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1.
Commun Biol ; 4(1): 1343, 2021 11 30.
Article in English | MEDLINE | ID: covidwho-1545654

ABSTRACT

The appearance of multiple new SARS-CoV-2 variants during the COVID-19 pandemic is a matter of grave concern. Some of these variants, such as B.1.617.2, B.1.1.7, and B.1.351, manifest higher infectivity and virulence than the earlier SARS-CoV-2 variants, with potential dramatic effects on the course of the pandemic. So far, analysis of new SARS-CoV-2 variants focused primarily on nucleotide substitutions and short deletions that are readily identifiable by comparison to consensus genome sequences. In contrast, insertions have largely escaped the attention of researchers although the furin site insert in the Spike (S) protein is thought to be a determinant of SARS-CoV-2 virulence. Here, we identify 346 unique inserts of different lengths in SARS-CoV-2 genomes and present evidence that these inserts reflect actual virus variance rather than sequencing artifacts. Two principal mechanisms appear to account for the inserts in the SARS-CoV-2 genomes, polymerase slippage and template switch that might be associated with the synthesis of subgenomic RNAs. At least three inserts in the N-terminal domain of the S protein are predicted to lead to escape from neutralizing antibodies, whereas other inserts might result in escape from T-cell immunity. Thus, inserts in the S protein can affect its antigenic properties and merit monitoring.

2.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Article in English | MEDLINE | ID: covidwho-1307383

ABSTRACT

Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.


Subject(s)
Adaptation, Physiological/genetics , Evolution, Molecular , SARS-CoV-2/genetics , Amino Acid Substitution , COVID-19/diagnosis , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , COVID-19 Testing , Coronavirus Nucleocapsid Proteins/genetics , Epistasis, Genetic , Genome, Viral/genetics , Humans , Immune Evasion/genetics , Mutation , Nuclear Localization Signals/genetics , Phosphoproteins/genetics , Phylogeny , Protein Interaction Domains and Motifs/genetics , SARS-CoV-2/classification , Selection, Genetic , Spike Glycoprotein, Coronavirus/genetics , Vaccination
3.
mBio ; 12(3): e0142321, 2021 06 29.
Article in English | MEDLINE | ID: covidwho-1280400

ABSTRACT

The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.


Subject(s)
Adenosine Triphosphate/analogs & derivatives , Antiviral Agents/pharmacology , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Guanosine Tetraphosphate/pharmacology , SARS-CoV-2/drug effects , Adenosine Triphosphate/pharmacology , COVID-19/drug therapy , Catalytic Domain/physiology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Humans , Ribosomes/metabolism
5.
medRxiv ; 2020 Nov 24.
Article in English | MEDLINE | ID: covidwho-955720

ABSTRACT

Background: While pathogens often evolve towards reduced virulence, many counterexamples are evident. When faced with a new pathogen, such as SARS-CoV-2, it is highly desirable to be able to forecast the case fatality rate (CFR) into the future. Considerable effort has been invested towards the development of a mathematical framework for predicting virulence evolution. Although these approaches accurately recapitulate some complex outcomes, most rely on an assumed trade-off between mortality and infectivity. It is often impractical to empirically validate this constraint for human pathogens. Results: Using a compartment model with parameters tuning the degree to which symptomatic individuals are isolated and the duration of immunity, we reveal kinetic constraints where the variation of multiple parameters in concert leads to decreased virulence and increased pathogen fitness, whereas independent variation of the parameters decreases pathogen fitness. Smallpox, SARS-CoV-2, and Influenza are analyzed as diverse representatives of human respiratory viruses. We show that highly virulent viruses, such as Smallpox, are likely often constrained by host behavior, whereas moderately virulent viruses, such as SARS-CoV-2, appear to be typically constrained by the relationship between the duration of immunity and CFR. Conclusions: The evolution of human respiratory epidemics appears to be often kinetically constrained and a reduction in virulence should not be assumed. Our findings imply that, without continued public health intervention, SARS-CoV-2 is likely to continue presenting a substantial disease burden. The existence of a parameter regime admitting endemic equilibrium suggests that herd immunity is unachievable. However, we demonstrate that even partial isolation of symptomatic individuals can have a major effect not only by reducing the number of fatalities in the short term but also by potentially changing the evolutionary trajectory of the virus towards reduced virulence.

6.
BMC Biol ; 18(1): 186, 2020 11 30.
Article in English | MEDLINE | ID: covidwho-949117

ABSTRACT

BACKGROUND: A crucial factor in mitigating respiratory viral outbreaks is early determination of the duration of the incubation period and, accordingly, the required quarantine time for potentially exposed individuals. At the time of the COVID-19 pandemic, optimization of quarantine regimes becomes paramount for public health, societal well-being, and global economy. However, biological factors that determine the duration of the virus incubation period remain poorly understood. RESULTS: We demonstrate a strong positive correlation between the length of the incubation period and disease severity for a wide range of human pathogenic viruses. Using a machine learning approach, we develop a predictive model that accurately estimates, solely from several virus genome features, in particular, the number of protein-coding genes and the GC content, the incubation time ranges for diverse human pathogenic RNA viruses including SARS-CoV-2. The predictive approach described here can directly help in establishing the appropriate quarantine durations and thus facilitate controlling future outbreaks. CONCLUSIONS: The length of the incubation period in viral diseases strongly correlates with disease severity, emphasizing the biological and epidemiological importance of the incubation period. Perhaps, surprisingly, incubation times of pathogenic RNA viruses can be accurately predicted solely from generic features of virus genomes. Elucidation of the biological underpinnings of the connections between these features and disease progression can be expected to reveal key aspects of virus pathogenesis.


Subject(s)
COVID-19/pathology , COVID-19/virology , Infectious Disease Incubation Period , SARS-CoV-2/genetics , Computer Simulation , Genome, Viral , Humans , Models, Biological , Mutation , Quarantine
7.
Proc Natl Acad Sci U S A ; 117(26): 15193-15199, 2020 06 30.
Article in English | MEDLINE | ID: covidwho-595720

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses an immediate, major threat to public health across the globe. Here we report an in-depth molecular analysis to reconstruct the evolutionary origins of the enhanced pathogenicity of SARS-CoV-2 and other coronaviruses that are severe human pathogens. Using integrated comparative genomics and machine learning techniques, we identify key genomic features that differentiate SARS-CoV-2 and the viruses behind the two previous deadly coronavirus outbreaks, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), from less pathogenic coronaviruses. These features include enhancement of the nuclear localization signals in the nucleocapsid protein and distinct inserts in the spike glycoprotein that appear to be associated with high case fatality rate of these coronaviruses as well as the host switch from animals to humans. The identified features could be crucial contributors to coronavirus pathogenicity and possible targets for diagnostics, prognostication, and interventions.


Subject(s)
Betacoronavirus/genetics , Evolution, Molecular , Genome, Viral , Nucleocapsid Proteins/genetics , Spike Glycoprotein, Coronavirus/genetics , Animals , Betacoronavirus/classification , Betacoronavirus/pathogenicity , Host Specificity , Humans , Machine Learning , Middle East Respiratory Syndrome Coronavirus/classification , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Mutagenesis, Insertional , Nuclear Localization Signals/genetics , Nucleocapsid Proteins/chemistry , Phylogeny , SARS-CoV-2 , Sequence Homology , Spike Glycoprotein, Coronavirus/chemistry , Virulence/genetics
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