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1.
Methods Mol Biol ; 2203: 187-204, 2020.
Article in English | MEDLINE | ID: covidwho-761353

ABSTRACT

Biotin-based proximity labeling circumvents major pitfalls of classical biochemical approaches to identify protein-protein interactions. It consists of enzyme-catalyzed biotin tags ubiquitously apposed on proteins located in close proximity of the labeling enzyme, followed by affinity purification and identification of biotinylated proteins by mass spectrometry. Here we outline the methods by which the molecular microenvironment of the coronavirus replicase/transcriptase complex (RTC), i.e., proteins located within a close perimeter of the RTC, can be determined by different proximity labeling approaches using BirAR118G (BioID), TurboID, and APEX2. These factors represent a molecular signature of coronavirus RTCs and likely contribute to the viral life cycle, thereby constituting attractive targets for the development of antiviral intervention strategies.


Subject(s)
Coronavirus/pathogenicity , Enzymes/genetics , Host-Pathogen Interactions/physiology , Proteomics/methods , Viral Proteins/metabolism , Animals , Ascorbate Peroxidases/genetics , Biotinylation , Carbon-Nitrogen Ligases/genetics , Cell Line , Coronavirus/genetics , Enzymes/metabolism , Escherichia coli Proteins/genetics , Fluorescent Antibody Technique , Microorganisms, Genetically-Modified , Repressor Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics
2.
J Virol ; 94(12)2020 06 01.
Article in English | MEDLINE | ID: covidwho-736044

ABSTRACT

The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that recently emerged in China is thought to have a bat origin, as its closest known relative (BatCoV RaTG13) was described previously in horseshoe bats. We analyzed the selective events that accompanied the divergence of SARS-CoV-2 from BatCoV RaTG13. To this end, we applied a population genetics-phylogenetics approach, which leverages within-population variation and divergence from an outgroup. Results indicated that most sites in the viral open reading frames (ORFs) evolved under conditions of strong to moderate purifying selection. The most highly constrained sequences corresponded to some nonstructural proteins (nsps) and to the M protein. Conversely, nsp1 and accessory ORFs, particularly ORF8, had a nonnegligible proportion of codons evolving under conditions of very weak purifying selection or close to selective neutrality. Overall, limited evidence of positive selection was detected. The 6 bona fide positively selected sites were located in the N protein, in ORF8, and in nsp1. A signal of positive selection was also detected in the receptor-binding motif (RBM) of the spike protein but most likely resulted from a recombination event that involved the BatCoV RaTG13 sequence. In line with previous data, we suggest that the common ancestor of SARS-CoV-2 and BatCoV RaTG13 encoded/encodes an RBM similar to that observed in SARS-CoV-2 itself and in some pangolin viruses. It is presently unknown whether the common ancestor still exists and, if so, which animals it infects. Our data, however, indicate that divergence of SARS-CoV-2 from BatCoV RaTG13 was accompanied by limited episodes of positive selection, suggesting that the common ancestor of the two viruses was poised for human infection.IMPORTANCE Coronaviruses are dangerous zoonotic pathogens; in the last 2 decades, three coronaviruses have crossed the species barrier and caused human epidemics. One of these is the recently emerged SARS-CoV-2. We investigated how, since its divergence from a closely related bat virus, natural selection shaped the genome of SARS-CoV-2. We found that distinct coding regions in the SARS-CoV-2 genome evolved under conditions of different degrees of constraint and are consequently more or less prone to tolerate amino acid substitutions. In practical terms, the level of constraint provides indications about which proteins/protein regions are better suited as possible targets for the development of antivirals or vaccines. We also detected limited signals of positive selection in three viral ORFs. However, we warn that, in the absence of knowledge about the chain of events that determined the human spillover, these signals should not be necessarily interpreted as evidence of an adaptation to our species.


