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1.
Mouse hepatitis virus (MHV) infection is found commonly in laboratory mice and this virus has been known to cause various diseases such as subclinical infection, enteritis, hepatitis, and encephalitis. Serological tests are used commonly to diagnose MHV infection. Complete MHV virions have been used primarily as antigens for serological diagnosis to date. To develop an antigen that is more specific, more sensitive, and easier to prepare for serological diagnosis, the antigenic sites in the MHV-nucleocapsid (N) protein were screened in this study. Sixteen antigenic linear sequences in the N protein were found using antisera obtained from mice infected naturally with MHV and a peptide array containing overlapping 10-mer peptides covering the entire N protein. From these antigenic sequences, two synthesized peptides, ILKKTTWADQTERGL and RFDSTLPGFETIMKVL, which were consistent with positions 24-38 and 357-372 of the N protein respectively, were used as antigens in ELISA. Evaluation of ELISA with these peptides revealed that both peptides were specific to anti-MHV antisera. Furthermore, ELISA performed using these peptides was more sensitive than commercial ELISA used for a screening sera from mice infected accidentally to MHV maintained in cages, suggesting that these peptides are useful for serological diagnosis of MHV infection.  相似文献   

2.
Envelope viruses maturate by macromolecule assembly and budding. To investigate these steps, we generated virus-like particles (VLPs) by co-expression of structural proteins of Sendai virus (SeV), a prototype of the family Paramyxoviridae. Simultaneous expression of matrix (M), nucleo- (N), fusion (F), and hemagglutinin-neuraminidase (HN) proteins resulted in the generation of VLPs that had morphology and density similar to those of authentic virus particles, although the efficiency of release from cells was significantly lower than that of the virus. By using this VLP formation as a model of virus budding, roles of individual proteins in budding were investigated. The M protein was a driving force of budding, and the F protein facilitated and the HN protein suppressed VLP release. Either of the glycoproteins, F or HN, as well as the N protein, significantly shifted density of VLPs to that of virus particles, suggesting that viral proteins bring about integrity of VLPs by protein-protein interactions. We further found that co-expression of a nonstructural protein, C, but not V, enhanced VLP release to a level comparable to that of virus particles, demonstrating that the C protein plays a role in virus budding.  相似文献   

3.
Herein, we show a direct relationship between the Hantaan virus (HTNV) nucleocapsid (N) protein and the modulation of apoptosis. We observed an increase in caspase-7 and -8, but not -9 in cells expressing HTNV N protein mutants lacking amino acids 270-330. Similar results were observed for the New World hantavirus, Andes virus. Nuclear factor kappa B (NF-κB) was sequestered in the cytoplasm after tumor necrosis factor receptor (TNFR) stimulation in cells expressing HTNV N protein. Further, TNFR stimulated cells expressing HTNV N protein inhibited caspase activation. In contrast, cells expressing N protein truncations lacking the region from amino acids 270-330 were unable to inhibit nuclear import of NF-κB and the mutants also triggered caspase activity. These results suggest that the HTNV circumvents host antiviral signaling and apoptotic response mediated by the TNFR pathway through host interactions with the N protein.  相似文献   

4.
汉滩病毒核衣壳蛋白C-端T细胞表位鉴定   总被引:3,自引:0,他引:3  
目的 鉴定汉滩病毒核衣壳蛋白 (HTNVNP)C 端T细胞表位 ,为肾综合征出血热(HFRS)发病机理、疫苗研制及抗病毒免疫反应研究奠定基础。方法 采用Ficoll密度梯度离心法分离HFRS恢复期患者外周血单个核细胞 (PBMC)。用IFN γELISPOT实验和T细胞增殖实验 ,测试 7名患者PBMC对 2 3条NPC 端合成多肽的T细胞应答。结果 IFN γELISPOT实验结果表明 ,2名供体(3、4 )可分别检测到对 5 1、70号 2条多肽特异性T细胞应答。在供体 3,70号肽特异性T细胞频率为4 5SFC 10 6 PBMC ;在供体 4 ,5 1号肽特异性T细胞频率为 82SFC 10 6 PBMC。T细胞增殖实验与ELISPOT结果基本一致 ,但 5 3号肽和 6 4号肽还可分别刺激供体 1和供体 4的T细胞增殖 ,而未能诱导IFN γ分泌。结论  5 1号和 70号多肽可能是NPC 端较强的T细胞表位。  相似文献   

