Structure of the Human Respiratory Syncytial Virus M2-1 Protein in Complex with a Short Positive-Sense Gene-End RNA

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人呼吸道合胞病毒M蛋白非复制型重组腺病毒的构建和鉴定

人呼吸道合胞病毒（human respiratory syncytial virus, RSV)基质蛋白（matrix protein,M）在RSV形态发生上具有重要作用,因含有CTL抗原表位,在疫苗研究上具有一定意义。为此,应用RT-PCR 方法从感染RSV的HEp-2 细胞中扩增获得M蛋白基因,构建了含M基因的非复制型重组腺病毒并进行表达和鉴定。基因序列分析显示RSV M基因仅有一处碱基发生错义突变。非复制型重组腺病毒DNA分子FGAd/RSVM转染293细胞,观察到细胞出现CPE,RT-PCR发现M基因有转录,Western blotting及间接免疫荧光分析检测到M蛋白。成功克隆A亚型RSV Long株M基因,并获得一株可表达A亚型RSV M蛋白的非复制型重组腺病毒FGAd/RSVM,可用于体内研究观察其免疫效果及免疫保护作用。     

抗呼吸道合胞病毒融合蛋白单链抗体的筛选及初步鉴定

目的：从大容量噬菌体抗体库中筛选人源性抗呼吸道合胞病毒F蛋白的单链抗体。方法：以RSV F蛋白为靶抗原,通过“吸附-洗涤-洗脱-扩增”过程从天然人源性噬菌体抗体库中筛选特异性抗F蛋白单链抗体。5轮筛选后,单克隆经ELISA检测,阳性克隆进行核酸序列分析,并将阳性克隆噬菌体感染E.coli HB2151,经IPTG诱导,制备抗RSV F蛋白的可溶性单链抗体,并进行Western及Dot blot分析。结果：经过筛选,获得了18株能与F蛋白特异性结合的阳性克隆,取OD值最高的克隆E4经测序并检索Kabat数据库分析,显示其基因与人免疫球蛋白可变区基因具有高度同源性,Western及Dot blot分析表明为单链抗体。结论：利用天然人源性噬菌体抗体库技术制备出高特异性的人源性抗RSV F蛋白单链抗体。     

Intrapatient Variation of the Respiratory Syncytial Virus Attachment Protein Gene

Intrapatient variability of the attachment (G) protein gene of respiratory syncytial virus (RSV) was examined using both population and single-genome sequencing. Samples from three patients infected with a group B virus variant which has a 60-nucleotide duplication in the G protein gene were examined. These samples were chosen because occasional mixed sequence bases were observed. In a minority of RSV genomes from these patients considerable variability was found, including point mutations, insertions, and deletions. Of particular note, the deletion of the exact portion of the gene which had been duplicated in some isolates was observed in viral RNAs from two patients.Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants and vulnerable adults (3, 9) and is unusual in that it can repeatedly reinfect individuals (5, 6). RSV isolates are classified into two groups, A and B, and the attachment (G) protein, a target for neutralizing antibodies, is the most variable of the viral proteins, showing considerable genetic and antigenic variability both within and between the groups (7, 8). The G protein is able to accommodate drastic changes, which have been observed both in culture during the selection of monoclonal antibody escape mutants (4, 12, 13) and in vivo with the emergence of new variants, including a group B strain with a duplication of 60 nucleotides (17). This strain with a 60-nucleotide duplication was first reported from Buenos Aires in 1999 (17) and then was subsequently detected in samples from 1998 in Madrid (16). The strain then became the dominant group B strain worldwide, indicating a selective advantage for this variant (16, 18). Thus, major genetic changes can be introduced into the G gene sequence while the virus replicates in its natural host, which can then be selected under favorable epidemiological conditions.Previous investigations of the genetic diversity of RSV exploited direct sequencing of PCR-amplified products (2), which represent the population average of the in vivo variants. Such sequences are derived from multiple copies of cDNA and represent the dominant sequence, and they thus do not allow detection of minority populations below about 20% prevalence (11). Information on intrapatient viral diversity during infections may therefore be missed, knowledge of which could be important in the overall understanding of the genetic diversity of this virus. We report here the analysis of individual RSV RNA molecules derived by single-genome amplification (SGA) and sequencing from clinical samples using a methodology developed for the analysis of HIV genomes (11, 14).RSV-positive samples were collected from infants admitted to Kilifi District Hospital, Kenya (10). Viral RNA extraction and cDNA synthesis were carried out as previously described (15). For population sequencing, a nested PCR was carried out on the cDNA using primers that amplified the ectodomain-coding part of the G protein gene, with the PCR product being directly sequenced. In the 2007-2008 RSV epidemic in Kilifi, group B viruses were predominant. By population sequencing of ∼100 group B samples, all were found to have the 60-nucleotide duplication observed in the Buenos Aires variant (data not shown). However, in some specimens there were some mixed bases at some positions, so the variability at the level of the single cDNA molecule was further investigated.Three samples that gave occasional mixed signals in the sequence chromatograms were further analyzed by SGA and sequencing. For SGA the cDNAs were serially diluted 3-fold up to 1:6,361. Ten nested PCRs were carried out on each dilution using Platinum high-fidelity PCR Supermix (Invitrogen) (containing Taq polymerase together with the proofreading enzyme Pyrococcus species GB-D polymerase). Based on the Poisson distribution, it has been shown that for a sample dilution yielding approximately 30% positive PCRs there is an 80% likelihood that each PCR is derived from a single cDNA molecule (11). For each of the identified endpoint dilutions, the cDNA was amplified in 80 separate nested PCRs using the high-fidelity enzyme and the positive reaction products sequenced. The nomenclature for the sequences reported in this paper is place of isolation (Kenya [Ken])/year of isolation/strain number. For SGA sequences an additional Roman number is given.The predicted length derived by population sequencing of the G proteins of the three samples examined by SGA was 310 amino acids, showing a 6-nucleotide deletion and a changed stop codon relative to the Buenos Aires strain (Fig. ​(Fig.1).1). The dominant sequences represented 60 to 88% of the sequences derived by SGA. The differences were due to point mutations, duplications, and deletions, as summarized in Table ​Table1;1; the consequences of these changes for the predicted length of the G protein are shown in Table ​Table22.Open in a separate windowFIG. 1.Nucleotide sequence alignment of part of the G protein gene (from nucleotide 400) of the sample 2 population sequence (Ken/08/80900) and a minority sequence (Ken/08/80900/ii), with the sequences of prototype group B strain CH18537 (accession number {"type":"entrez-nucleotide","attrs":{"text":"M17213","term_id":"333942","term_text":"M17213"}}M17213) and Buenos Aires strain BA/3833/99B (accession number {"type":"entrez-nucleotide","attrs":{"text":"AY333362","term_id":"33355616","term_text":"AY333362"}}AY333362). This shows the duplication of 60 nucleotides in the Kenyan and Buenos Aires viruses relative to CH18537 and the loss of the same 60 nucleotides in the Kenyan minority sequence. Termination codons are underlined.

