post-transcriptional modifications in the viral life cycle. This new knowledge enhances our understanding of virus-host dynamics and could inform the development of targeted therapeutic strategies.

A single-dose of intranasal vaccination with a live-attenuated SARS-CoV-2 vaccine candidate promotes protective mucosal and systemic immunity

An attenuated SARS-CoV-2 virus with modified viral transcriptional regulatory sequences and deletion of open-reading frames 3, 6, 7 and 8 (∆3678) was previously reported to protect hamsters from SARS-CoV-2 infection and transmission. Here we report that a single-dose intranasal vaccination of ∆3678 protects K18-hACE2 mice from wild-type or variant SARS-CoV-2 challenge. Compared with wild-type virus infection, the ∆3678 vaccination induces equivalent or higher levels of lung and systemic T cell, B cell, IgA, and IgG responses. The results suggest ∆3678 as an attractive mucosal vaccine candidate to boost pulmonary immunity against SARS-CoV-2.

Comparative analysis of bioinformatics tools to characterize SARS-CoV-2 subgenomic RNAs.

During the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), positive-sense genomic RNA and subgenomic RNAs (sgRNAs) are synthesized by a discontinuous process of transcription characterized by a template switch, regulated by transcription-regulating sequences (TRS). Although poorly known about makeup and dynamics of sgRNAs population and function of its constituents, next-generation sequencing approaches with the help of bioinformatics tools have made a significant contribution to expand the knowledge of sgRNAs in SARS-CoV-2. For this scope to date, Periscope, LeTRS, sgDI-tector, and CORONATATOR have been developed. However, limited number of studies are available to compare the performance of such tools. To this purpose, we compared Periscope, LeTRS, and sgDI-tector in the identification of canonical (c-) and noncanonical (nc-) sgRNA species in the data obtained with the Illumina ARTIC sequencing protocol applied to SARS-CoV-2-infected Caco-2 cells, sampled at different time points. The three software showed a high concordance rate in the identification and in the quantification of c-sgRNA, whereas more differences were observed in nc-sgRNA. Overall, LeTRS and sgDI-tector result to be adequate alternatives to Periscope to analyze Fastq data from sequencing platforms other than Nanopore.

A novel chimeric vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) I and II elicits neutralizing antibodies against both types.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important virus within the swine industry. The virus causes respiratory disease and reproductive failure. Two species of PRRSV-I and II are co-dominant, yet no effective vaccination strategy has been developed to protect against these two types. With an aim to develop a chimeric vaccine strain to protect against both types, in this study, a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) type I and II was rescued using reverse genetics for the first time. Four chimeric infectious clones were designed based on the genomic arrangement of the structural proteins. However, only the clone carrying the transcriptional regulatory sequence (TRS) and ORF6 of a PRRSV-I and ORF6 of a PRRSV-II generated a viable recombinant virus, suggesting that concurrent expression of ORF6 from both parental viruses is essential for the recovery of type I and II chimeric PRRSV. The chimeric virus showed significantly lower replication ability than its parental strains in vitro, which was improved by serial passaging. In vivo, groups of pigs were inoculated with either the chimeric virus, one of the parental strains, or PBS. The chimeric virus replicated in pig tissue and was detected in serum 7 days post-inoculation. Serum neutralization tests indicated that pigs inoculated with the chimeric virus elicited neutralizing antibodies that inhibited infection with strains of both species and with greater coverage than the parental viruses. In conclusion, the application of this technique to construct a chimeric PRRSV holds promise for the development of a highly effective modified live vaccine candidate. This is particularly significant since there are currently no approved commercial divalent vaccines available to combat PRRSV-I and II co-infections.

ALAN as an omic tool to build gene signatures and to stratify patients.