5.
To catalyze RNA synthesis, the Sendai virus P-L RNA polymerase complex first binds the viral nucleocapsid (NC) template through an interaction of the P subunit with NP assembled with the genome RNA. For replication, the polymerase utilizes an NP(0)-P complex as the substrate for the encapsidation of newly synthesized RNA which involves both NP-RNA and NP-NP interactions. Previous studies showed that the C-terminal 124 amino acids of NP (aa 401-524) contain the P-NC binding site. To further delineate the amino acids important for this interaction, C-terminal truncations and site-directed mutations in NP were characterized for their replication activity and protein-protein interactions. This C-terminal region was found in fact to be necessary for several different protein interactions. The C-terminal 492-524 aa were nonessential for the complete activity of the protein. Deletion of amino acids 472-491, however, abolished replication activity due to a specific defect in the formation of the NP(0)-P complex. Binding of the P protein of the polymerase complex to NC required aa 462-471 of NP, while self-assembly of NP into NC required aa 440-461. Site-directed mutations from aa 435 to 491 showed, however, that the charged amino acids in this region were not essential for these defects.  相似文献   

6.
We prepared the chimeric recombinant Sendai virus [rSeV(Ppi)] by replacing the P gene of the Z strain with that of pi strain for analyzing the function of Ppi, Vpi and Cpi proteins. Intriguingly, HA production by rSeV(Ppi) is significantly lower at 38°C than at 32°C, showing that virus growth of rSeV(Ppi) is slightly suppressed at 38°C. However, the main phenotypes of SeVpi, a marked temperature sensitivity as viral replication and an ability of establishing persistent infection, are not explained by the Ppi, Vpi and Cpi proteins. The V and C proteins form inclusion bodies in L929 cells infected with rSeV(Ppi) and incubated at 38°C. L929 cells infected with rSeV(Ppi) and L929 cells stably expressing the Cpi protein show resistance to interferon-β at 32 and 38°C, indicating that the Cpi protein per se is not temperature-sensitive to inhibition of IFN signaling. The complete genome sequences of Sendai virus (SeV) pi and parent Nagoya strains were determined. Fifty nucleic acid substitutions are found in the genome sequence of SeV pi strain in comparison with Nagoya strain. There are three nucleic acid substitutions in the leader sequence, while the trailer, intergenic, gene-end and gene-start sequences of both strains are completely identical. Deletions and insertions of nucleotide are not found. There are 32 amino acid substitutions in Sendai virus pi strain. The specific amino acid substitutions unique to the SeVpi are 18. Information about the complete genome sequences of SeVpi strain is important to totally understand the persistent infection and lower pathogenicity of SeV.Machiko Nishio and Ai Nagata have contributed equally.  相似文献   

7.
Retroviral nucleocapsid (NC) proteins are small Gag-derived products containing one or two zinc finger motifs that mediate genomic RNA packaging into virions. In this study, we addressed the role of the feline immunodeficiency virus (FIV) NC protein in the late stages of virus replication by analyzing the assembly phenotype of FIV NC mutant viruses and the RNA binding activity of a panel of recombinant FIV NC mutant proteins. Substitution of serine for the first cysteine residue in the NC proximal zinc finger was sufficient to impair both virion assembly and genomic RNA binding. A similar defective phenotype with respect to particle formation and RNA binding was observed when the basic residues Lys28 and Lys29 in the region connecting both zinc fingers were replaced by alanine. In contrast, mutation of the first cysteine residue in the distal zinc finger had no effect on virion production and allowed substantial RNA binding activity of the mutant NC protein. Moreover, this NC mutant virus exhibited wild-type replication kinetics in the feline MYA-1 T-cell line. Interestingly, amino acid substitutions disrupting the highly conserved PSAP and LLDL motifs present in the C-terminus of the FIV NC abrogated virion formation without affecting the NC RNA binding activity. Our results indicate that the proximal zinc finger of the FIV NC is more important for virion production and genomic RNA binding than the distal motif. In addition, this study suggests that assembly domains in the FIV NC C-terminus may be functionally equivalent to those present in the p6 domain of the Gag polyprotein of primate lentiviruses.  相似文献   

