Mosaic evolution of the severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome (SARS) is a deadly form of pneumonia caused by a novel coronavirus, a viral family responsible for mild respiratory tract infections in a wide variety of animals including humans, pigs, cows, mice, cats, and birds. Analyses to date have been unable to identify the precise origin of the SARS coronavirus. We used Bayesian, neighbor-joining, and split decomposition phylogenetic techniques on the SARS virus replicase, surface spike, matrix, and nucleocapsid proteins to reveal the evolutionary origin of this recently emerging infectious agent. The analyses support a mammalian-like origin for the replicase protein, an avian-like origin for the matrix and nucleocapsid proteins, and a mammalian-avian mosaic origin for the host-determining spike protein. A bootscan recombination analysis of the spike gene revealed high nucleotide identity between the SARS virus and a feline infectious peritonitis virus throughout the gene, except for a 200- base-pair region of high identity to an avian sequence. These data support the phylogenetic analyses and suggest a possible past recombination event between mammalian-like and avian-like parent viruses. This event occurred near a region that has been implicated to be the human receptor binding site and may have been directly responsible for the switch of host of the SARS coronavirus from animals to humans.     

Molecular biology of severe acute respiratory syndrome coronavirus

The worldwide epidemic of severe acute respiratory syndrome (SARS) in 2003 was caused by a novel coronavirus called SARS-CoV. Coronaviruses and their closest relatives possess extremely large plus-strand RNA genomes and employ unique mechanisms and enzymes in RNA synthesis that separate them from all other RNA viruses. The SARS epidemic prompted a variety of studies on multiple aspects of the coronavirus replication cycle, yielding both rapid identification of the entry mechanisms of SARS-CoV into host cells and valuable structural and functional information on SARS-CoV proteins. These recent advances in coronavirus research have important implications for the development of anti-SARS drugs and vaccines.     

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.     

免疫系统：严重急性呼吸综合征冠状病毒2型感染后的一把双刃剑

免疫系统是人体内的一把双刃剑,它一方面能清除侵染的各类病原体,但另一方面其异常调控又能在人体中引发各类免疫性疾病,甚至导致死亡。本文将简要讨论人体免疫系统与新的冠状病毒﹝即严重急性呼吸综合征冠状病毒2型(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)〕感染的相互关系。一方面免疫系统能全方位地预防病毒感染,进化出一整套从分子到细胞、从短期到长期的病毒清除机制;另一方面,免疫系统又可能引发“细胞因子风暴”,给SARS-CoV-2的感染患者带来负面作用。本文还将讨论受到广泛关注的免疫相关的治疗策略,着重探讨抗体依赖的增强效应(antibody-dependent enhancement, ADE)可能给疫苗研发带来的困难与挑战。     

Characterization of severe acute respiratory syndrome coronavirus membrane protein

The coronavirus membrane protein (M) is the key player in the assembly of virions at intracellular membranes between endoplasmic-reticulum and Golgi-complex. Using a newly established human monoclonal anti-M antibody we detected glycosylated and nonglycosylated membrane-associated M in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected cells and in purified virions. Further analyses revealed that M contained a single N-glycosylation site at asparagine 4. Recombinant M was transported to the plasma membrane and gained complex-type N-glycosylation. In SARS-CoV infected cells and in purified virions, however, N-glycosylation of M remained endoglycosidase H-sensitive suggesting that trimming of the N-linked sugar side chain is inhibited.     

Sumoylation of the nucleocapsid protein of severe acute respiratory syndrome coronavirus

Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes a highly basic nucleocapsid (N) protein of 422 amino acids. Similar to other coronavirus N proteins, SARS-CoV N protein is predicted to be phosphorylated and may contain nuclear localization signals, serine/arginine-rich motif, RNA binding domain and regions responsible for self-association and homo-oligomerization. In this study, we demonstrate that the protein is posttranslationally modified by covalent attachment to the small ubiquitin-like modifier. The major sumoylation site was mapped to the (62)lysine residue of the N protein. Further expression and characterization of wild type N protein and K62A mutant reveal that sumoylation of the N protein drastically promotes its homo-oligomerization, and plays certain roles in the N protein-mediated interference of host cell division. This is the first report showing that a coronavirus N protein undergoes posttranslational modification by sumoylation, and the functional implication of this modification in the formation of coronavirus ribouncleoprotein complex, virion assembly and virus-host interactions.     

Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nsp1, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nsp1 (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a approximately 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nsp1/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.     

Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus

Nitric oxide (NO) is an important signaling molecule between cells which has been shown to have an inhibitory effect on some virus infections. The purpose of this study was to examine whether NO inhibits the replication cycle of the severe acute respiratory syndrome coronavirus (SARS CoV) in vitro. We found that an organic NO donor, S-nitroso-N-acetylpenicillamine, significantly inhibited the replication cycle of SARS CoV in a concentration-dependent manner. We also show here that NO inhibits viral protein and RNA synthesis. Furthermore, we demonstrate that NO generated by inducible nitric oxide synthase, an enzyme that produces NO, inhibits the SARS CoV replication cycle.     

Conformational states of the severe acute respiratory syndrome coronavirus spike protein ectodomain

The severe acute respiratory syndrome coronavirus enters cells through the activities of a spike protein (S) which has receptor-binding (S1) and membrane fusion (S2) regions. We have characterized four sequential states of a purified recombinant S ectodomain (S-e) comprising S1 and the ectodomain of S2. They are S-e monomers, uncleaved S-e trimers, cleaved S-e trimers, and dissociated S1 monomers and S2 trimer rosettes. Lowered pH induces an irreversible transition from flexible, L-shaped S-e monomers to clove-shaped trimers. Protease cleavage of the trimer occurs at the S1-S2 boundary; an ensuing S1 dissociation leads to a major rearrangement of the trimeric S2 and to formation of rosettes likely to represent clusters of elongated, postfusion trimers of S2 associated through their fusion peptides. The states and transitions of S suggest conformational changes that mediate viral entry into cells.     

值得关切的严重急性呼吸综合征冠状病毒2型变异株

自2019年12月2019冠状病毒病暴发流行以来,严重急性呼吸综合征冠状病毒 2 型已经产生了1万个以上的变异株。其中有些可能获得更强的传染性,有的致病性得以提高,有的或许不能被现有的检测试剂检测出来,还有的也许能够逃逸疫苗的免疫保护作用。世界卫生组织于2021年5月31日发布了针对这些变异株的新的命名系统。本文对当前世界上流行较广的4个变异株进行综述,包括最近在广州市引起小暴发的δ变异株。     

Mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a distant member of the Group 2 coronaviruses, has recently been identified as the etiological agent of severe acute respiratory syndrome (SARS). The genome of SARS-CoV contains four structural genes that are homologous to genes found in other coronaviruses, as well as six subgroup-specific open reading frames (ORFs). ORF3 encodes a predicted 154-amino-acid protein that lacks similarity to any known protein, and is designated 3b in this article. We reported previously that SARS-CoV 3b is predominantly localized in the nucleolus, and induces G0/G1 arrest and apoptosis in transfected cells. In this study, we show that SARS-CoV 3b fused with EGFP at its N- or C- terminus co-localized with a mitochondria-specific marker in some transfected cells. Mutation analysis of SARS-CoV 3b revealed that the domain spanning amino acids 80 to 138 was essential for its mitochondria localization. These results provide new directions for studies of the role of SARS-CoV 3b protein in SARS pathogenesis.     

Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.     

In vitro inhibition of severe acute respiratory syndrome coronavirus by chloroquine

We report on chloroquine, a 4-amino-quinoline, as an effective inhibitor of the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) in vitro. Chloroquine is a clinically approved drug effective against malaria. We tested chloroquine phosphate for its antiviral potential against SARS-CoV-induced cytopathicity in Vero E6 cell culture. Results indicate that the IC50 of chloroquine for antiviral activity (8.8 +/- 1.2 microM) was significantly lower than its cytostatic activity; CC50 (261.3 +/- 14.5 microM), yielding a selectivity index of 30. The IC50 of chloroquine for inhibition of SARS-CoV in vitro approximates the plasma concentrations of chloroquine reached during treatment of acute malaria. Addition of chloroquine to infected cultures could be delayed for up to 5h postinfection, without an important drop in antiviral activity. Chloroquine, an old antimalarial drug, may be considered for immediate use in the prevention and treatment of SARS-CoV infections.     

Tea crude extracts effectively inactivate severe acute respiratory syndrome coronavirus 2

It is well known that black and green tea extracts, particularly polyphenols, have antimicrobial activity against various pathogenic microbes including viruses. However, there is limited data on the antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which emerged rapidly in China in late 2019 and which has been responsible for coronavirus disease 2019 (COVID-19) pandemic globally. In this study, 20 compounds and three extracts were obtained from black and green tea and found that three tea extracts showed significant antiviral activity against SARS-CoV-2, whereby the viral titre decreased about 5 logs TCID₅₀ per ml by 1·375 mg ml⁻¹ black tea extract and two-fold diluted tea bag infusion obtained from black tea when incubated at 25°C for 10 s. However, when concentrations of black and green tea extracts were equally adjusted to 344 µg ml⁻¹, green tea extracts showed more antiviral activity against SARS-CoV-2. This simple and highly respected beverage may be a cheap and widely acceptable means to reduce SARS-CoV-2 viral burden in the mouth and upper gastrointestinal and respiratory tracts in developed as well as developing countries.     