1563 Background: Understanding aberrant gene behavior is critical towards devising clinical therapeutics or gene based diagnostic panels. Clustering and AI/ML have clinical utility, but they do not contextualize changes in gene behavior that result from differences in cell type, microenvironment, or prior treatments. Here, we developed the Algorithm for Linking Activity Networks (ALAN), an informatics algorithm that allows rapid functional comparisons of all detectable genes between clinically annotated patient cohorts. ALAN outputs quantify gene behavior without any additional inputs and can be applied on all forms of clinical omic data. We tested the utility of ALAN to identify the molecular features of resistance and novel precision therapeutics. Methods: We applied the ALAN algorithm in prostate tissue using whole-transcriptome sequencing (WTS) data from SU2C/PCF (n = 209, metastatic prostate cancer), TCGA (n = 493, primary prostate cancer), and GTEX (n = 245, benign prostate tissue). We conducted informatics analysis of whole-exome sequencing (WES) data from SU2C/PCF (n = 444 mPC), Fred Hutch (n = 130 mPC), and MCTP (n = 61 mPC) to identify mPC patients with amplifications in RSPO2. We scored an ALAN gene signature in a patient with single-cell RNA sequencing pre and post AR inhibition (n = 112, 83). We examined ChIP-seq data to evaluate binding of AR and Co-factors at the CD276 transcription regulatory sequences. Results: In mPCs, ALAN unbiasedly identified genes that prior studies have implicated as resistance mechanisms towards enzalutamide. In this process, ALAN nominated RSPO2, a secreted protein, as a potential resistant gene that has never been targeted in mPC. In recent studies, we found RSPO2 amplifications in 19.7-28.5% of mPC. We utilized ALAN outputs to build a novel gene signature that was exclusively enriched (p-value < 0.0001) in the transcriptome collected from single cells of a post-resistant mPC sample. We also utilized ALAN to stratify the optimal mPC patients in receiving precision therapies against B7-H3 ( CD276). B7-H3 is an immune regulatory molecule in the B7 superfamily that includes PD-L1 and CTLA-4 and has not been targeted in mPC. B7-H3 was unlike other B7 family members and exhibited robust association with AR co-factors and AR signaling. We confirmed ALAN findings as multiple B7-H3 transcriptional enhancers and promoters were bound by AR and AR-cofactors. Conclusions: ALAN is an algorithm that can be readily deployed on existing clinical omic data to build gene signatures, identify resistant genes, and risk stratify patients. ALAN nominated a novel therapeutic target, RSPO2, which is recurrently amplified in mPC patients. ALAN predicted that mPCs with high baseline levels of AR activity would be optimal candidates for B7-H3 precision therapeutics. Altogether, ALAN outputs should be considered for making clinical decisions that would benefit by comprehensive comparisons of all detectable genes.

Metatranscriptomics analysis reveals a novel transcriptional and translational landscape during Middle East respiratory syndrome coronavirus infection

Among all RNA viruses, coronavirus RNA transcription is the most complex and involves a process termed "discontinuous transcription" that results in the production of a set of 3'-nested, co-terminal genomic and subgenomic RNAs during infection. While the expression of the classic canonical set of subgenomic RNAs depends on the recognition of a 6- to 7-nt transcription regulatory core sequence (TRS), here, we use deep sequence and metagenomics analysis strategies and show that the coronavirus transcriptome is even more vast and more complex than previously appreciated and involves the production of leader-containing transcripts that have canonical and noncanonical leader-body junctions. Moreover, by ribosome protection and proteomics analyses, we show that both positive- and negative-sense transcripts are translationally active. The data support the hypothesis that the coronavirus proteome is much vaster than previously noted in the literature.

Selective translational control of cellular and viral mRNAs by RPS3 mRNA binding.

RPS3, a universal core component of the 40S ribosomal subunit, interacts with mRNA at the entry channel. Whether RPS3 mRNA-binding contributes to specific mRNA translation and ribosome specialization in mammalian cells is unknown. Here we mutated RPS3 mRNA-contacting residues R116, R146 and K148 and report their impact on cellular and viral translation. R116D weakened cap-proximal initiation and promoted leaky scanning, while R146D had the opposite effect. Additionally, R146D and K148D displayed contrasting effects on start-codon fidelity. Translatome analysis uncovered common differentially translated genes of which the downregulated set bears long 5'UTR and weak AUG context, suggesting a stabilizing role during scanning and AUG selection. We identified an RPS3-dependent regulatory sequence (RPS3RS) in the sub-genomic 5'UTR of SARS-CoV-2 consisting of a CUG initiation codon and a downstream element that is also the viral transcription regulatory sequence (TRS). Furthermore, RPS3 mRNA-binding residues are essential for SARS-CoV-2 NSP1-mediated inhibition of host translation and for its ribosomal binding. Intriguingly, NSP1-induced mRNA degradation was also reduced in R116D cells, indicating that mRNA decay occurs in the ribosome context. Thus, RPS3 mRNA-binding residues have multiple translation regulatory functions and are exploited by SARS-CoV-2 in various ways to influence host and viral mRNA translation and stability.

One toolkit to bring them all, and in silico analyze them.

One toolkit to bring them all, and in silico analyze them.

Regulation of adhesin synthesis in Aggregatibacter actinomycetemcomitans.

