Mosaic structure of human coronavirus NL63, one thousand years of evolution

Before the SARS outbreak only two human coronaviruses (HCoV) were known: HCoV-OC43 and HCoV-229E. With the discovery of SARS-CoV in 2003, a third family member was identified. Soon thereafter, we described the fourth human coronavirus (HCoV-NL63), a virus that has spread worldwide and is associated with croup in children. We report here the complete genome sequence of two HCoV-NL63 clinical isolates, designated Amsterdam 57 and Amsterdam 496. The genomes are 27,538 and 27,550 nucleotides long, respectively, and share the same genome organization. We identified two variable regions, one within the 1a and one within the S gene, whereas the 1b and N genes were most conserved. Phylogenetic analysis revealed that HCoV-NL63 genomes have a mosaic structure with multiple recombination sites. Additionally, employing three different algorithms, we assessed the evolutionary rate for the S gene of group Ib coronaviruses to be approximately 3 x 10(-4) substitutions per site per year. Using this evolutionary rate we determined that HCoV-NL63 diverged in the 11th century from its closest relative HCoV-229E.     

人冠状病毒NL63棘突蛋白的研究进展

20世纪60年代以前,人们普遍认为只有2种人冠状病毒(HCoV)HCoV-229E和HCoV-OC43感染人类,2002～2003年出现的由SARS-CoV引发的严重急性呼吸综合征(SARS)的流行使人冠状病毒又一次成为研究的焦点,2004年又发现了一种新的人冠状病毒HCoV-NL63。在此,主要就HCoV-NL63,特别是其主要结构蛋白——棘突蛋白的研究进展做一简要综述。     

Characterization and Complete Genome Sequence of Human Coronavirus NL63 Isolated in China

Human coronavirus NL63 (HCoV-NL63) was first discovered in Amsterdam in 2004 and was identified as a new human respiratory coronavirus. We here report the first complete genome sequence of HCoV-NL63 strain CBJ 037 isolated in 2008 from a patient with bronchitis in Beijing, China.     

Human coronavirus NL63, a new respiratory virus

From the mid-1960s onwards, it was believed that only two human coronavirus species infect humans: HCoV-229E and HCoV-OC43. Then, in 2003, a novel member of the coronavirus family was introduced into the human population: SARS-CoV, causing an aggressive lung disease. Fortunately, this virus was soon expelled from the human population, but it quickly became clear that the human coronavirus group contains more members then previously assumed, with HCoV-NL63 identified in 2004. Despite its recent discovery, ample results from HCoV-NL63 research have been described. We present an overview of the publications on this novel coronavirus.     

The Nucleocapsid Protein of Human Coronavirus NL63

Human coronavirus (HCoV) NL63 was first described in 2004 and is associated with respiratory tract disease of varying severity. At the genetic and structural level, HCoV-NL63 is similar to other members of the Coronavirinae subfamily, especially human coronavirus 229E (HCoV-229E). Detailed analysis, however, reveals several unique features of the pathogen. The coronaviral nucleocapsid protein is abundantly present in infected cells. It is a multi-domain, multi-functional protein important for viral replication and a number of cellular processes. The aim of the present study was to characterize the HCoV-NL63 nucleocapsid protein. Biochemical analyses revealed that the protein shares characteristics with homologous proteins encoded in other coronaviral genomes, with the N-terminal domain responsible for nucleic acid binding and the C-terminal domain involved in protein oligomerization. Surprisingly, analysis of the subcellular localization of the N protein of HCoV-NL63 revealed that, differently than homologous proteins from other coronaviral species except for SARS-CoV, it is not present in the nucleus of infected or transfected cells. Furthermore, no significant alteration in cell cycle progression in cells expressing the protein was observed. This is in stark contrast with results obtained for other coronaviruses, except for the SARS-CoV.     