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein

The worldwide outbreak of severe acute respiratory syndrome (SARS) was shown to be associated with a novel coronavirus (CoV) now called SARS CoV. We report here the generation of SARS CoV S protein-pseudotyped murine leukemia virus (MLV) vector particles. The wild-type S protein pseudotyped MLV vectors, although at a low efficiency. Partial deletion of the cytoplasmic tail of S dramatically increased infectivity of pseudotypes, with titers only two- to threefold lower than those of pseudotypes generated in parallel with the vesicular stomatitis virus G protein. S-pseudotyped MLV particles were used to analyze viral tropism. MLV(SARS) pseudotypes and wild-type SARS CoV displayed similar cell types and tissue and host restrictions, indicating that the expression of a functional receptor is the major restraint in permissiveness to SARS CoV infection. Efficient gene transfer could be detected in Vero and CaCo2 cells, whereas the level of gene marking of 293T, HeLa, and HepG2 cells was only slightly above background levels. A cat cell line and a dog cell line were not susceptible. Interestingly, PK-15, a porcine kidney cell line, and primary porcine kidney cells were also highly permissive for SARS S pseudotypes and wild-type SARS CoV. This finding suggests that swine may be susceptible to SARS infection and may be a source for infection of humans. Taken together, these results indicate that MLV(SARS) pseudotypes are highly valuable for functional studies of viral tropism and entry and, in addition, can be a powerful tool for the development of therapeutic entry inhibitors without posing a biohazard to human beings.     

Modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro

The clinical picture of severe acute respiratory syndrome (SARS) is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome. However, the events that lead to the recruitment of leukocytes are poorly understood. To study the cellular response in the acute phase of SARS coronavirus (SARS-CoV)-host cell interaction, we investigated the induction of chemokines, adhesion molecules, and DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin) by SARS-CoV. Immunohistochemistry revealed neutrophil, macrophage, and CD8 T-cell infiltration in the lung autopsy of a SARS patient who died during the acute phase of illness. Additionally, pneumocytes and macrophages in the patient's lung expressed P-selectin and DC-SIGN. In in vitro study, we showed that the A549 and THP-1 cell lines were susceptible to SARS-CoV. A549 cells produced CCL2/monocyte chemoattractant protein 1 (MCP-1) and CXCL8/interleukin-8 (IL-8) after interaction with SARS-CoV and expressed P-selectin and VCAM-1. Moreover, SARS-CoV induced THP-1 cells to express CCL2/MCP-1, CXCL8/IL-8, CCL3/MIP-1alpha, CXCL10/IP-10, CCL4/MIP-1beta, and CCL5/RANTES, which attracted neutrophils, monocytes, and activated T cells in a chemotaxis assay. We also demonstrated that DC-SIGN was inducible in THP-1 as well as A549 cells after SARS-CoV infection. Our in vitro experiments modeling infection in humans together with the study of a lung biopsy of a patient who died during the early phase of infection demonstrated that SARS-CoV, through a dynamic interaction with lung epithelial cells and monocytic cells, creates an environment conducive for immune cell migration and accumulation that eventually leads to lung injury.     

Antibodies at work in the time of severe acute respiratory syndrome coronavirus 2

In view of devastating effects of COVID-19 on human life, there is an urgent need for the licened vaccines or therapeutics for the SARS-CoV-2 infection. Age-old passive immunization with protective antibodies to neutralize the virus is one of the strategies for emergency prophylaxis and therapy for coronavirus disease 2019 (COVID-19). In this review, the authors discuss up-to-date advances in immune-based therapy for COVID-19. The use of convalescent plasma therapy as the first line of defense to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been established, with encouraging results. Monoclonal antibodies (mAbs) that bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or block the interaction between SARS-CoV-2 RBD and the human angiotensin-converting enzyme 2 receptor have been found to be very promising as a countermeasure for tackling the SARS-CoV-2 infection, and clinical trials are underway. Considering the counterproductive antibody-dependent enhancement of the virus, mAbs therapy that is safe and efficacious, even in people with underlying conditions, will be a significant breakthrough. In addition, emerging immunotherapeutic interventions using nanobodies and cellular immunotherapy are promising avenues for tackling the COVID-19 pandemic. The authors also discuss the implication of mAbs as mediators of cytokine storm syndrome to modify the immune response of COVID-19 patients, thus reducing the fatality rate of COVID-19 infection.