Aggregatibacter actinomycetemcomitans is a gram-negative bacterium associated with periodontal disease and a variety of disseminated extra-oral infections. Tissue colonization is mediated by fimbriae and non-fimbriae adhesins resulting in the formation of a sessile bacterial community or biofilm, which confers enhanced resistance to antibiotics and mechanical removal. The environmental changes experienced by A. actinomycetemcomitans during infection are detected and processed by undefined signaling pathways that alter gene expression. In this study, we have characterized the promoter region of the extracellular matrix protein adhesin A (EmaA), which is an important surface adhesin in biofilm biogenesis and disease initiation using a series of deletion constructs consisting of the emaA intergenic region and a promotor-less lacZ sequence. Two regions of the promoter sequence were found to regulate gene transcription and in silico analysis indicated the presence of multiple transcriptional regulatory binding sequences. Analysis of four regulatory elements, CpxR, ArcA, OxyR, and DeoR, was undertaken in this study. Inactivation of arcA, the regulator moiety of the ArcAB two-component signaling pathway involved in redox homeostasis, resulted in a decrease in EmaA synthesis and biofilm formation. Analysis of the promoter sequences of other adhesins identified binding sequences for the same regulatory proteins, which suggests that these proteins are involved in the coordinate regulation of adhesins required for colonization and pathogenesis.

Simultaneous expression of three reporter proteins from a porcine reproductive and respiratory syndrome virus-based vector

The mechanism of discontinuous transcription for the synthesis of a series of sub-genomic mRNAs to express the structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) potentially allows for the simultaneous expression of multiple foreign genes. This can occur by insertion of multiple novel independent transcription units between the ORF sequences of the PRRSV genome. Here, an expression cassette consisting of a red fluorescent protein (RFP) gene flanked at its 3′ end by transcription-regulating sequences (TRS) and an expression cassette consisting of an iLOV gene flanked at its 5′ end by TRS, was constructed. The resulting expression cassette containing a RFP and an iLOV gene were introduced between ORF1b and 2 as well as ORF7 and 3′UTR, respectively, in an infectious PRRSV cDNA clone. Transfection of the resulting clone (pGX-12RFP-73iLOV) into cells resulted in the recovery of a recombinant virus (rGX-12RFP-73iLOV). Simultaneous expression of RFP and iLOV was observed in MARC-145 cells infected with rGX-RFP-iLOV. To test the ability of the PRRSV genome to express all three reporter genes simultaneously, an expression cassette containing the Gluc gene and one containing the iLOV gene were also inserted in between ORF1b and 2 as well as ORF7 and 3′UTR, respectively. This was performed in a recently obtained infectious PRRSV cDNA clone carrying a RFP gene in nsp2. Transfection of the construct (pGX-R-Gluc-iLOV) carrying the three reporter genes into cells allowed the rescue of the recombinant reporter virus (rGX-R-Gluc-iLOV) which showed similar growth characteristics to the parental virus but yielded 100-fold less infectious viruses. Fluorescence microscopy of cells infected with rGX-R-Gluc-iLOV demonstrated the presence of both RFP and iLOV genes. Gluc activities in supernatants harvested at different time points from cells infected with recombinant viruses carrying Gluc showed increased levels of Gluc activity as the infection progressed. This indicated that Gluc gene as well as its activity were acceptable parameters to monitor viral propagation. Our results indicate that it is possible to introduce at least three foreign proteins simultaneously in a PRRSV-based vector and such studies will prove invaluable in our future understanding of these viruses.

Perceptions into causes and consequences of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) variants are emerging worldwide and pathogenicity varies widely from no symptoms to death. The SARS‐CoV‐2 is evolving as lineages like Alpha, Beta, Gamma, Epsilon, Iota, Delta, and Omicron in the course of time. The main reasons for such viral evolution are (a) the imperfect nature of SARS‐CoV‐2 RNA polymerase, and viral exonuclease mediated proofreading functions resulting in the generation of mutations in viral genomes; (b) fusions of the 5′ leader sequence to unexpected 3′ sites, and transcription regulatory sequences (TRSs) in subgenomic RNAs (sgRNAs), which result in the generation of structural variants and novel open reading frames; (c) these viruses are combated by the host type I interferons (IFNs). In such a process IFNs upregulate viral RNA editing APOBEC3G/F and ADAR1 genes, which induce mutations in viral genomes. These factors play important roles in causing viral evolution and the emergence of more efficient SARS‐CoV‐2 genomes, which escape the host immune defense system, and vaccine‐elicited antibodies and impede therapeutic strategies. The main challenges we now face are how to control future SARS‐CoV‐2 evolution, the elimination of their deleterious side effects, and the onset of new diseases as aftermaths of SARS‐CoV‐2 infections. Preventive measures like (a) the development of broadly neutralizing antibodies and novel vaccines, therapies based on genomics and proteomics data will help in avoiding, and/or minimizing SARS‐CoV‐2 infections; (b) targeted therapies, application of patient‐based precision medicine methodology can help in achieving the goal and avoiding unwanted deleterious side effects and the onset of SARS‐CoV‐2 infections mediated several diseases in future.

