Differential enrichment of yeast DNA in SARS-CoV-2 and related genomes supports synthetic origin hypothesis

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be also considered. Results: Here we show that the genomes from SARS-CoV-2 and from SARS-CoV-1 are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for virus replication, host cell invasion, and host immune response. Specifically, for SARS-CoV-2, we identify two sites: one at the start of the viral replicase domain, and the other at the end of the spike gene past its critical domain junction; for SARS-CoV-1, one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain. As yeast is not a natural host for this virus family, we propose a directed passage model for viral constructs, including virus replicase, in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Model and data together allow us to infer a hypothetical tripartite genome assembly scheme for the synthetic biogenesis of SARS-CoV-2 and SARS-CoV-1. Conclusions: These results provide evidence that the genome sequences of SARS-CoV-1, SARS-CoV-2, but not that of RaTG13, BANAL-20-52 and all other closest SARS coronavirus family members identified, are carriers of distinct homology signals that might point to large-scale genomic editing during a passage of directed replication and chromosomal integration inside genetically modified yeast cells.

Differential enrichment of yeast DNA in SARS-CoV-2 and related genomes supports synthetic origin hypothesis

Background: Knowledge about the origin of SARS-CoV-2 is necessary for both a biological and epidemiological understanding of the COVID-19 pandemic. Evidence suggests that a proximal evolutionary ancestor of SARS-CoV-2 belongs to the bat coronavirus family. However, as further evidence for a direct zoonosis remains limited, alternative modes of SARS-CoV-2 biogenesis should be also considered. Results: Here we show that genomes from SARS-CoV-2 and from closely related coronaviruses are differentially enriched with short chromosomal sequences from the yeast S. cerevisiae at focal positions that are known to be critical for virus replication, host cell invasion, and host immune response. Specifically, for SARS-CoV-2, we identify two sites: one at the start of the viral replicase domain, and the other at the end of the spike gene past its critical domain junction; for SARS-CoV-1, one at the start of the RNA dependent RNA polymerase gene, and the other at the start of the spike protein’s receptor binding domain. As yeast is not a natural host for this virus family, we propose a directed passage model for viral constructs, including virus replicase, in yeast cells based on co-transformation of virus DNA plasmids carrying yeast selectable genetic markers followed by intra-chromosomal homologous recombination through gene conversion. Highly differential sequence homology data across yeast chromosomes congruent with chromosomes harboring specific auxotrophic markers further support this passage model. Model and data together allow us to infer a hypothetical tripartite genome assembly scheme for the synthetic biogenesis of SARS-CoV-2 and SARS-CoV-1. Conclusions: These results provide evidence that the genome sequences of SARS-CoV-1, SARS-CoV-2, but not that of RaTG13 and all other closest SARS coronavirus family members identified, are carriers of distinct homology signals that might point to large-scale genomic editing during a passage of directed replication and chromosomal integration inside genetically modified yeast cells.

NbPsbO1 Interacts Specifically with Bamboo Mosaic Virus Subgenomic RNA Promoter and Is Required for Efficient BaMV SgRNA Transcription

Many positive-strand (+) RNA viruses produce subgenomic RNAs (sgRNAs) in the infection cycle through the combined activities of viral replicase and host proteins. However, knowledge about host proteins involved in direct sgRNA promoter recognition is limited. Here, in the partially purified replicase complexes from Bamboo mosaic virus (BaMV)-infected tissue, we have identified Nicotiana benthamiana Photosystem II oxygen-evolving complex protein, NbPsbO1, which specifically interacted with the promoter of sgRNA but not that of genomic RNA (gRNA). Silencing of NbPsbO1 expression suppressed BaMV accumulation in N. benthamiana protoplasts without affecting viral gRNA replication. Overexpression of wild-type NbPsbO1 stimulated BaMV sgRNA accumulation. Fluorescent microscopy examination revealed that the fluorescence associated with NbPsbO1 was redistributed from chloroplast granal thylakoids to stroma in BaMV-infected cells. Overexpression of a mis-localized mutant of NbPsbO1, dTPPsbO1-T7, inhibited BaMV RNA accumulation in N. benthamiana , whereas overexpression of an NbPsbO1 derivative, sPsbO1-T7, designed to be targeted to chloroplast stroma, upregulated sgRNA level. Furthermore, depletion of NbPsbO1 in BaMV RdRp preparation significantly inhibited sgRNA synthesis in vitro , but exerted no effect on (+) or (-) gRNA synthesis, which indicates that NbPsbO1 is required for efficient sgRNA synthesis. These results reveal a novel role for NbPsbO1 in the selective enhancement of BaMV sgRNA transcription, most likely via direct interaction with the sgRNA promoter. IMPORTANCE Production of subgenomic RNAs (sgRNAs) for efficiently translating of downstream viral proteins is one of the major strategies adapted for viruses that contain multicistronic RNA genome. Both viral genomic RNA (gRNA) replication and sgRNA transcription rely on the combined activities of viral replicase and host proteins, which recognize promoter regions for the initiation of RNA synthesis. However, compared to the cis -acting elements involved in the regulation of sgRNA synthesis, the host factors involved in sgRNA promoter recognition mostly remain to be elucidated. Here, we found a chloroplast protein, NbPsbO1, which specifically interacts with Bamboo mosaic virus (BaMV) sgRNA promoter. We showed that NbPsbO1 is relocated to the BaMV replication site in BaMV infected cells, and demonstrated that NbPsbO1 is required for efficient BaMV sgRNA transcription, but exerts no effect on gRNA replication. This study provides a new insight into the regulating mechanism of viral gRNA and sgRNA synthesis.

