SARS-CoV-2 is the seventh coronavirus known to infect humans; SARSCoV, MERS-CoV and SARS-CoV-2 can cause severe disease, whereas HKU1, NL63, OC43 and 229E are associated with mild symptoms6. Here we review what can be deduced about the origin of SARS-CoV-2 from comparative analysis of genomic data. We offer a perspective on the notable features of the SARS-CoV-2 genome and discuss scenarios by which they could have arisen. Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus.

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The proximal origin of SARS-CoV-2.

A newly described coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of coronavirus disease 2019 (COVID-19), has infected over 2.3 million people, led to the death of more than 160,000 individuals and caused worldwide social and economic disruption1,2. There are no antiviral drugs with proven clinical efficacy for the treatment of COVID-19, nor are there any vaccines that prevent infection with SARS-CoV-2, and efforts to develop drugs and vaccines are hampered by the limited knowledge of the molecular details of how SARS-CoV-2 infects cells. Here we cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins that physically associated with each of the SARS-CoV-2 proteins using affinity-purification mass spectrometry, identifying 332 high-confidence protein–protein interactions between SARS-CoV-2 and human proteins. Among these, we identify 66 druggable human proteins or host factors targeted by 69 compounds (of which, 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found two sets of pharmacological agents that displayed antiviral activity: inhibitors of mRNA translation and predicted regulators of the sigma-1 and sigma-2 receptors. Further studies of these host-factor-targeting agents, including their combination with drugs that directly target viral enzymes, could lead to a therapeutic regimen to treat COVID-19. A human–SARS-CoV-2 protein interaction map highlights cellular processes that are hijacked by the virus and that can be targeted by existing drugs, including inhibitors of mRNA translation and predicted regulators of the sigma receptors.

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A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

Coronaviruses (CoVs), enveloped positive-sense RNA viruses, are characterized by club-like spikes that project from their surface, an unusually large RNA genome, and a unique replication strategy. Coronaviruses cause a variety of diseases in mammals and birds ranging from enteritis in cows and pigs and upper respiratory disease in chickens to potentially lethal human respiratory infections. Here we provide a brief introduction to coronaviruses discussing their replication and pathogenicity, and current prevention and treatment strategies. We also discuss the outbreaks of the highly pathogenic Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and the recently identified Middle Eastern Respiratory Syndrome Coronavirus (MERS-CoV).

/pdf/coronaviruses-an-overview-of-their-replication-and-4lyvr9vdqs.pdf

Coronaviruses: An Overview of Their Replication and Pathogenesis

The recent emergence of a novel coronavirus (2019-nCoV), which is causing an outbreak of unusual viral pneumonia in patients in Wuhan, a central city in China, is another warning of the risk of CoVs posed to public health. In this minireview, we provide a brief introduction of the general features of CoVs and describe diseases caused by different CoVs in humans and animals. This review will help understand the biology and potential risk of CoVs that exist in richness in wildlife such as bats.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jmv.25681

Emerging coronaviruses: Genome structure, replication, and pathogenesis.

The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding fu...

Structure, Function, and Evolution of Coronavirus Spike Proteins.

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.

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Continuous and Discontinuous RNA Synthesis in Coronaviruses

Severe acute respiratory syndrome (SARS) is a respiratory disease characterized by an atypical pneumonia, caused by a novel coronavirus (CoV) (13, 15, 27, 28, 35, 41, 45). The disease was reported for the first time in Guangdong Province, China, at the end of 2002, and spread to 32 countries in the course of a few months. After July 2003, only 4 community-acquired SARS cases were reported in China, but there have been at least as many reports of laboratory-acquired infections, with secondary spread in one case. Efficacious therapy is not available for this life-threatening disease that caused a mortality of about 10% in the epidemic of 2002 to 2003. Therefore, it is of interest to engineer attenuated SARS-CoVs as safer research tools.

SARS-CoV is an enveloped virus of the Coronaviridae family, group 2, and has a single-stranded, positive-sense 29.7-kb RNA genome (18, 48). Coronaviruses replicate in the cell cytoplasm and encode a nested set of mRNA molecules of different sizes. Viral genome expression begins with the translation of two large polyproteins, pp1a and pp1ab, including the viral replicase genes (50). Expression of the open reading frame (ORF) 1ab involves a ribosomal frameshifting into the −1 frame just upstream of the ORF 1a translation termination codon (4). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield functional components of the membrane-bound replicase complex (59). The replicase complex is involved in genome replication and transcription of subgenomic mRNAs (sgmRNAs), encoding structural proteins, such as the spike (S), envelope (E), membrane (M), and nucleocapsid (N), and a set of nonstructural proteins, whose sequence and number differ between the different species of coronavirus (47). In the case of SARS-CoV, ORFs 3a and 7a encode additional structural proteins (22, 25, 46). Among human CoVs (HCoVs), such as HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, and Hong Kong University 1-CoV, SARS-CoV causes the most severe disease (52, 54).

The virion envelope of CoVs contains at least three structural proteins, S, E, and M, embedded in the membrane. SARS-CoV has an additional structural membrane protein, 3a (25, 46). CoV M and E proteins are key factors for virus assembly and budding (6-8, 11, 14, 30). In fact, expression of these proteins in cell lines results in the production of virus-like particles (VLPs) (3, 8, 23, 53). In the case of SARS-CoV, there are conflicting reports on the proteins necessary for the formation of VLPs. Some reports describe the requirement of E and M for the efficient assembly of pseudoparticles in insect cells (21, 36), whereas others suggest that E protein is not essential for SARS-CoV-like particle formation in mammalian cells and propose that M and N proteins play a major role in morphogenesis (23).