TABLE 1.

Diversity in the SGA-derived sequences

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2^nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2^nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2^nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.     

Antigenic Structure of Human Respiratory Syncytial Virus Fusion Glycoprotein

New series of escape mutants of human respiratory syncytial virus were prepared with monoclonal antibodies specific for the fusion (F) protein. Sequence changes selected in the escape mutants identified two new antigenic sites (V and VI) recognized by neutralizing antibodies and a group-specific site (I) in the F1 chain of the F molecule. The new epitopes, and previously identified antigenic sites, were incorporated into a refined prediction of secondary-structure motifs to generate a detailed antigenic map of the F glycoprotein.     

The Respiratory Syncytial Virus Matrix Protein Possesses a Crm1-Mediated Nuclear Export Mechanism

The respiratory syncytial virus (RSV) matrix (M) protein is localized in the nucleus of infected cells early in infection but is mostly cytoplasmic late in infection. We have previously shown that M localizes in the nucleus through the action of the importin β1 nuclear import receptor. Here, we establish for the first time that M''s ability to shuttle to the cytoplasm is due to the action of the nuclear export receptor Crm1, as shown in infected cells, and in cells transfected to express green fluorescent protein (GFP)-M fusion proteins. Specific inhibition of Crm1-mediated nuclear export by leptomycin B increased M nuclear accumulation. Analysis of truncated and point-mutated M derivatives indicated that Crm1-dependent nuclear export of M is attributable to a nuclear export signal (NES) within residues 194 to 206. Importantly, inhibition of M nuclear export resulted in reduced virus production, and a recombinant RSV carrying a mutated NES could not be rescued by reverse genetics. That this is likely to be due to the inability of a nuclear export deficient M to localize to regions of virus assembly is indicated by the fact that a nuclear-export-deficient GFP-M fails to localize to regions of virus assembly when expressed in cells infected with wild-type RSV. Together, our data suggest that Crm1-dependent nuclear export of M is central to RSV infection, representing the first report of such a mechanism for a paramyxovirus M protein and with important implications for related paramyxoviruses.The Pneumovirus respiratory syncytial virus (RSV) within the Paramyxoviridae family is the most common cause of lower-respiratory-tract disease in infants (7). The negative-sense single-strand RNA genome of RSV encodes two nonstructural and nine structural proteins, comprising the envelope glycoproteins (F, G, and SH), the nucleocapsid proteins (N, P, and L), the nucleocapsid-associated proteins (M2-1 and M2-2), and the matrix (M) protein (1, 7, 11). Previously, we have shown that M protein localizes in the nucleus at early stages of infection, but later in infection it is localized mainly in the cytoplasm, in association with nucleocapsid-containing cytoplasmic inclusions (13, 16). The M proteins of other negative-strand viruses, such as Sendai virus, Newcastle disease virus, and vesicular stomatitis virus (VSV), have also been observed in the nucleus at early stages of infection (32, 40, 48). Interestingly, the M proteins of all of these viruses, including RSV, play major roles in virus assembly, which take place in the cytoplasm and at the cell membrane (11, 12, 24, 34, 36, 39), but the mechanisms by which trafficking between the nucleus and cytoplasm occurs are unknown.The importin β family member Crm1 (exportin 1) is known to mediate nuclear export of proteins bearing leucine-rich nuclear export signals (NES) (8, 9, 18, 19, 37, 42, 43), such as the human immunodeficiency virus type 1 Rev protein (4). In the case of the influenza virus matrix (M1) protein, binding to the influenza virus nuclear export protein, which possesses a Crm1-recognized NES, appears to be responsible for its export from the nucleus, bound to the influenza virus RNA (3).We have recently shown that RSV M localizes in the nucleus through a conventional nuclear import pathway dependent on the nuclear import receptor importin β1 (IMPβ1) and the guanine nucleotide-binding protein Ran (14). In the present study, we show for the first time that RSV M possesses a Crm1-dependent nuclear export pathway, based on experiments using the specific inhibitor leptomycin B (LMB) (25), both in RSV-infected cells and in green fluorescent protein (GFP)-M fusion protein-expressing transfected cells. We use truncated and point-mutated M derivatives to map the Crm1-recognized NES within the M sequence and show that Crm1-dependent nuclear export is critical to the RSV infectious cycle, since LMB treatment early in infection, inhibiting M export from the nucleus, reduces RSV virion production and a recombinant RSV carrying a NES mutation in M was unable to replicate, probably because M deficient in nuclear export could not localize to areas of virus assembly, as shown in RSV-infected cells transfected to express GFP-M. This is the first report of a Crm1-mediated nuclear export pathway for a paramyxovirus M protein, with implications for the trafficking and function of other paramyxovirus M proteins.     

