MERS冠状病毒3C样蛋白酶是一种新的细胞自噬诱导蛋白

冠状病毒（Coronavirus, CoV）3C样蛋白酶（3CLpro）在冠状病毒复制过程中起重要作用,是一种重要的潜在抗病毒药物候选靶标。细胞自噬是宿主重要抗病毒防御机制之一,但目前冠状病毒诱导细胞自噬及其机制还不很清楚。本研究以人类新发高致病性冠状病毒 --中东呼吸综合征冠状病毒（MERS CoV）为研究对象,探讨人类冠状病毒感染与细胞自噬的关系。通过免疫荧光法检测发现,MERS 3CLpro引起细胞内eGFP-LC3B绿色荧光点状聚集,同时MERS 3CLpro诱导自噬标志蛋白微管相关蛋白1-轻链3基 (LC3-II)表达增多,表明MERS 3CLpro可激活细胞自噬。进一步研究发现,MERS 3CLpro诱导细胞自噬体形成而阻断或抑制自噬溶酶体形成,即MERS 3CLpro诱导不完全细胞自噬效应,而且MERS 3CLpro诱导细胞自噬具有时间依赖性且不依赖于其蛋白酶催化活性。此外发现SARS CoV和NL63 CoV等其它人类冠状病毒3CLpro也具有诱导细胞自噬效应,表明3CLpro诱导细胞自噬可能是人类冠状病毒所具有的一种普遍生物学特性。本研究首次发现冠状病毒蛋白酶3CLpro能诱导宿主细胞自噬,是一种新型冠状病毒来源的宿主细胞自噬诱导蛋白,这一发现拓展了对人类冠状病毒蛋白酶功能的新认识,为研究冠状病毒与宿主抗病毒天然免疫以及以病毒蛋白酶为靶标的抗病毒药物研究提供了理论基础。     

Live, attenuated coronavirus vaccines through the directed deletion of group-specific genes provide protection against feline infectious peritonitis

Feline infectious peritonitis (FIP) is a fatal immunity-mediated disease caused by mutants of a ubiquitous coronavirus. Since previous attempts to protect cats under laboratory and field conditions have been largely unsuccessful, we used our recently developed system of reverse genetics (B. J. Haijema, H. Volders, and P. J. M. Rottier, J. Virol. 77:4528-4538, 2003) for the development of a modified live FIP vaccine. With this objective, we deleted the group-specific gene cluster open reading frame 3abc or 7ab and obtained deletion mutant viruses that not only multiplied well in cell culture but also showed an attenuated phenotype in the cat. At doses at which the wild-type virus would be fatal, the mutants with gene deletions did not cause any clinical symptoms. They still induced an immune response, however, as judged from the high levels of virus-neutralizing antibodies. The FIP virus (FIPV) mutant lacking the 3abc cluster and, to a lesser extent, the mutant missing the 7ab cluster, protected cats against a lethal homologous challenge; no protection was obtained with the mutant devoid of both gene clusters. Our studies show that the deletion of group-specific genes from the coronavirus genome results in live attenuated candidate vaccines against FIPV. More generally, our approach may allow the development of vaccines against infections with other pathogenic coronaviruses, including that causing severe acute respiratory syndrome in humans.     

猪流行性腹泻病毒通过病毒蛋白酶激活Caspase-3诱导细胞凋亡

冠状病毒是一大类能够引起呼吸系统疾病,从而威胁人类健康的病毒．目前,对冠状病毒诱导细胞凋亡及其机制研究甚少.本研究以动物冠状病毒 猪流行性腹泻病毒(PEDV) 为模型探讨冠状病毒诱导细胞凋亡效应及其可能作用机制. 通过流式细胞术检测发现感染PEDV病毒后细胞凋亡率明显升高,且PEDV诱导细胞凋亡呈时间和剂量依赖性(P<0.05或P<0.01);进一步研究发现,冠状病毒木瓜样蛋白酶（PLP）在病毒引起凋亡过程中起重要作用.实验发现,转染PEDV-PLP质粒后,caspase-3活化体表达水平明显升高. 提示冠状病毒PLP蛋白酶通过激活caspase-3在病毒诱导细胞凋亡过程中起着关键作用. 以上结果为研究人类冠状病毒PLP蛋白功能及其通过细胞凋亡调节宿主抗病毒天然免疫机制提供重要基础.     

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Does Not Replicate in Syrian Hamsters

In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters.     