北京地区人冠状病毒NL63 N和E蛋白基因序列及生物信息学分析

为了解北京地区新近发现的新型冠状病毒-人冠状病毒NL63(Human coronavirus NL63,HCoV-NL63)的N和E蛋白编码基因的特征,从经RT-PCR检测阳性的临床标本中扩增得到的HCoV-NL63 N蛋白和E蛋白编码基因序列,分别克隆至pCF-T和pUCm-T载体中并进行测序,同时运用生物信息学的方法,对北京HCoV-NL63阳性标本BJ8081 N和E蛋白编码基因的核苷酸和氨基酸序列与HCoV-NL63原型株及其他几种冠状病毒的N和E蛋白编码基因的核苷酸和氨基酸序列进行比较分析和种系进化分析;用SOPMA方法对BJ8081 N和E蛋白的二级结构进行了预测分析,并对N和E蛋白的其他生物学特性进行了预测分析.经序列比对分析发现,BJ8081 N蛋白氨基酸序列在78～85肽段(FYYLGTGP)内与所比较的其他冠状病毒N蛋白相应位置的氨基酸序列完全相同,提示此区段可能为包括HCoV-NL63在内的所有冠状病毒N蛋白的保守区域.在BJ8081 N蛋白氨基酸序列的100～121肽段可能是和基因组RNA相结合的位置;在BJ8081 E蛋白的15～37位氨基酸可能是E蛋白的跨膜区域.研究对BJ8081 N蛋白和E蛋白的编码基因序列进行了测定和生物信息学分析,为今后对HCoV-NL63的进一步深入研究奠定了基础.     

Pan-coronavirus fusion inhibitors as the hope for today and tomorrow

The last two decades of the 21st century have seen emerging zoonotic coronavirus(CoV)diseases,including severe acute respiratory syndrome(SARS)(Holmes 2003),Middle East respiratory syndrome(MERS)(Graham et al.,2013)and coronavirus disease 2019(COVID-19)(Jiang et al.,2020).     

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。     

Coronavirus 229E susceptibility in man-mouse hybrids is located on human chromosome 15

Human coronavirus 229E, n enveloped, RNA-containing virus, causes respiratory illness in man and is serologically related to murine coronavirus JHM, which causes acute and chronic demyelination in rodents. 229E displays a species-specific host range restriction whose genetic basis was studied in human-mouse hybrids. 229E replicated in human WI-38 cells but not in three mouse cell lines tested (RAG, LM/TK-, and A9). Human coronavirus sensitivity (HCVS) was expressed as a dominant phenotype in hybrids, indicating that mouse cells do not actively suppress 229E replication. HCVS segregated concordantly with the human chromosome 15 enzyme markers mannose phosphate isomerase (MPI) and the muscle form of pyruvate kinase (PKM2), and analysis of hybrids containing an X/15 translocation [t(X;15)(p11;q11)] localized HCVS to the q11 leads to qter region of chromosome 15. HCVS might code for a specific surface receptor, allowing 229E to be absorbed to and received within the host cell.     

人冠状病毒HCoV-NL63和HCoV-HKU1常规RT-PCR与实时荧光定量RT-PCR检测方法的建立及应用比较

分别设计HCoV-NL63和HCoV-HKU1特异的引物与荧光标记探针,并合成含靶基因的模板RNA,建立常规RT-PCR方法与实时荧光定量RT-PCR方法,对其灵敏性、特异性和可重复性以及用于临床样本的适用性等进行平行比较评价.结果表明:这两种方法皆可对HCoV-NL63或HCoV-HKU1进行特异性诊断,其中荧光定量RT-PCR方法检测灵敏度均可达10拷贝/25μL反应体积,不同批次重复检测结果间的变异系数均小于5%.上述方法应用于158份临床鼻咽拭子标本,其中荧光定量RT-PCR方法检出6份HCoV-NL63阳性标本,5份HCoV-HKU1阳性标本,而常规RT-PCR方法则分别检出HCoV-NL63阳性与HCoV-HKU1阳性各3份.对常规RT-PCR方法获得的阳性样品进行序列分析证实上述方法的可靠性.本实验成功建立了可用于临床标本检测的人冠状病毒HCoV-NL63和HCoV-HKU1常规RT-PCR方法与实时荧光定量RT-PCR检测方法,并初步证实荧光定量RT-PCR检测方法检出率明显高于常规RT-PCR方法,这为开展HCoV-NL63和HCoV-HKU1的流行监测及临床早期诊断提供了有效技术手段.     