8.
The large (about 2200 amino acids) L polymerase protein of nonsegmented negative-strand RNA viruses (order Mononegavirales) has six conserved sequence regions (“domains”) postulated to constitute the specific enzymatic activities involved in viral mRNA synthesis, 5′-end capping, cap methylation, 3′ polyadenylation, and genomic RNA replication. Previous studies with vesicular stomatitis virus identified amino acid residues within the L protein domain VI required for mRNA cap methylation. In our recent study we analyzed four amino acid residues within domain VI of the Sendai virus L protein and our data indicated that there could be differences in L protein sequence requirements for cap methylation in two different families of Mononegavirales — rhabdoviruses and paramyxoviruses. In this study, we conducted a more comprehensive mutational analysis by targeting the entire SeV L protein domain VI, creating twenty-four L mutants, and testing these mutations for their effects on viral mRNA synthesis, cap methylation, viral genome replication and virus growth kinetics. Our analysis identified several residues required for successful cap methylation and virus replication and clearly showed the importance of the K-D-K-E tetrad and glycine-rich motif in the SeV cap methylation. This study is the first extensive sequence analysis of the L protein domain VI in the family Paramyxoviridae, and it confirms structural and functional similarity of this domain across different families of the order Mononegavirales.  相似文献   

9.
目的:探讨汉滩病毒核衣壳蛋白(HTNV-NP)诱生的特异性细胞免疫的分子基础,阐明HFRS的发病机理。方法:分离HFRS患者的PBMC,建立HTNV-NP特异性的CTL克隆,同时将克隆有HINV S基因不同片段的真核表达载体转染患者自身的B淋巴母细胞系(BLCL),建立CTL靶细胞系,并进行细胞杀伤试验。结果:建立的特异性CTL克隆对表达完整NP、NP羧基和氨基端肽段的靶细胞均有比较明显的杀伤效应,平均杀伤率分别为50.2%、39.0%和25.4%。结论:NTNV-NP优势T细胞表位可能主要位于病毒核蛋白的羧基端。  相似文献   

10.
目的制备发热伴血小板减少综合征布尼亚病毒(SFTSV)核衣壳蛋白(NP)的单克隆抗体,并探讨其在该病诊断中的作用。方法克隆NP基因,构建原核表达载体pComb3XSS-NP,并转化大肠杆菌Top10F′,经诱导表达,纯化获得6HIS-NP融合蛋白。以该融合蛋白免疫Balb/c小鼠,经杂交瘤技术制备NP的鼠源单克隆抗体。经鉴定后,选择1株1C8-C6进行大量制备、纯化,并偶联辣根过氧化物酶(HRP),用该酶标抗体对SFTSV感染人的急性期血清进行Western blot检测。结果成功表达、纯化SFTSV重组的NP。经小鼠免疫、细胞融合和ELISA筛选后,共获得4株鼠源单克隆抗体。具较高滴度的1C8-C6酶标抗体能与SFTSV感染人的急性期血清中的NP在Western blot水平上结合,而与汉坦病毒、登革热病毒感染人急性期血清及正常人血清不反应。结论 SFTSV NP的表达及其单抗的制备为该病的实验室诊断奠定了基础。  相似文献   

11.
The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-beta induction through inhibition of IRF-3 activation, in viral pathogenesis. Mice infected with rSeV mutants, deficient in V expression or expressing V lacking the C-terminus, had several-fold higher IFN activity levels in the lungs than those in wild-type virus-infected mice, and the mutant viruses were rapidly excluded from the lung from the early phase of infection before induction of acquired immunity. In addition, the unique early clearance of the mutants did not occur in IRF-3 knockout (KO) mice. However, high titers of IFN were detected even in the infected KO mice. Furthermore, early clearance of the mutant viruses was also observed in IFN signaling-deficient mice, IFN-alpha/beta receptor KO mice and STAT1 KO mice. These results indicate that SeV V protein counteracts IRF-3-mediated innate antiviral immunity for efficient virus replication and pathogenesis in mice, but it is not IFN.  相似文献   