Natural history of a recurrent feline coronavirus infection and the role of cellular immunity in survival and disease

We describe the natural history, viral dynamics, and immunobiology of feline infectious peritonitis (FIP), a highly lethal coronavirus infection. A severe recurrent infection developed, typified by viral persistence and acute lymphopenia, with waves of enhanced viral replication coinciding with fever, weight loss, and depletion of CD4+ and CD8+ T cells. Our combined observations suggest a model for FIP pathogenesis in which virus-induced T-cell depletion and the antiviral T-cell response are opposing forces and in which the efficacy of early T-cell responses critically determines the outcome of the infection. Rising amounts of viral RNA in the blood, consistently seen in animals with end-stage FIP, indicate that progression to fatal disease is the direct consequence of a loss of immune control, resulting in unchecked viral replication. The pathogenic phenomena described here likely bear relevance to other severe coronavirus infections, in particular severe acute respiratory syndrome, for which multiphasic disease progression and acute T-cell lymphopenia have also been reported. Experimental FIP presents a relevant, safe, and well-defined model to study coronavirus-mediated immunosuppression and should provide an attractive and convenient system for in vivo testing of anticoronaviral drugs.     

Intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence.

Cats infected with virulent feline coronavirus strains develop feline infectious peritonitis, an invariably fatal, immunologically mediated disease; avirulent strains cause either clinically inapparent infection or mild enteritis. Four virulent coronavirus isolates and five avirulent isolates were assessed by immunofluorescence and virus titration for their ability to infect and replicate in feline peritoneal macrophages in vitro. The avirulent coronaviruses infected fewer macrophages, produced lower virus titers, were less able to sustain viral replication, and spread less efficiently to other susceptible macrophages than the virulent coronaviruses. Thus, the intrinsic resistance of feline macrophages may play a pivotal role in the outcome of coronavirus infection in vivo.     

Recombination and Coronavirus Defective Interfering RNAs

Naturally occurring defective interfering RNAs have been found in 4 of 14 coronavirus species. They range in size from 2.2 kb to approximately 25 kb, or 80% of the 30-kb parent virus genome. The large DI RNAs do not in all cases appear to require helper virus for intracellular replication and it has been postulated that they may on their own function as agents of disease. Coronavirus DI RNAs appear to arise by internal deletions (through nonhomologous recombination events) on the virus genome or on DI RNAs of larger size by a polymerase strand-switching (copy-choice) mechanism. In addition to their use in the study of virus RNA replication and virus assembly, coronavirus DI RNAs are being used in a major way to study the mechanism of a high-frequency, site-specific RNA recombination event that leads to leader acquisition during virus replication (i.e., the leader fusion event that occurs during synthesis of subgenomic mRNAs, and the leader-switching event that can occur during DI RNA replication), a distinguishing feature of coronaviruses (and arteriviruses). Coronavirus DI RNAs are also being engineered as vehicles for the generation of targeted recombinants of the parent virus genome.     

Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagyinducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM’s inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.     

SARS virus: The beginning of the unraveling of a new coronavirus

Severe acute respiratory syndrome (SARS) virus caused a severe outbreak in several regions of the world in 2003. The virus is a novel coronavirus, which may have an origin in wild animals such as civet cats in southern China. Its genome structure, gene expression pattern and protein profiles are similar to those of other coronaviruses. However, distinct patterns of several open reading frames in the SARS virus genome may contribute to its severe virulence. The potential mutability of the coronavirus genome may pose problems in the control of future SARS outbreaks. The mechanism of SARS pathogenesis may involve both direct viral cytocidal effects on the target cells and immune-mediated mechanisms. The life cycle of the SARS virus is largely unknown; however, based on the analogy with other coronaviruses, several potential targets for antiviral development are identified. Vaccines offer an important preventive measure for possible future recurrences of SARS, but the prospect for their development is still unknown because of the uncertainty regarding the role of immune responses in SARS virus pathogenesis. The comparative studies of other coronaviruses offer insights into the understanding of SARS virus.     

Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.     

Coronaviruses: emerging and re-emerging pathogens in humans and animals

The severe acute respiratory syndrome coronavirus (SARS-CoV) and recently emerged Middle East respiratory syndrome coronavirus (MERS-CoV) epidemics have proven the ability of coronaviruses to cross species barrier and emerge rapidly in humans. Other coronaviruses such as porcine epidemic diarrhea virus (PEDV) are also known to cause major disease epidemics in animals wiith huge economic loss. This special issue in Virology Journal aims to highlight the advances and key discoveries in the animal origin, viral evolution, epidemiology, diagnostics and pathogenesis of the emerging and re-emerging coronaviruses in both humans and animals.     