Culturing the Unculturable: Human Coronavirus HKU1 Infects,Replicates, and Produces Progeny Virions in Human Ciliated Airway Epithelial Cell Cultures

Culturing newly identified human lung pathogens from clinical sample isolates can represent a daunting task, with problems ranging from low levels of pathogens to the presence of growth suppressive factors in the specimens, compounded by the lack of a suitable tissue culture system. However, it is critical to develop suitable in vitro platforms to isolate and characterize the replication kinetics and pathogenesis of recently identified human pathogens. HCoV-HKU1, a human coronavirus identified in a clinical sample from a patient with severe pneumonia, has been a major challenge for successful propagation on all immortalized cells tested to date. To determine if HCoV-HKU1 could replicate in in vitro models of human ciliated airway epithelial cell cultures (HAE) that recapitulate the morphology, biochemistry, and physiology of the human airway epithelium, the apical surfaces of HAE were inoculated with a clinical sample of HCoV-HKU1 (Cean1 strain). High virus yields were found for several days postinoculation and electron micrograph, Northern blot, and immunofluorescence data confirmed that HCoV-HKU1 replicated efficiently within ciliated cells, demonstrating that this cell type is infected by all human coronaviruses identified to date. Antiserum directed against human leukocyte antigen C (HLA-C) failed to attenuate HCoV-HKU1 infection and replication in HAE, suggesting that HLA-C is not required for HCoV-HKU1 infection of the human ciliated airway epithelium. We propose that the HAE model provides a ready platform for molecular studies and characterization of HCoV-HKU1 and in general serves as a robust technology for the recovery, amplification, adaptation, and characterization of novel coronaviruses and other respiratory viruses from clinical material.About 335 new or emerging infectious diseases have been identified since 1940 (23), and while many threaten human health, the global economy, and national security, respiratory pathogens are of particular public health concern. Using modern methods, several previously unknown viruses have been identified, including respiratory pathogens (1, 18, 27, 54, 57), yet research remains restricted to prevalence and disease association studies since a virus culture system is oftentimes lacking. Immortalized tissue culture cells are adapted to growth in laboratory conditions and, as such, display altered gene expression patterns, which may not be optimal for the replication of fastidious viruses. Primary cell-differentiated culture models provide alternative in vitro model systems closer in nature to the in vivo host tissue environment for infection studies and amplification of pathogens for further characterization. Here, we use an in vitro model of human ciliated airway epithelial cell cultures (HAE) that mimic the properties of the cartilaginous airway epithelium (17) to culture the previously unculturable human coronavirus HKU1 (HCoV-HKU1).Coronaviruses are important pathogens of humans and animals, causing a range of symptoms depending on the host. Following the severe acute respiratory syndrome (SARS)-CoV epidemic, several new strains of human coronaviruses were identified by molecular techniques, including HCoV-NL63, identified in the Netherlands from an infant with bronchiolitis (54), and HCoV-HKU1, identified in an adult patient with severe pneumonia in Hong Kong (57). HCoV-NL63 has been demonstrated to infect and replicate in both conventional immortalized cells and human ciliated airway cell cultures, producing sufficient amounts of virus for characterization studies of viral replication and pathogenesis and the successful development of an infectious clone (3, 13, 22, 41). In contrast, little is known about HCoV-HKU1, as no in vitro replication model has been identified to date, limiting further investigations of the virus.Clinical isolates of previously isolated human coronaviruses have been adapted to replicate in standard transformed cell culture; for example, SARS-CoV and HCoV-NL63 replicate efficiently in epithelial monkey kidney cells (VeroE6 and LLC-MK2), HCoV-OC43 in BHK21 cells, and HCoV-229E in MRC5 cells (14, 24, 35, 47, 54, 59). Despite the successful amplification of these human coronaviruses in cell lines, all attempts to date to culture a clinical isolate of HCoV-HKU1 have failed. No HCoV-HKU1 genomic replication was observed after inoculation of standard cell lines previously utilized for virus propagation, including RD (human rhabdomyosarcoma cells), HRT-18 (colorectal adenocarcinoma cells), HEp-2 (human epithelial carcinoma cells), MRC-5 (human lung fibroblast cells), A549 (human lung epithelial adenocarcinoma cells), Caco2 (human colorectal adenocarcinoma cells), Huh-7 (human hepatoma cells), B95a (marmoset B-lymphoblastoid cells), mixed neuron-glia culture, LLC-MK2 (rhesus monkey kidney cells), FRhK-4 (rhesus monkey kidney cells), BSC-1 (African green monkey kidney cells), Vero E6 (African green monkey kidney cells), MDCK (Madin-Darby canine kidney cells), I13.35 (murine macrophage cells), and L929 (murine fibroblast cells) (57).Here, we use human ciliated airway epithelial cell cultures to successfully propagate HCoV-HKU1 for the first time in vitro. In this culture model, HCoV-HKU1 genome copy numbers increased by several logs over the initial three-day incubation period and electron micrograph, Northern blot, and immunofluorescence data confirmed HKU1 replication in HAE and that ciliated cells were the preferential target for virus infection, the same cell type infected by all human coronaviruses tested so far in these model systems.     

SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells

Viruses require specific cellular receptors to infect their target cells. Angiotensin-converting enzyme 2 (ACE2) is a cellular receptor for two divergent coronaviruses, SARS coronavirus (SARS-CoV) and human coronavirus NL63 (HCoV-NL63). In addition to hostcell receptors, lysosomal cysteine proteases are required for productive infection by some viruses. Here we show that SARS-CoV, but not HCoV-NL63, utilizes the enzymatic activity of the cysteine protease cathepsin L to infect ACE2-expressing cells. Inhibitors of cathepsin L blocked infection by SARS-CoV and by a retrovirus pseudotyped with the SARS-CoV spike (S) protein but not infection by HCoV-NL63 or a retrovirus pseudotyped with the HCoV-NL63 S protein. Expression of exogenous cathepsin L substantially enhanced infection mediated by the SARS-CoV S protein and by filovirus GP proteins but not by the HCoV-NL63 S protein or the vesicular stomatitis virus G protein. Finally, an inhibitor of endosomal acidification had substantially less effect on infection mediated by the HCoV-NL63 S protein than on that mediated by the SARS-CoV S protein. Our data indicate that two coronaviruses that utilize a common receptor nonetheless enter cells through distinct mechanisms.     

Involvement of Aminopeptidase N (CD13) in Infection of Human Neural Cells by Human Coronavirus 229E

Attachment to a cell surface receptor can be a major determinant of virus tropism. Previous studies have shown that human respiratory coronavirus HCV-229E uses human aminopeptidase N (hAPN [CD13]) as its cellular receptor for infection of lung fibroblasts. Although human coronaviruses are recognized respiratory pathogens, occasional reports have suggested their possible neurotropism. We have previously shown that human neural cells, including glial cells in primary cultures, are susceptible to human coronavirus infection in vitro (A. Bonavia, N. Arbour, V. W. Yong, and P. J. Talbot, J. Virol. 71:800–806, 1997). However, the only reported expression of hAPN in the nervous system is at the level of nerve synapses. Therefore, we asked whether hAPN is utilized as a cellular receptor for infection of these human neural cell lines. Using flow cytometry, we were able to show the expression of hAPN on the surfaces of various human neuronal and glial cell lines that are susceptible to HCV-229E infection. An hAPN-specific monoclonal antibody (WM15), but not control antibody, inhibited the attachment of radiolabeled HCV-229E to astrocytic, neuronal, and oligodendrocytic cell lines. A correlation between the apparent amount of cell surface hAPN and the level of virus attachment was observed. Furthermore, the presence of WM15 inhibited virus infection of these cell lines, as detected by indirect immunofluorescence. These results indicate that hAPN (CD13) is expressed on neuronal and glial cell lines in vitro and serves as the receptor for infection by HCV-229E. This further strengthens the neurotropic potential of this human respiratory virus.     

Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63

Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease cis-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.     

Core structure of S2 from the human coronavirus NL63 spike glycoprotein

Human coronavirus NL63 (HCoV-NL63) has recently been identified as a causative agent of acute respiratory tract illnesses in infants and young children. The HCoV-NL63 spike (S) protein mediates virion attachment to cells and subsequent fusion of the viral and cellular membranes. This viral entry process is a primary target for vaccine and drug development. HCoV-NL63 S is expressed as a single-chain glycoprotein and consists of an N-terminal receptor-binding domain (S1) and a C-terminal transmembrane fusion domain (S2). The latter contains two highly conserved heptad-repeat (HR) sequences that are each extended by 14 amino acids relative to those of the SARS coronavirus or the prototypic murine coronavirus, mouse hepatitis virus. Limited proteolysis studies of the HCoV-NL63 S2 fusion core identify an alpha-helical domain composed of a trimer of the HR segments N57 and C42. The crystal structure of this complex reveals three C42 helices entwined in an oblique and antiparallel manner around a central triple-stranded coiled coil formed by three N57 helices. The overall geometry comprises distinctive high-affinity conformations of interacting cross-sectional layers of the six helices. As a result, this structure is unusually stable, with an apparent melting temperature of 78 degrees C in the presence of the denaturant guanidine hydrochloride at 5 M concentration. The extended HR regions may therefore be required to prime the group 1 S glycoproteins for their fusion-activating conformational changes during viral entry. Our results provide an initial basis for understanding an intriguing interplay between the presence or absence of proteolytic maturation among the coronavirus groups and the membrane fusion activity of their S glycoproteins. This study also suggests a potential strategy for the development of improved HCoV-NL63 fusion inhibitors.     

Human respiratory coronavirus OC43: genetic stability and neuroinvasion

The complete genome sequences of the human coronavirus OC43 (HCoV-OC43) laboratory strain from the American Type Culture Collection (ATCC), and a HCoV-OC43 clinical isolate, designated Paris, were obtained. Both genomes are 30,713 nucleotides long, excluding the poly(A) tail, and only differ by 6 nucleotides. These six mutations are scattered throughout the genome and give rise to only two amino acid substitutions: one in the spike protein gene (I958F) and the other in the nucleocapsid protein gene (V81A). Furthermore, the two variants were shown to reach the central nervous system (CNS) after intranasal inoculation in BALB/c mice, demonstrating neuroinvasive properties. Even though the ATCC strain could penetrate the CNS more effectively than the Paris 2001 isolate, these results suggest that intrinsic neuroinvasive properties already existed for the HCoV-OC43 ATCC human respiratory isolate from the 1960s before it was propagated in newborn mouse brains. It also demonstrates that the molecular structure of HCoV-OC43 is very stable in the environment (the two variants were isolated ca. 40 years apart) despite virus shedding and chances of persistence in the host. The genomes of the two HCoV-OC43 variants display 71, 53.1, and 51.2% identity with those of mouse hepatitis virus A59, severe acute respiratory syndrome human coronavirus Tor2 strain (SARS-HCoV Tor2), and human coronavirus 229E (HCoV-229E), respectively. HCoV-OC43 also possesses well-conserved motifs with regard to the genome sequence of the SARS-HCoV Tor2, especially in open reading frame 1b. These results suggest that HCoV-OC43 and SARS-HCoV may share several important functional properties and that HCoV-OC43 may be used as a model to study the biology of SARS-HCoV without the need for level three biological facilities.     

Protease-mediated entry via the endosome of human coronavirus 229E

Human coronavirus 229E, classified as a group I coronavirus, utilizes human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.