Subject(s)
Betacoronavirus/genetics , Evolution, Molecular , Selection, Genetic , Amino Acid Sequence , Animals , Betacoronavirus/classification , Chiroptera/virology , Coronavirus Infections/virology , Genome, Viral/genetics , Humans , Models, Molecular , Open Reading Frames/genetics , Pandemics , Phylogeny , Pneumonia, Viral/virology , Viral Proteins/chemistry , Viral Proteins/genetics
3.
Virol J ; 17(1): 131, 2020 08 27.
Article in English | MEDLINE | ID: covidwho-733041

ABSTRACT

BACKGROUND: The Covid19 infection is caused by the SARS-CoV-2 virus, a novel member of the coronavirus (CoV) family. CoV genomes code for a ORF1a / ORF1ab polyprotein and four structural proteins widely studied as major drug targets. The genomes also contain a variable number of open reading frames (ORFs) coding for accessory proteins that are not essential for virus replication, but appear to have a role in pathogenesis. The accessory proteins have been less well characterized and are difficult to predict by classical bioinformatics methods. METHODS: We propose a computational tool GOFIX to characterize potential ORFs in virus genomes. In particular, ORF coding potential is estimated by searching for enrichment in motifs of the X circular code, that is known to be over-represented in the reading frames of viral genes. RESULTS: We applied GOFIX to study the SARS-CoV-2 and related genomes including SARS-CoV and SARS-like viruses from bat, civet and pangolin hosts, focusing on the accessory proteins. Our analysis provides evidence supporting the presence of overlapping ORFs 7b, 9b and 9c in all the genomes and thus helps to resolve some differences in current genome annotations. In contrast, we predict that ORF3b is not functional in all genomes. Novel putative ORFs were also predicted, including a truncated form of the ORF10 previously identified in SARS-CoV-2 and a little known ORF overlapping the Spike protein in Civet-CoV and SARS-CoV. CONCLUSIONS: Our findings contribute to characterizing sequence properties of accessory genes of SARS coronaviruses, and especially the newly acquired genes making use of overlapping reading frames.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , Open Reading Frames , SARS Virus/genetics , Viral Regulatory and Accessory Proteins/genetics , Animals , Codon , Computational Biology , Evolution, Molecular , Genes, Viral , Humans , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Viral Matrix Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Regulatory and Accessory Proteins/chemistry
4.
PLoS Pathog ; 16(8): e1008718, 2020 08.
Article in English | MEDLINE | ID: covidwho-717612

ABSTRACT

APOBEC3 enzymes are innate immune effectors that introduce mutations into viral genomes. These enzymes are cytidine deaminases which transform cytosine into uracil. They preferentially mutate cytidine preceded by thymidine making the 5'TC motif their favored target. Viruses have evolved different strategies to evade APOBEC3 restriction. Certain viruses actively encode viral proteins antagonizing the APOBEC3s, others passively face the APOBEC3 selection pressure thanks to a depleted genome for APOBEC3-targeted motifs. Hence, the APOBEC3s left on the genome of certain viruses an evolutionary footprint. The aim of our study is the identification of these viruses having a genome shaped by the APOBEC3s. We analyzed the genome of 33,400 human viruses for the depletion of APOBEC3-favored motifs. We demonstrate that the APOBEC3 selection pressure impacts at least 22% of all currently annotated human viral species. The papillomaviridae and polyomaviridae are the most intensively footprinted families; evidencing a selection pressure acting genome-wide and on both strands. Members of the parvoviridae family are differentially targeted in term of both magnitude and localization of the footprint. Interestingly, a massive APOBEC3 footprint is present on both strands of the B19 erythroparvovirus; making this viral genome one of the most cleaned sequences for APOBEC3-favored motifs. We also identified the endemic coronaviridae as significantly footprinted. Interestingly, no such footprint has been detected on the zoonotic MERS-CoV, SARS-CoV-1 and SARS-CoV-2 coronaviruses. In addition to viruses that are footprinted genome-wide, certain viruses are footprinted only on very short sections of their genome. That is the case for the gamma-herpesviridae and adenoviridae where the footprint is localized on the lytic origins of replication. A mild footprint can also be detected on the negative strand of the reverse transcribing HIV-1, HIV-2, HTLV-1 and HBV viruses. Together, our data illustrate the extent of the APOBEC3 selection pressure on the human viruses and identify new putatively APOBEC3-targeted viruses.