Origins and Evolution of the Primate Hepatitis B Virus

Recent interest in the origins and subsequent evolution of the hepatitis B virus (HBV) has strengthened with the discovery of ancient HBV sequences in fossilized remains of humans dating back to the Neolithic period around 7,000 years ago. Metagenomic analysis identified a number of African non-human primate HBV sequences in the oldest samples collected, indicating that human HBV may have at some stage, evolved in Africa following zoonotic transmissions from higher primates. Ancestral genotype A and D isolates were also discovered from the Bronze Age, not in Africa but rather Eurasia, implying a more complex evolutionary and migratory history for HBV than previously recognized. Most full-length ancient HBV sequences exhibited features of inter genotypic recombination, confirming the importance of recombination and the mutation rate of the error-prone viral replicase as drivers for successful HBV evolution. A model for the origin and evolution of HBV is proposed, which includes multiple cross-species transmissions and favors subsequent recombination events that result in a pathogen and can successfully transmit and cause persistent infection in the primate host.

Exploring the binding efficacy of ivermectin against the key proteins of SARS-CoV-2 pathogenesis: an in silico approach

Aim: COVID-19 is currently the biggest threat to mankind. Recently, ivermectin (a US FDA-approved antiparasitic drug) has been explored as an anti-SARS-CoV-2 agent. Herein, we have studied the possible mechanism of action of ivermectin using in silico approaches. Materials & methods: Interaction of ivermectin against the key proteins involved in SARS-CoV-2 pathogenesis were investigated through molecular docking and molecular dynamic simulation. Results: Ivermectin was found as a blocker of viral replicase, protease and human TMPRSS2, which could be the biophysical basis behind its antiviral efficiency. The antiviral action and ADMET profile of ivermectin was on par with the currently used anticorona drugs such as hydroxychloroquine and remdesivir. Conclusion: Our study enlightens the candidature of ivermectin as an effective drug for treating COVID-19.

Targeting novel structural and functional features of coronavirus protease nsp5 (3CLpro, Mpro) in the age of COVID-19

Coronavirus protease nsp5 (M pro , 3CL pro ) remains a primary target for coronavirus therapeutics due to its indispensable and conserved role in the proteolytic processing of the viral replicase polyproteins. In this review, we discuss the diversity of known coronaviruses, the role of nsp5 in coronavirus biology, and the structure and function of this protease across the diversity of known coronaviruses, and evaluate past and present efforts to develop inhibitors to the nsp5 protease with a particular emphasis on new and mostly unexplored potential targets of inhibition. With the recent emergence of pandemic SARS-CoV-2, this review provides novel and potentially innovative strategies and directions to develop effective therapeutics against the coronavirus protease nsp5.

Nonstructural protein 7 and 8 complexes of SARS-CoV-2

The pandemic outbreak of coronavirus disease 2019 (COVID-19) across the world has led to millions of infection cases and caused a global public health crisis. Current research suggests that SARS-CoV-2 is a highly contagious coronavirus that spreads rapidly through communities. To understand the mechanisms of viral replication, it is imperative to observe coronavirus viral replicase, a huge protein complex comprising up to 16 viral nonstructural and associated host proteins, which is the most promising antiviral target for inhibiting viral genome replication and transcription. Recently, several components of the viral replicase complex in SARS-CoV-2 have been solved to provide a basis for the design of new antiviral therapeutics. Here, we report the crystal structure of the SARS-CoV2 nsp7-8 tetramer, which comprises two copies of each protein representing nsp7’s full-length and the C-terminus of nsp8 owing to N-terminus proteolysis during the process of crystallization. We also identified a long helical extension and highly flexible N-terminal domain of nsp8, which is preferred for interacting with single-stranded nucleic acids.