重组呼吸道合胞病毒G蛋白疫苗的免疫安全性研究

将呼吸道合胞病毒(respiratory syncytical virus,RSV)G蛋白与CpG佐剂共同免疫小鼠,分析RSV G蛋白免疫原性及安全性。前期纯化的G_(CX3C)和G_(CTL)两种蛋白分别与CpG佐剂混合,在0、2、4周以肌肉注射方式免疫昆明小鼠,免疫结束后检测小鼠肺部匀浆液中的IFN-γ、IL-4及Ig E等指标。同时末次免疫结束后,小鼠用滴度105pfu的RSV进行攻毒,解剖分离小鼠肺部,制备病理切片进行观察。G_(CTL)蛋白免疫组肺部匀浆液中的IL-4以及IgE低于G_(CX3C)蛋白免疫组。CpG组及GCTL+CpG组肺组织匀浆液中的INF-γ含量以及INF-γ/IL-4比值显著高于其他组(P0.05),且ELISPOT实验表明CpG佐剂能够促使分泌INF-γ的脾淋巴细胞数量增多。攻毒后,通过肺部组织切片观察,发现PBS和CpG免疫组肺部病变极为严重,G_(CTL)+CpG组的病变程度比G_(CX3C)+CpG组严重,而G_(CX3C)组的病变情况比G_(CTL)组严重。这些结果表明,重组G_(CTL)蛋白能够降低动物免疫应答的Th2型极化,具有良好的安全性。     

Respiratory Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial and Immune Cell Responses

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory infection in infants and young children and causes disease in the elderly and persons with compromised cardiac, pulmonary, or immune systems. Despite the high morbidity rates of RSV infection, no highly effective treatment or vaccine is yet available. The RSV G protein is an important contributor to the disease process. A conserved CX3C chemokine-like motif in G likely contributes to the pathogenesis of disease. Through this motif, G protein binds to CX3CR1 present on various immune cells and affects immune responses to RSV, as has been shown in the mouse model of RSV infection. However, very little is known of the role of RSV CX3C-CX3CR1 interactions in human disease. In this study, we use an in vitro model of human RSV infection comprised of human peripheral blood mononuclear cells (PBMCs) separated by a permeable membrane from human airway epithelial cells (A549) infected with RSV with either an intact CX3C motif (CX3C) or a mutated motif (CX4C). We show that the CX4C virus induces higher levels of type I/III interferon (IFN) in A549 cells, increased IFN-α and tumor necrosis factor alpha (TNF-α) production by human plasmacytoid dendritic cells (pDCs) and monocytes, and increased IFN-γ production in effector/memory T cell subpopulations. Treatment of CX3C virus-infected cells with the F(ab′)₂ form of an anti-G monoclonal antibody (MAb) that blocks binding to CX3CR1 gave results similar to those with the CX4C virus. Our data suggest that the RSV G protein CX3C motif impairs innate and adaptive human immune responses and may be important to vaccine and antiviral drug development.     

Respiratory Syncytial Virus Infection of the Newborn

In an outbreak of respiratory syncytial (R.S.) virus infection in a maternity hospital the respiratory illness was of a mild nature and the virus was not found in infants without respiratory symptoms. This confirms the suggestion that R.S. virus can infect infants at a very early age. Rapid diagnosis was achieved by applying the direct fluorescent antibody technique to cells in nasal secretions. This proved to be more sensitive than culture techniques where there was delay between the onset of respiratory symptoms and submission of specimens to the laboratory.