Subject(s)
Cytidine Deaminase/metabolism , Genome, Viral/genetics , Host-Pathogen Interactions/genetics , Selection, Genetic/genetics , Virus Replication/genetics , Coronaviridae/genetics , Humans , Immunity, Innate/immunology , Papillomaviridae/genetics , Parvoviridae/genetics , Polyomaviridae/genetics , Viral Proteins/genetics
5.
Clin Immunol ; 219: 108572, 2020 10.
Article in English | MEDLINE | ID: covidwho-713545

ABSTRACT

Human Leukocyte Antigen (HLA) includes a large set of genes with important actions in immune response against viral infection. Numerous studies have revealed the existence of significant associations between certain HLA alleles and the susceptibility and prognosis of different infectious diseases. In this pilot study we analyse the binding affinity between 66 class I HLA alleles and SARS-CoV-2 viral peptides, and its association with the severity of the disease. A total of 45 Spanish patients with mild, moderate and severe SARS-CoV-2 infection were typed for HLA class I; after that, we analysed if an in silico model of HLA I-viral peptide binding affinity and classical HLA supertypes could be correlated to the severity of the disease. Our results suggest that patients with mild disease present Class I HLA molecules with a higher theoretical capacity for binding SARS-Cov-2 peptides and showed greater heterozygosity when comparing them with moderate and severe groups. In this regard, identifying HLA-SARS-CoV-2 peptides binding differences between individuals would help to clarify the heterogeneity of clinical responses to the disease and will also be useful to guide a personalized treatment according to its particular risk.


Subject(s)
Betacoronavirus/pathogenicity , Coronavirus Infections/genetics , Histocompatibility Antigens Class I/genetics , Host-Pathogen Interactions/immunology , Pneumonia, Viral/genetics , Viral Proteins/genetics , Adult , Aged , Alleles , Betacoronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/virology , Disease Progression , Female , Gene Expression , Gene Frequency , Histocompatibility Antigens Class I/classification , Histocompatibility Antigens Class I/immunology , Humans , Immunity, Innate , Male , Middle Aged , Pandemics , Peptides/genetics , Peptides/immunology , Pilot Projects , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Protein Binding , Severity of Illness Index , Spain , Viral Proteins/immunology
6.
Arch Virol ; 165(10): 2373-2377, 2020 Oct.
Article in English | MEDLINE | ID: covidwho-695405

ABSTRACT

In situ hybridization (ISH) and immunohistochemistry (IHC) are essential tools to characterize SARS-CoV-2 infection and tropism in naturally and experimentally infected animals and also for diagnostic purposes. Here, we describe three RNAscope®-based ISH assays targeting the ORF1ab, spike, and nucleocapsid genes and IHC assays targeting the spike and nucleocapsid proteins of SARS-CoV-2.


Subject(s)
Betacoronavirus/genetics , Betacoronavirus/isolation & purification , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Animals , Antibodies, Monoclonal , Antibodies, Viral , Antisense Elements (Genetics)/genetics , Chlorocebus aethiops , Coronavirus Infections/virology , Genes, Viral , Humans , Immunohistochemistry/methods , In Situ Hybridization/methods , Nucleocapsid Proteins/genetics , Nucleocapsid Proteins/metabolism , Pandemics , Pneumonia, Viral/virology , RNA, Viral/metabolism , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolism
7.
Nat Commun ; 11(1): 3810, 2020 07 30.
Article in English | MEDLINE | ID: covidwho-690732

ABSTRACT

The pandemic of COVID-19 has posed an unprecedented threat to global public health. However, the interplay between the viral pathogen of COVID-19, SARS-CoV-2, and host innate immunity is poorly understood. Here we show that SARS-CoV-2 induces overt but delayed type-I interferon (IFN) responses. By screening 23 viral proteins, we find that SARS-CoV-2 NSP1, NSP3, NSP12, NSP13, NSP14, ORF3, ORF6 and M protein inhibit Sendai virus-induced IFN-ß promoter activation, whereas NSP2 and S protein exert opposite effects. Further analyses suggest that ORF6 inhibits both type I IFN production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect. Finally, we find that IFN-ß treatment effectively blocks SARS-CoV-2 replication. In summary, our study shows that SARS-CoV-2 perturbs host innate immune response via both its structural and nonstructural proteins, and thus provides insights into the pathogenesis of SARS-CoV-2.