12.
Cevik B  Smallwood S  Moyer SA 《Virology》2007,363(1):189-197
The RNA dependent RNA polymerase of Sendai virus consists of a complex of the large (L) and phosphoprotein (P) subunits where L is thought to be responsible for all the catalytic activities necessary for viral RNA synthesis. We previously showed that the L protein forms an oligomer [Smallwood, S., Cevik, B., Moyer, S.A., 2002. Intragenic complementation and oligomerization of the L subunit of the Sendai virus RNA polymerase. Virology 304, 235-245] and mapped the L oligomerization domain between amino acids 1 and 174 of the protein [Cevik, B., Smallwood, S., Moyer, S.A., 2003. The oligomerization domain resides at the very N-terminus of the Sendai virus L RNA polymerase protein. Virology 313, 525-536]. An internal deletion encompassing amino acids 20 to 178 of the L protein lost polymerase activity but still formed an L-L oligomer. The first 25 amino acids of paramyxovirus L proteins are highly conserved and site-directed mutagenesis within this region eliminated the biological activity of the L protein but did not have any effect on P-L or L-L interactions. Moreover deletion of amino acids 2-18 in L abolished biological activity, but again the L-L binding was normal demonstrating that the oligomerization domain of L protein resides in two N-terminal regions of the protein. Therefore, sequences between both aa 2-19 and aa 20-178 can independently mediate Sendai L oligomerization, however, both are required for the activity of the protein.  相似文献   

13.
Nishio M  Tsurudome M  Ito M  Kawano M  Komada H  Ito Y 《Virology》2003,314(1):110-124
It is commonly accepted that the temperature-sensitive phenotype of Sendai virus (SeV) persistently infected cells is caused by the M and/or HN proteins. Expression level of the L, M, HN, and V proteins is extremely low in L929 cells persistently infected with SeVpi (L929/SeVpi cells) incubated at 38°C. The HN protein quickly disappears in L929/SeVpi cells following a temperature shift up to 38°C, and pulse-chase experiments show that the Lpi, HNpi, and Mpi proteins are unstable at 38°C. Following a temperature shift either upward or downward, M protein is translocated into the nucleus and then localizes to the perinuclear region. None of virus-specific polypeptides are detected in the cells primarily infected with SeVpi and incubated at 38°C and virus proteins are not pulse-labeled at 38°C, indicating that temperature-sensitive step is at an early stage of infection. The Mpi protein is transiently located in the nucleus of the SeVpi primarily infected cells. Recombinant SeVs possessing the HNpi or/and Mpi proteins are not temperature-sensitive. The HN protein is expressed at very low levels and the F protein localizes to the perinuclear region in rSeV(Mpi)-infected cells incubated at 38°C for 18 h. rSeVs having the Mpi protein exhibit lower cytotoxicity and are incapable of establishing persistent infection. Amino acid 116 of the Mpi protein is related to the nuclear translocation and lower cytopathogenesis, whereas aa183 is involved in the interaction between M protein and viral glycoproteins.  相似文献   

14.
Sendai virus NP gene codes for a 524 amino acid NP protein   总被引:4,自引:0,他引:4  
The complete nucleoprotein (NP) gene sequences of the Sendai virus Fushimi and 6/94 strains were determined. For both viruses an open reading frame of 524 amino acids can be predicted for the NP proteins. By comparing the sequences with others reported in the literature, the 5 noncoding region and the middle third of the coding region were found to be highly conserved. The carboxyl terminal part carries nine amino acid changes and a completely different sequence of the carboxyl terminus with a seven amino acid extension. This carboxyl terminus of the Sendai virus NP protein was confirmed using tryptic peptide sequence analysis.  相似文献   

