The Coronavirus Replication-Transcription Complex.

Replication of the coronavirus genome requires continuous RNA synthesis, whereas transcription is a discontinuous process unique among RNA viruses. Transcription includes a template switch during the synthesis of subgenomic negative-strand RNAs to add a copy of the leader sequence. Coronavirus transcription is regulated by multiple factors, including the extent of base-pairing between transcription-regulating sequences of positive and negative polarity, viral and cell protein-RNA binding, and high-order RNA-RNA interactions. Coronavirus RNA synthesis is performed by a replication-transcription complex that includes viral and cell proteins that recognize cis-acting RNA elements mainly located in the highly structured 5' and 3' untranslated regions. In addition to many viral nonstructural proteins, the presence of cell nuclear proteins and the viral nucleocapsid protein increases virus amplification efficacy. Coronavirus RNA synthesis is connected with the formation of double-membrane vesicles and convoluted membranes. Coronaviruses encode proofreading machinery, unique in the RNA virus world, to ensure the maintenance of their large genome size.

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.

SARS-coronavirus (SARS-CoV) replication and transcription are mediated by a replication/transcription complex (RTC) of which virus-encoded, non-structural proteins (nsps) are the primary constituents. The 16 SARS-CoV nsps are produced by autoprocessing of two large precursor polyproteins. The RTC is believed to be associated with characteristic virus-induced double-membrane structures in the cytoplasm of SARS-CoV-infected cells. To investigate the link between these structures and viral RNA synthesis, and to dissect RTC organization and function, we isolated active RTCs from infected cells and used them to develop the first robust assay for their in vitro activity. The synthesis of genomic RNA and all eight subgenomic mRNAs was faithfully reproduced by the RTC in this in vitro system. Mainly positive-strand RNAs were synthesized and protein synthesis was not required for RTC activity in vitro. All RTC activity, enzymatic and putative membrane-spanning nsps, and viral RNA cosedimented with heavy membrane structures. Furthermore, the pelleted RTC required the addition of a cytoplasmic host factor for reconstitution of its in vitro activity. Newly synthesized subgenomic RNA appeared to be released, while genomic RNA remained predominantly associated with the RTC-containing fraction. RTC activity was destroyed by detergent treatment, suggesting an important role for membranes. The RTC appeared to be protected by membranes, as newly synthesized viral RNA and several replicase/transcriptase subunits were protease- and nuclease-resistant and became susceptible to degradation only upon addition of a non-ionic detergent. Our data establish a vital functional dependence of SARS-CoV RNA synthesis on virus-induced membrane structures.

The SARS-Coronavirus PLnc domain of nsp3 as a replication/transcription scaffolding protein

Coronavirus, with a 31-kb RNA genome, replicates its own RNA and transcribes subgenomic mRNAs by complex mechanisms. Viral RNA synthesis is regulated by multiple RNA regions, which appear to interact either directly or indirectly. Multiple cellular proteins bind to these regions and may undergo additional protein-protein interactions. These findings suggest that coronavirus RNA synthesis is carried out on a ribonucleoprotein via a mechanism that involves both viral and cellular proteins associated with viral RNA, similar to DNA-dependent RNA transcription. This mode of RNA synthesis may be applicable to most RNA viruses.

The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle.