Subject(s)
Betacoronavirus/physiology , Coronavirus Infections/virology , Immune Evasion , Interferon Type I/metabolism , Pneumonia, Viral/virology , Signal Transduction , Betacoronavirus/genetics , Betacoronavirus/immunology , Betacoronavirus/metabolism , Cell Line , Coronavirus Infections/immunology , Humans , Immunity, Innate , Interferon-beta/genetics , Interferon-beta/metabolism , Interferon-beta/pharmacology , Mutation , Open Reading Frames , Pandemics , Pneumonia, Viral/immunology , Promoter Regions, Genetic , Signal Transduction/drug effects , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication/drug effects
8.
Front Immunol ; 11: 1581, 2020.
Article in English | MEDLINE | ID: covidwho-688990

ABSTRACT

To ultimately combat the emerging COVID-19 pandemic, it is desired to develop an effective and safe vaccine against this highly contagious disease caused by the SARS-CoV-2 coronavirus. Our literature and clinical trial survey showed that the whole virus, as well as the spike (S) protein, nucleocapsid (N) protein, and membrane (M) protein, have been tested for vaccine development against SARS and MERS. However, these vaccine candidates might lack the induction of complete protection and have safety concerns. We then applied the Vaxign and the newly developed machine learning-based Vaxign-ML reverse vaccinology tools to predict COVID-19 vaccine candidates. Our Vaxign analysis found that the SARS-CoV-2 N protein sequence is conserved with SARS-CoV and MERS-CoV but not from the other four human coronaviruses causing mild symptoms. By investigating the entire proteome of SARS-CoV-2, six proteins, including the S protein and five non-structural proteins (nsp3, 3CL-pro, and nsp8-10), were predicted to be adhesins, which are crucial to the viral adhering and host invasion. The S, nsp3, and nsp8 proteins were also predicted by Vaxign-ML to induce high protective antigenicity. Besides the commonly used S protein, the nsp3 protein has not been tested in any coronavirus vaccine studies and was selected for further investigation. The nsp3 was found to be more conserved among SARS-CoV-2, SARS-CoV, and MERS-CoV than among 15 coronaviruses infecting human and other animals. The protein was also predicted to contain promiscuous MHC-I and MHC-II T-cell epitopes, and the predicted linear B-cell epitopes were found to be localized on the surface of the protein. Our predicted vaccine targets have the potential for effective and safe COVID-19 vaccine development. We also propose that an "Sp/Nsp cocktail vaccine" containing a structural protein(s) (Sp) and a non-structural protein(s) (Nsp) would stimulate effective complementary immune responses.


Subject(s)
Betacoronavirus , Coronavirus Infections , Machine Learning , Pandemics , Pneumonia, Viral , Viral Vaccines , Animals , Betacoronavirus/genetics , Betacoronavirus/immunology , Coronavirus Infections/epidemiology , Coronavirus Infections/genetics , Coronavirus Infections/immunology , Coronavirus Infections/prevention & control , Epitopes, B-Lymphocyte/genetics , Epitopes, B-Lymphocyte/immunology , Humans , Immunogenicity, Vaccine , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunology , Pandemics/prevention & control , Pneumonia, Viral/epidemiology , Pneumonia, Viral/genetics , Pneumonia, Viral/immunology , Pneumonia, Viral/prevention & control , Viral Proteins/genetics , Viral Proteins/immunology , Viral Vaccines/genetics , Viral Vaccines/immunology
9.
J Med Virol ; 92(6): 584-588, 2020 06.
Article in English | MEDLINE | ID: covidwho-685102

ABSTRACT

Last December 2019, a new virus, named novel Coronavirus (COVID-2019) causing many cases of severe pneumonia was reported in Wuhan, China. The virus knowledge is limited and especially about COVID-2019 pathogenesis. The Open Reading Frame 1ab (ORF1ab) of COVID-2019 has been analyzed to evidence the presence of mutation caused by selective pressure on the virus. For selective pressure analysis fast-unconstrained Bayesian approximation (FUBAR) was used. Homology modelling has been performed by SwissModel and HHPred servers. The presence of transmembrane helical segments in Coronavirus ORF1ab non structural protein 2 (nsp2) and nsp3 was tested by TMHMM, MEMSAT, and MEMPACK tools. Three-dimensional structures have been analyzed and displayed using PyMOL. FUBAR analysis revealed the presence of potential sites under positive selective pressure (P < .05). Position 723 in the COVID-2019 has a serine instead a glycine residue, while at aminoacidic position 1010 a proline instead an isoleucine. Significant (P < .05) pervasive negative selection in 2416 sites (55%) was found. The positive selective pressure could account for some clinical features of this virus compared with severe acute respiratory syndrome (SARS) and Bat SARS-like CoV. The stabilizing mutation falling in the endosome-associated-protein-like domain of the nsp2 protein could account for COVID-2019 high ability of contagious, while the destabilizing mutation in nsp3 proteins could suggest a potential mechanism differentiating COVID-2019 from SARS. These data could be helpful for further investigation aimed to identify potential therapeutic targets or vaccine strategy, especially in the actual moment when the epidemic is ongoing and the scientific community is trying to enrich knowledge about this new viral pathogen.