Repurposing existing drugs: identification of SARS-CoV-2 3C-like protease inhibitors

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19). Since its emergence, the COVID-19 pandemic has not only distressed medical services but also caused economic upheavals, marking urgent the need for effective therapeutics. The experience of combating SARS-CoV and MERS-CoV has shown that inhibiting the 3-chymotrypsin-like protease (3CLpro) blocks the replication of the virus. Given the well-studied properties of FDA-approved drugs, identification of SARS-CoV-2 3CLpro inhibitors in an FDA-approved drug library would be of great therapeutic value. Here, we screened a library consisting of 774 FDA-approved drugs for potent SARS-CoV-2 3CLpro inhibitors, using an intramolecularly quenched fluorescence (IQF) peptide substrate. Ethacrynic acid, naproxen, allopurinol, butenafine hydrochloride, raloxifene hydrochloride, tranylcypromine hydrochloride, and saquinavir mesylate have been found to block the proteolytic activity of SARS-CoV-2 3CLpro. The inhibitory activity of these repurposing drugs against SARS-CoV-2 3CLpro highlights their therapeutic potential for treating COVID-19 and other Betacoronavirus infections.     

Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer,Integral Membrane and Nucleocapsid Proteins of Feline,Canine and Porcine Coronaviruses

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU-2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.     

Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses

Both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. Expeditious research on the severe acute respiratory syndrome (SARS) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell-death regulation during infection. Apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from SARS patients. Viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. Occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. The diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. As a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell-death pathway. Continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets.     

新型冠状病毒感染与复制的进展

冠状病毒是一类具有囊膜包裹的线性单股正链RNA病毒,在自然界广泛存在,可引起不同程度的呼吸性传染病。新型冠状病毒是一种新发突发病毒,对各类人群均易感。截止目前,该病已经在世界范围内广泛流行,对公共卫生安全构成极大的威胁。文中从冠状病毒及新型冠状病毒的基因组特征、关键蛋白、对宿主的感染和复制的角度加以综述,旨在为获得病毒侵染宿主细胞致病机制的探究提供理论依据,也为特异的抗病毒药物的研发提供基础支持。     

Chimeric Exchange of Coronavirus nsp5 Proteases (3CLpro) Identifies Common and Divergent Regulatory Determinants of Protease Activity

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp''s 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp''s at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.     

Sequence analysis of the nucleocapsid gene of feline coronaviruses circulating in Italy

Molecular analysis of the N genes of feline coronaviruses (FCoV) strains detected in naturally infected cats were carried out to investigate the genetic diversity among these viruses. Phylogeny showed a general clustering trend on the basis of geographic origin rather than on virulence characteristics. The analysis of the pattern of nucleotide substitutions disclosed "hot spots" sites which may represent immunological domains. In conclusion, our results demonstrate that the N gene does not carry mutations associated with the pathotypical switch FECV --> FIPV. During persistent infection, the individual qualitative immune response might address the accumulations of mutations in the N gene and the development of FIP.     

新型人冠状病毒HCoV—NL63和HCoV-HKU1的研究进展

多年来,人们公认的人冠状病毒包括HCoV-229E和HCoV-0C43等2株。但2002-2003年,由一种新型人冠状病毒SARS-CoV所引发的全球范围的严重急性呼吸综合征（SARS）的流行,使多国蒙受巨大损失,由此,冠状病毒又成为研究的焦点。随着分子生物学技术的发展,2004-2005年,又发现了2种新型人冠状病毒HCoV-NL63和HCoV-HKU1。在此,就这2种病毒的发现、流行情况,及其与疾病的相关性做一简要综述。     

Cinanserin is an inhibitor of the 3C-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro

The 3C-like proteinase (3CLpro) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is one of the most promising targets for anti-SARS-CoV drugs due to its crucial role in the viral life cycle. In this study, a database containing structural information of more than 8,000 existing drugs was virtually screened by a docking approach to identify potential binding molecules of SARS-CoV 3CLpro. As a target for screening, both a homology model and the crystallographic structure of the binding pocket of the enzyme were used. Cinanserin (SQ 10,643), a well-characterized serotonin antagonist that has undergone preliminary clinical testing in humans in the 1960s, showed a high score in the screening and was chosen for further experimental evaluation. Binding of both cinanserin and its hydrochloride to bacterially expressed 3CLpro of SARS-CoV and the related human coronavirus 229E (HCoV-229E) was demonstrated by surface plasmon resonance technology. The catalytic activity of both enzymes was inhibited with 50% inhibitory concentration (IC50) values of 5 microM, as tested with a fluorogenic substrate. The antiviral activity of cinanserin was further evaluated in tissue culture assays, namely, a replicon system based on HCoV-229E and quantitative test assays with infectious SARS-CoV and HCoV-229E. All assays revealed a strong inhibition of coronavirus replication at nontoxic drug concentrations. The level of virus RNA and infectious particles was reduced by up to 4 log units, with IC50 values ranging from 19 to 34 microM. These findings demonstrate that the old drug cinanserin is an inhibitor of SARS-CoV replication, acting most likely via inhibition of the 3CL proteinase.