Background: Coronaviruses are a group of related viruses that cause diseases in mammals and birds. In humans, coronaviruses cause respiratory tract infections that can range from mild to lethal. Mild illnesses include some cases of the common cold, while more lethal varieties can cause SARS, MERS, and COVID-19. The outbreak was identified in Wuhan, China, in December 2019, declared to be a Public Health Emergency of International Concern on 30 January 2020, and recognized as a pandemic on 11 March 2020. Introduction: Coronaviruses are the subfamily Orthocoronavirinae, within the family of Coronaviridae; order Nidovirales, and realm Riboviria. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses is approximately from 26 to 32kilobases. Coronaviruses were first discovered in the 1930s and Human coronaviruses were discovered in the 1960s. The earliest ones studied were from human patients with the common cold, which were later named human coronavirus 229E and human coronavirus OC43. Other human coronaviruses have since been identified, including SARS-CoV in 2003, HCoV NL63 in 2004, HKU1 in 2005, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Most of these have involved serious respiratory tract infections. Discussions & Results: Based on our multidisciplinary research, we have found the major cause and some treatments methods for fighting this powerful pathogen. The prime cause of COVID-19 is pushing the mitochondrial to lose MMP. A loss of the MMP by any mechanism leads to functional and structural collapse of the mitochondria and cell death. Mitophagy plays an important role in maintaining mitochondrial homeostasis, but can also eliminate healthy mitochondria in cases such as cell starvation, viral invasion, and erythroid cell differentiation. The mitochondrial fusion and fission are highly dynamic. Viruses specially COVID-19, interfere with these processes to distort mitochondrial dynamic to facilitate their proliferation. Thus, interfering with these processes promotes the interference of different cellular signaling pathways. The severe acute respiratory syndrome coronavirus (SARS-CoV) escapes the innate immune response by translocating its ORF-9b to mitochondria and promotes proteosomal degradation of dynamin-like protein (Drp1) leading to mitochondrial fission. We also researched on Ultrasonic Energy to destroy the virus which leads to positive results but it needs more future research. The most destructive way of viruses is to enhance Reactive Oxygen Species (ROS) and free radicals in human contaminated cell which cause inflammation in a host cell. ELF-EMF converts free radicals into less active molecules and eliminates them into two pathways which have been discussed in the discussion part. Using ELF-EMF affects the second pathway that relies on the activity of the catalase and superoxide dismutase enzymes which is the most effective pathway. For the best result of treatment, is the use of lowfrequency magnetic fields (LFMF) plus EMF-ELF which penetrate into deeper tissues, cells and mitochondria. We also have gone through many researches since 1920 and found if we emit the frequency as the same frequency of COVID-19, can cause resonance in the virus and destroy it. So we measured the SARS-CoV-2 frequency by Cyclotron and calculated the frequencyof the virus is 30 KHz-500 KHz. Conclusion: COVID-19 (SARS-CoV-2) is one of the most complex viruses which have been discovered since 2020. Until today, there has been no Antiviral Drug which can be useful in the treatment of this infectious disease has been discovered till today. COVID-19 genomic sequence containing SARS-CoV, MERS-CoV and Influenza A. Therefore; there is a high possibility of continuing COVID-19 even in summer. To gain the best result in treatment, we should use low-frequency magnetic fields (LFMF) plus EMF which penetrate into deeper tissues, cells and mitochondria in order to reduce ROS and Inflammation. In order to destroy SARS-CoV-2 virus in environment and also in infected individuals, we should use ELF-EMF plus LFMF. We also have gone through many researches since 1920 and found if we emit the frequency as the same frequency of COVID-19, it can cause resonance in the virus and destroy it. So we measured the SARS-CoV-2 frequency by Cyclotron and calculated the frequency of the virus that id is 30 KHz-500 KHz. The differences in the frequencies are due to the size of the virus which is from 26 to 32 Kilobases.

Several host proteins have been identified that bind to the cis-acting sequences of mouse hepatitis virus (MHV) RNAs (Furuya and Lai, 1993; Yu and Leibowitz, 1995; Zhang and Lai, 1995; Li et al., 1997, 1999). The first among those is the heterogeneous nuclear ribonucleoprotein (hnRNP) Al. In normal mammalian cells, hnRNP-Al is predominantly localized in the nucleus, but it shuttles between the nucleus and the cytoplasm (Dreyfuss et al., 1993). It interacts with a number of RNAs, particularly pre-mRNA and snRNAs, and a number of proteins such as itself, other hnRNPs and some serine-arginine (SR)-rich proteins through its amino terminal RNA-binding domains and the carboxyl terminal glycine-rich domain, respectively, and thus is associated with the RNP complex (Dreyfuss et al. 1993; Buvoli et al., 1988; Cartegni et al., 1996). hnRNP A1 has been implicated in promoting RNA-reannealing (Buvoli et al., 1992) and in regulating alternative RNA-splicing (Dreyfuss et al., 1993). Its involvement in regulation of viral RNA processing of human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV)-2 has also been suggested (Black et al, 1995, 1996). Recent evidence indicates that hnRNP Al is required for inhibition of HIV-1 pre-mRNA splicing (Caputi et al., 1999). Previously, we found that hnRNP Al specifically binds to the cis-acting sequences (the negative-strand leader and intergenic [IG] sequence) of MHV RNA (Furuya and Lai, 1993; Zhang and Lai, 1995). Site-directed mutagenesis of the IG sequence indicated that the extent of mRNA transcription correlates with the efficiency of hnRNP A1-binding to the IG sequence (Zhang and Lai, 1995). In MHV-infected cells, hnRNP A1 is translocated from the nucleus to the cytoplasm (Li et al., 1997). These findings suggest that hnRNP Al may play a role in regulation of MHV RNA replication and transcription. However, to date, no direct evidence for a functional role of hnRNP Al in MHV RNA synthesis has been reported.KeywordsHelper VirusRecombinant Vaccinia VirusHeterogeneous Nuclear RibonucleoproteinMouse Hepatitis VirusIntracellular Viral RNAsThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Coronaviruses are important human and animal pathogens causing respiratory and gastrointestinal infections ranging from mild to severe. Two highly pathogenic human coronaviruses with high fatality rates, SARS‐CoV and MERS‐CoV, have emerged in different parts of the world in the recent past causing major public health concerns. Several unique features of coronaviruses make them likely the candidates for zoonotic transmissions into the human population: (1) their exceptionally large RNA genomes allow for increased plasticity and diversity, (2) their presence in mammalian (bat) and avian reservoir species with specialised immune functions and increased mobility facilitates their geographical spread and (3) their sophisticated molecular mechanisms of viral entry, replication and assembly involving a number of poorly studied viral accessory proteins complicates the development of suitable therapeutic strategies. Key Concepts Coronaviruses contain the largest known viral RNA genomes. Bats and birds are major reservoirs for coronaviruses. Two highly pathogenic human coronaviruses, SARS‐CoV and MERS‐CoV, have emerged since the early 2000s. Coronavirus spike proteins are activated by a variety of proteases, often in a two‐step proteolysis process. Many coronaviruses utilise exopeptidases as primary cellular receptors.