Subject(s)
Betacoronavirus/genetics , SARS Virus/genetics , Viral Nonstructural Proteins/chemistry , Viral Proteins/chemistry , Betacoronavirus/pathogenicity , Coronavirus Infections/virology , Female , Gene Expression , Humans , Male , Models, Molecular , Mutation , Pandemics , Pneumonia, Viral/virology , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , SARS Virus/pathogenicity , Selection, Genetic , Structural Homology, Protein , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism
11.
Adv Virus Res ; 107: 383-416, 2020.
Article in English | MEDLINE | ID: covidwho-679455

ABSTRACT

Since the end of 2019, the global COVID-19 outbreak has once again made coronaviruses a hot topic. Vaccines are hoped to be an effective way to stop the spread of the virus. However, there are no clinically approved vaccines available for coronavirus infections. Reverse genetics technology can realize the operation of RNA virus genomes at the DNA level and provide new ideas and strategies for the development of new vaccines. In this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. An efficient reverse genetics platform is useful for obtaining the ideal attenuated strain to prepare an attenuated live vaccine.


Subject(s)
Betacoronavirus/immunology , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Vaccines, Synthetic/immunology , Viral Proteins/genetics , Viral Proteins/immunology , Viral Vaccines/immunology , Coronavirus Infections/immunology , Genome, Viral/genetics , Humans , Pneumonia, Viral/immunology , RNA, Viral/genetics , Reverse Genetics/methods
12.
Jpn J Infect Dis ; 73(4): 304-307, 2020 07 22.
Article in English | MEDLINE | ID: covidwho-678395

ABSTRACT

During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Humans , Japan , Pandemics , Reverse Transcriptase Polymerase Chain Reaction/methods , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
13.
Int J Mol Med ; 46(3): 957-964, 2020 Sep.
Article in English | MEDLINE | ID: covidwho-676117

ABSTRACT

Reverse transcription­quantitative polymerase chain reaction (RT­qPCR) is the gold standard method for the diagnosis of COVID­19 infection. Due to pre­analytical and technical limitations, samples with low viral load are often misdiagnosed as false­negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT­qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID­19 were analyzed by droplet digital PCR (ddPCR) and RT­qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)­approved probe for the SARS­CoV­2 N gene were used. SYBR­Green RT­qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT­qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT­qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10­fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT­qPCR for the diagnosis of COVID­19 infection in false­negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID­19 and the follow­up of positive patients until complete remission.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Humans , Nucleocapsid Proteins/genetics , Pandemics , Sensitivity and Specificity , Spike Glycoprotein, Coronavirus/genetics , Viral Proteins/genetics
14.
Euro Surveill ; 25(26)2020 07.
Article in English | MEDLINE | ID: covidwho-639489

ABSTRACT

Following SARS-CoV-2 emergence in China, a specific surveillance was implemented in France. Phylogenetic analysis of sequences retrieved through this surveillance suggests that detected initial introductions, involving non-clade G viruses, did not seed local transmission. Nevertheless, identification of clade G variants subsequently circulating in the country, with the earliest from a patient who neither travelled to risk areas nor had contact with travellers, suggests that SARS-CoV-2 might have been present before the first recorded local cases.


Subject(s)
Coronavirus Infections/genetics , Coronavirus/genetics , Disease Outbreaks/prevention & control , Sentinel Surveillance , Betacoronavirus , Coronavirus/classification , Coronavirus/isolation & purification , Coronavirus Infections/diagnosis , Coronavirus Infections/epidemiology , Coronavirus Infections/transmission , France/epidemiology , Genome, Viral/genetics , Humans , Pandemics/prevention & control , Phylogeny , Pneumonia, Viral/diagnosis , Pneumonia, Viral/epidemiology , Pneumonia, Viral/transmission , RNA, Viral/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis , Viral Proteins/genetics
15.
Int J Mol Sci ; 21(13)2020 Jul 07.
Article in English | MEDLINE | ID: covidwho-639407

ABSTRACT

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.