Intragenomic rearrangements involving 5′-untranslated region segments in SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses

BackgroundVariation of the betacoronavirus SARS-CoV-2 has been the bane of COVID-19 control. Documented variation includes point mutations, deletions, insertions, and recombination among closely or distantly related coronaviruses. Here, we describe yet another aspect of genome variation by beta- and alphacoronaviruses that was first documented in an infectious isolate of the betacoronavirus SARS-CoV-2, obtained from 3 patients in Hong Kong that had a 5′-untranslated region segment at the end of the ORF6 gene that in its new location translated into an ORF6 protein with a predicted modified carboxyl terminus. While comparing the amino acid sequences of translated ORF8 genes in the GenBank database, we found a subsegment of the same 5′-UTR-derived amino acid sequence modifying the distal end of ORF8 of an isolate from the United States and decided to carry out a systematic search.MethodsUsing the nucleotide and in the case of SARS-CoV-2 also the translated amino acid sequence in three reading frames of the genomic termini of coronaviruses as query sequences, we searched for 5′-UTR sequences in regions other than the 5′-UTR in SARS-CoV-2 and reference strains of alpha-, beta-, gamma-, and delta-coronaviruses.ResultsWe here report numerous genomic insertions of 5′-untranslated region sequences into coding regions of SARS-CoV-2, other betacoronaviruses, and alphacoronaviruses, but not delta- or gammacoronaviruses. To our knowledge this is the first systematic description of such insertions. In many cases, these insertions would change viral protein sequences and further foster genomic flexibility and viral adaptability through insertion of transcription regulatory sequences in novel positions within the genome. Among human Embecorivus betacoronaviruses, for instance, from 65% to all of the surveyed sequences in publicly available databases contain inserted 5′-UTR sequences.ConclusionThe intragenomic rearrangements involving 5′-untranslated region sequences described here, which in several cases affect highly conserved genes with a low propensity for recombination, may underlie the generation of variants homotypic with those of concern or interest and with potentially differing pathogenic profiles. Intragenomic rearrangements thus add to our appreciation of how variants of SARS-CoV-2 and other beta- and alphacoronaviruses may arise.

Identification of consensus hairpin loop structure among the negative sense subgenomic RNAs of SARS-CoV-2

BackgroundSARS-CoV-2 is the causative agent of worldwide pandemic disease coronavirus disease 19. SARS-CoV-2 bears positive sense RNA genome that has organized and complex pattern of replication/transcription process including the generation of subgenomic RNAs. Transcription regulatory sequences have important role in the pausing of replication/transcription and generation of subgenomic RNAs.ResultsIn the present bioinformatics analysis, a consensus secondary structure was identified among negative sense subgenomic RNAs of SARS-CoV-2. This consensus region is present at the adjacent of initiation codon.ConclusionsThis study proposed that consensus structured domain could involve in mediating the long pausing of replication/transcription complex and responsible for subgenomic RNA production.

Physiological concentrations of glucocorticoids induce pathological DNA double-strand breaks.

Steroid hormones induce the transcription of target genes by activating nuclear receptors. Early transcriptional response to various stimuli, including hormones, involves the active catalysis of topoisomerase II (TOP2) at transcription regulatory sequences. TOP2 untangles DNAs by transiently generating double-strand breaks (DSBs), where TOP2 covalently binds to DSB ends. When TOP2 fails to rejoin, called "abortive" catalysis, the resulting DSBs are repaired by tyrosyl-DNA phosphodiesterase 2 (TDP2) and non-homologous end-joining (NHEJ). A steroid, cortisol, is the most important glucocorticoid, and dexamethasone (Dex), a synthetic glucocorticoid, is widely used for suppressing inflammation in clinics. We here revealed that clinically relevant concentrations of Dex and physiological concentrations of cortisol efficiently induce DSBs in G1 phase cells deficient in TDP2 and NHEJ. The DSB induction depends on glucocorticoid receptor (GR) and TOP2. Considering the specific role of TDP2 in removing TOP2 adducts from DSB ends, induced DSBs most likely represent stalled TOP2-DSB complexes. Inhibition of RNA polymerase II suppressed the DSBs formation only modestly in the G1 phase. We propose that cortisol and Dex frequently generate DSBs through the abortive catalysis of TOP2 at transcriptional regulatory sequences, including promoters or enhancers, where active TOP2 catalysis occurs during early transcriptional response.