Coronaviruses (CoV) encode sixteen non-structural proteins (nsps), most of which form the replication-transcription complex (RTC). The RTC contains a core composed of one nsp12 RNA-dependent RNA polymerase (RdRp), two nsp8s and one nsp7. The core RTC recruits other nsps to synthesize all viral RNAs within the infected cell. While essential for viral replication, the mechanism by which the core RTC assembles into a processive polymerase remains poorly understood. We show that the core RTC preferentially assembles by first having nsp12-polymerase bind to the RNA template, followed by the subsequent association of nsp7 and nsp8. Once assembled on the RNA template, the core RTC requires hundreds of seconds to undergo a conformational change that enables processive elongation. In the absence of RNA, the (apo-)RTC requires several hours to adopt its elongation-competent conformation. We propose that this obligatory activation step facilitates the recruitment of additional nsp’s essential for efficient viral RNA synthesis and may represent a promising target for therapeutic interventions.

Coronaviruses: Molecular Biology

Coronaviruses: Molecular Biology (Coronaviridae)

Porcine Epidemic Diarrhea Virus Variants with High Pathogenicity, China

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus from the Nidovirales order that causes reproductive failure and respiratory disease in pigs and poses a constant threat to the global pig industry. The PRRSV-encoded nonstructural protein 11 (nsp11) is a nidovirus-specific endoribonuclease (NendoU) that is conserved throughout the Arteriviridae and Coronaviridae families. Previously, our research and that of others demonstrated that PRRSV nsp11 inhibits type I interferon (IFN) production through NendoU activity-dependent mechanisms. Here, we found that PRRSV nsp11 also inhibited IFN-stimulated response element (ISRE) promoter activity and subsequent transcription of IFN-stimulated genes (ISGs). Detailed analysis showed that nsp11 targeted interferon regulatory factor 9 (IRF9), but not transducer and activator of transcription 1 (STAT1) or STAT2, key molecules in the type I IFN signaling pathway. Furthermore, the nsp11-IRF9 interaction impaired the formation and nuclear translocation of the transcription factor complex IFN-stimulated gene factor 3 (ISGF3) in both nsp11-overexpressed and PRRSV-infected cells. Importantly, nsp11 mutations (H129A, H144A, and K173A) that ablate NendoU activity or its cell cytotoxicity also interacted with IRF9 and retained the ability to block IFN signaling, indicating that the nsp11-IRF9 interaction is independent of NendoU activity or cell cytotoxicity of nsp11. Taking the results together, our study demonstrated that PRRSV nsp11 antagonizes type I IFN signaling by targeting IRF9 via a NendoU activity-independent mechanism, and this report describes a novel strategy evolved by PRRSV to counteract host innate antiviral responses, revealing a potential new function for PRRSV nsp11 in type I IFN signaling.IMPORTANCE The nidovirus-specific endoribonuclease (NendoU) encoded by PRRSV nonstructural protein 11 (nsp11) is a unique NendoU of nidoviruses that infect vertebrates; thus, it is an attractive target for the development of antinidovirus drugs. Previous studies have revealed that the NendoU of nidoviruses, including porcine reproductive and respiratory syndrome virus (PRRSV) and human coronavirus 229E (HCoV-229E), acts as a type I interferon (IFN) antagonist. Here, for the first time, we demonstrated that overexpression of PRRSV nsp11 also inhibits IFN signaling by targeting the C-terminal interferon regulatory factor (IRF) association domain of IRF9. This interaction impaired the ability of IRF9 to form the transcription factor complex IFN-stimulated gene factor 3 (ISGF3) and to act as a signaling protein of IFN signaling. Collectively, our data identify IRF9 as a natural target of PRRSV NendoU and reveal a novel mechanism evolved by an arterivirus to counteract innate immune signaling.

Mature mRNAs of Trypanosoma brucei have a common 5' terminal sequence of 35 nucleotides. This is acquired by an unknown mechanism from the 5' end of a separately transcribed precursor RNA of about 140 nt called the mini-exon-derived RNA or medRNA. We have investigated the nature of the 5' ends of mature mRNAs and of the medRNA by chemical decapping and enzymic recapping. We infer that a 5' cap is present on both of these RNAs and conclude that the mini-exon-derived RNA donates its 5' cap along with the mini-exon sequence to the pre-mRNA. Using nuclear run-on experiments we show that medRNA synthesis is much more sensitive to alpha-amanitin than 5S RNA synthesis and only slightly less sensitive than tubulin gene transcription. This result, together with the presence of a cap at the 5' end of the medRNA indicates that the mini-exon is transcribed by an RNA polymerase II type enzyme. Our experiments also confirm the existence of a second minor medRNA of about 125 nt and show the presence of other small capped RNAs possibly analogous to the small nuclear RNAs of other organisms.