Subject(s)
Betacoronavirus/genetics , Genome, Viral , MicroRNAs/metabolism , RNA, Viral/chemistry , 3' Untranslated Regions , Base Sequence , Coronavirus Infections/pathology , Coronavirus Infections/virology , Databases, Genetic , Humans , MicroRNAs/chemistry , MicroRNAs/genetics , Mutation , Nucleic Acid Conformation , Pandemics , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , RNA Splice Sites , RNA Splicing , Sequence Alignment , Viral Nonstructural Proteins/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolism
16.
Mol Biol Evol ; 37(9): 2463-2464, 2020 09 01.
Article in English | MEDLINE | ID: covidwho-638189

ABSTRACT

Identifying the origin of SARS-CoV-2, the etiological agent of the current COVID-19 pandemic, may help us to avoid future epidemics of coronavirus and other zoonoses. Several theories about the zoonotic origin of SARS-CoV-2 have recently been proposed. Although Betacoronavirus found in Rhinolophus bats from China have been broadly implicated, their genetic dissimilarity to SARS-CoV-2 is so high that they are highly unlikely to be its direct ancestors. Thus, an intermediary host is suspected to link bat to human coronaviruses. Based on genomic CpG dinucleotide patterns in different coronaviruses from different hosts, it was suggested that SARS-CoV-2 might have evolved in a canid gastrointestinal tract prior to transmission to humans. However, similar CpG patterns are now reported in coronaviruses from other hosts, including bats themselves and pangolins. Therefore, reduced genomic CpG alone is not a highly predictive biomarker, suggesting a need for additional biomarkers to reveal intermediate hosts or tissues. The hunt for the zoonotic origin of SARS-CoV-2 continues.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/epidemiology , Genome, Viral , Pandemics , Pneumonia, Viral/epidemiology , Viral Proteins/genetics , Zoonoses/epidemiology , Animals , Betacoronavirus/classification , Betacoronavirus/pathogenicity , Chiroptera/virology , Coronavirus Infections/transmission , Coronavirus Infections/virology , CpG Islands , Eutheria/virology , Evolution, Molecular , Gene Expression , Mutation , Pneumonia, Viral/transmission , Pneumonia, Viral/virology , Reassortant Viruses/classification , Reassortant Viruses/genetics , Reassortant Viruses/pathogenicity , Recombination, Genetic , Viral Proteins/metabolism , Zoonoses/transmission , Zoonoses/virology
17.
Ann Agric Environ Med ; 27(2): 175-183, 2020 Jun 19.
Article in English | MEDLINE | ID: covidwho-614683

ABSTRACT

Coronaviruses (CoVs) are positive-strand RNA viruses with the largest genome among all RNA viruses. They are able to infect many host, such as mammals or birds. Whereas CoVs were identified 1930s, they became known again in 2003 as the agents of the Severe Acute Respiratory Syndrome (SARS). The spike protein is thought to be essential in the process of CoVs entry, because it is associated with the binding to the receptor on the host cell. It is also involved in cell tropism and pathogenesis. Receptor recognition is the crucial step in the infection. CoVs are able to bind a variety of receptors, although the selection of receptor remains unclear. Coronaviruses were initially believed to enter cells by fusion with the plasma membrane. Further studies demonstrated that many of them involve endocytosis through clathrin-dependent, caveolae-dependent, clathrin-independent, as well as caveolae-independent mechanisms. The aim of this review is to summarise current knowledge about coronaviruses, focussing especially on CoVs entry into the host cell. Advances in understanding coronaviruses replication strategy and the functioning of the replicative structures are also highlighted. The development of host-directed antiviral therapy seems to be a promising way to treat infections with SARS-CoV or other pathogenic coronaviruses. There is still much to be discovered in the inventory of pro- and anti-viral host factors relevant for CoVs replication. The latest pandemic danger, originating from China, has given our previously prepared work even more of topicality.