The CoV E protein is present in virions in low copy number as a transmembrane protein (30, 34, 42). E protein contains a short (7 to 9 amino acid) hydrophilic amino terminus region and a 21- to 29-amino-acid hydrophobic region, followed by a hydrophilic carboxy terminus (6, 7). The SARS-CoV E protein contains an unusually short, palindromic transmembrane helical hairpin predicted around a previously unidentified pseudo-center of symmetry (2, 26). This hairpin possibly modifies lipid bilayers by increasing their curvature and likely plays a pivotal role in viral budding.

CoV E proteins share several characteristics with proteins of other viruses that function as ion channels, having a highly hydrophobic domain that forms at least one amphipathic α-helix that oligomerizes to form a putative ion-conductive pore in membranes (51). SARS-CoV and murine hepatitis virus (MHV) E proteins alter membrane permeability (31-33). Furthermore, HCoV-229E, MHV, SARS-CoV, and infectious bronchitis virus (IBV) E proteins form ion channels that are more permeable to monovalent cations than to monovalent anions (55, 56). The transmissible gastroenteritis virus (TGEV) E protein is essential for the production of recombinant infectious virus (9, 39). In contrast, MHV E protein is critical but not essential for virus replication because an MHV mutant in which the E gene has been deleted grows in cell culture to titers that are at least 3 orders of magnitude lower than recombinant wild-type viruses (30).

In this article, we report the generation of a recombinant SARS-CoV (rSARS-CoV) mutant in which expression of the E gene was abolished in a SARS-CoV cDNA clone that was assembled as a bacterial artificial chromosome (BAC). Although maximal virus titers reached by this defective virus were lower than those of the recombinant wild-type virus, the ΔE virus infected different cell lines, indicating that E protein is important but not essential for SARS-CoV replication. This rSARS-CoV-ΔE virus was attenuated in vivo, making it a safer research tool and a promising vaccine candidate.

A Severe Acute Respiratory Syndrome Coronavirus That Lacks the E Gene Is Attenuated In Vitro and In Vivo

The construction of cDNA clones encoding large-size RNA molecules of biological interest, like coronavirus genomes, which are among the largest mature RNA molecules known to biology, has been hampered by the instability of those cDNAs in bacteria. Herein, we show that the application of two strategies, cloning of the cDNAs into a bacterial artificial chromosome and nuclear expression of RNAs that are typically produced within the cytoplasm, is useful for the engineering of large RNA molecules. A cDNA encoding an infectious coronavirus RNA genome has been cloned as a bacterial artificial chromosome. The rescued coronavirus conserved all of the genetic markers introduced throughout the sequence and showed a standard mRNA pattern and the antigenic characteristics expected for the synthetic virus. The cDNA was transcribed within the nucleus, and the RNA translocated to the cytoplasm. Interestingly, the recovered virus had essentially the same sequence as the original one, and no splicing was observed. The cDNA was derived from an attenuated isolate that replicates exclusively in the respiratory tract of swine. During the engineering of the infectious cDNA, the spike gene of the virus was replaced by the spike gene of an enteric isolate. The synthetic virus replicated abundantly in the enteric tract and was fully virulent, demonstrating that the tropism and virulence of the recovered coronavirus can be modified. This demonstration opens up the possibility of employing this infectious cDNA as a vector for vaccine development in human, porcine, canine, and feline species susceptible to group 1 coronaviruses.

https://www.pnas.org/content/pnas/97/10/5516.full.pdf

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans , indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans , whereas it would only survive a single virus infection cycle in vivo . This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

/pdf/engineering-a-replication-competent-propagation-defective-2z1n0k7yi1.pdf

Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

Infection by different coronaviruses (CoVs) causes alterations in the transcriptional and translational patterns, cell cycle, cytoskeleton, and apoptosis pathways of the host cells. In addition, CoV infection may cause inflammation, alter immune and stress responses, and modify the coagulation pathways. The balance between the up- and downregulated genes could explain the pathogenesis caused by these viruses. We review specific aspects of CoV-host interactions. CoV genome replication takes place in the cytoplasm in a membrane-protected microenvironment and may control the cell machinery by locating some of their proteins in the host cell nucleus. CoVs initiate translation by cap-dependent and cap-independent mechanisms. CoV transcription involves a discontinuous RNA synthesis (template switching) during the extension of a negative copy of the subgenomic mRNAs. The requirement for base-pairing during transcription has been formally demonstrated in arteriviruses and CoVs. CoV N proteins have RNA chaperone activity that may help initiate template switching. Both viral and cellular proteins are required for replication and transcription, and the role of selected proteins is addressed.

https://www.annualreviews.org/doi/pdf/10.1146/annurev.micro.60.080805.142157

Fernando Almazán

Papers

Continuous and Discontinuous RNA Synthesis in Coronaviruses

A Severe Acute Respiratory Syndrome Coronavirus That Lacks the E Gene Is Attenuated In Vitro and In Vivo

Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome

Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

Biochemical Aspects of Coronavirus Replication and Virus-Host Interaction