Subject(s)
Cell Membrane/virology , Coronavirus Infections/virology , Coronavirus/physiology , Virus Internalization , Animals , Coronavirus/genetics , Humans , Viral Proteins/genetics , Viral Proteins/metabolism , Viral Tropism
18.
Mol Cell Proteomics ; 19(9): 1503-1522, 2020 09.
Article in English | MEDLINE | ID: covidwho-616588

ABSTRACT

As the COVID-19 pandemic continues to spread, thousands of scientists around the globe have changed research direction to understand better how the virus works and to find out how it may be tackled. The number of manuscripts on preprint servers is soaring and peer-reviewed publications using MS-based proteomics are beginning to emerge. To facilitate proteomic research on SARS-CoV-2, the virus that causes COVID-19, this report presents deep-scale proteomes (10,000 proteins; >130,000 peptides) of common cell line models, notably Vero E6, Calu-3, Caco-2, and ACE2-A549 that characterize their protein expression profiles including viral entry factors such as ACE2 or TMPRSS2. Using the 9 kDa protein SRP9 and the breast cancer oncogene BRCA1 as examples, we show how the proteome expression data can be used to refine the annotation of protein-coding regions of the African green monkey and the Vero cell line genomes. Monitoring changes of the proteome on viral infection revealed widespread expression changes including transcriptional regulators, protease inhibitors, and proteins involved in innate immunity. Based on a library of 98 stable-isotope labeled synthetic peptides representing 11 SARS-CoV-2 proteins, we developed PRM (parallel reaction monitoring) assays for nano-flow and micro-flow LC-MS/MS. We assessed the merits of these PRM assays using supernatants of virus-infected Vero E6 cells and challenged the assays by analyzing two diagnostic cohorts of 24 (+30) SARS-CoV-2 positive and 28 (+9) negative cases. In light of the results obtained and including recent publications or manuscripts on preprint servers, we critically discuss the merits of MS-based proteomics for SARS-CoV-2 research and testing.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/genetics , Host-Pathogen Interactions/genetics , Pneumonia, Viral/genetics , Proteomics/methods , Viral Proteins/genetics , A549 Cells , Amino Acid Sequence , Animals , BRCA1 Protein/genetics , BRCA1 Protein/metabolism , Betacoronavirus/pathogenicity , Caco-2 Cells , Case-Control Studies , Chlorocebus aethiops , Coronavirus Infections/pathology , Coronavirus Infections/virology , Gene Expression Regulation , Gene Ontology , Humans , Indicators and Reagents , Molecular Sequence Annotation , Open Reading Frames , Pandemics , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/pathology , Pneumonia, Viral/virology , Proteomics/instrumentation , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Signal Recognition Particle/genetics , Signal Recognition Particle/metabolism , Signal Transduction , Vero Cells , Viral Proteins/classification , Viral Proteins/metabolism , Virus Internalization
20.
Virus Res ; 286: 198074, 2020 09.
Article in English | MEDLINE | ID: covidwho-611212

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human coronavirus causing the pandemic of severe pneumonia (Coronavirus Disease 2019, COVID-19). SARS-CoV-2 is highly pathogenic in human, having posed immeasurable public health challenges to the world. Innate immune response is critical for the host defense against viral infection and the dysregulation of the host innate immune responses probably aggravates SARS-CoV-2 infection, contributing to the high morbidity and lethality of COVID-19. It has been reported that some coronavirus proteins play an important role in modulating innate immunity of the host, but few studies have been conducted on SARS-CoV-2. In this study, we screened the viral proteins of SARS-CoV-2 and found that the viral ORF6, ORF8 and nucleocapsid proteins were potential inhibitors of type I interferon signaling pathway, a key component for antiviral response of host innate immune. All the three proteins showed strong inhibition on type I interferon (IFN-ß) and NF-κB-responsive promoter, further examination revealed that these proteins were able to inhibit the interferon-stimulated response element (ISRE) after infection with Sendai virus, while only ORF6 and ORF8 proteins were able to inhibit the ISRE after treatment with interferon beta. These findings would be helpful for the further study of the detailed signaling pathway and unveil the key molecular player that may be targeted.


Subject(s)
Betacoronavirus/genetics , Host-Pathogen Interactions/genetics , Interferon-beta/genetics , NF-kappa B/genetics , Nucleocapsid Proteins/genetics , Viral Proteins/genetics , Betacoronavirus/immunology , Gene Expression Regulation , Genes, Reporter , HEK293 Cells , Host-Pathogen Interactions/immunology , Humans , Immunity, Innate , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/immunology , Interferon-beta/immunology , Luciferases/genetics , Luciferases/metabolism , NF-kappa B/immunology , Nucleocapsid Proteins/immunology , Plasmids/chemistry , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Response Elements , Sendai virus/genetics , Sendai virus/immunology , Signal Transduction , Transfection/methods , Viral Proteins/immunology
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