15.
目的寻找HeLa细胞表面与人CD59特异性结合的短肽序列。方法分别用非转基因HeLa细胞和转染CD59基因的HeLa细胞的裂解蛋白进行5轮亲和筛选,并通过竞争结合实验,然后用ELISA方法筛选噬菌体阳性克隆。提取单克隆DNA测序,并对其进行DNA序列分析推导出短肽序列。结果随机挑选的16个单克隆中有10个克隆对HeLa细胞有特异性结合力,经测序得到一条高度同源的多肽序列。结论通过噬菌体随机肽库对肿瘤细胞进行全细胞蛋白筛选,得到了噬菌体多肽能高特异性与肿瘤细胞结合的短肽序列,为下一步设计肿瘤逃逸相关的CD59活性位点的短肽药物提供了参考依据。  相似文献   

16.
The hantavirus nucleocapsid (N) protein is an important immunogen that stimulates a strong and cross-reactive immune response in humans and rodents. A large proportion of the response to N protein has been found to target its N-terminus. However, the exact nature of this bias towards the N-terminus is not yet fully understood. We characterized six monoclonal antibodies (mAbs) against the N protein of Montano virus (MTNV), a Mexican hantavirus. Five of these mAbs recognized eight American hantaviruses and six European and Asian hantaviruses, but not the Soricomorpha-borne Thottapalayam hantavirus. The N protein-reactive binding regions of the five mAbs were mapped to discontinuous epitopes within the N-terminal 13-51 amino acid residues, while a single serotype-specific mAb was mapped to residues 1-25 and 49-75. Our findings suggest that discontinuous epitopes at the N-terminus are conserved, at least in rodent-borne hantaviruses, and that they contribute considerably to N protein cross-reactivity.  相似文献   

17.
Irie T  Nagata N  Yoshida T  Sakaguchi T 《Virology》2008,374(2):495-505
The Sendai virus (SeV) C proteins are a nested set of four accessory proteins, C', C, Y1, and Y2, encoded on the P mRNA from an alternate reading frame. The C proteins are multifunctional proteins involved in viral pathogenesis, inhibition of viral RNA synthesis, counteracting the innate immune response of the host cell, inhibition of virus-induced apoptosis, and facilitating virus-like particle (VLP)/virus budding. Among these functions, the roles for pathogenesis and counteracting host cell interferon (IFN) responses have been studied extensively, but the others are less well understood. In this paper, we found that the C proteins contributed in many ways to the efficient production of infectious virus particles by using a series of SeV recombinants without one or more C protein expression. Knockout of both C' and C protein expression resulted in reduced virus release despite higher viral protein synthesis in the cells. Interestingly, for the viruses without C' and C, or all four C protein expression, non-infectious virions containing antigenomic RNAs were produced predominantly compared to genomic RNA-containing infectious virions, due to aberrant viral RNA synthesis. Our results demonstrate for the first time that the C proteins regulate balance of viral genome and antigenome RNA synthesis for efficient production of infectious virus particles in the course of virus infection.  相似文献   

18.
Cytoplasmic actins have been found interacting with viral proteins and identified in virus particles. We analyzed by confocal microscopy the cytoplasmic β- and γ-actin patterns during the course of Sendai virus infections in polarized cells. We observed a spectacular remodeling of the β-cytoplasmic actin which correlated with productive viral multiplication. Conversely, suppression of M during the course of a productive infection resulted in the decrease of particle production and the absence of β-actin remodeling. As concomitant suppression of β- and γ-actins resulted as well in reduction of virus particle production, we propose that Sendai virus specifically induces actin remodeling in order to promote efficient virion production. Beta- and γ-cytoplasmic actin recruitment could substitute for that of the endosomal sorting complex required for transport (ESCRT) mobilized by other enveloped viruses but apparently not used by Sendai virus.  相似文献   

19.
Puumala virus (PUUV) is a hantavirus that causes a mild form of hemorrhagic fever with renal syndrome in northern and central Europe, and in large parts of Russia. The nucleocapsid (N) protein encoded by hantaviruses plays an important role in the life-cycle of these viruses, and one important function for the N-protein is to oligomerize, surround and protect the viral RNAs. We have identified amino- and carboxy-terminal regions involved in PUUV N–N interactions, which comprise amino acids 100–120 and 330–405. Our findings strengthen the hypothesis that the amino-terminus of the N-protein of hantaviruses holds a more regulatory function regarding N–N interactions, while conserved residues in the carboxy-terminal region, F335 together with F336 and W392, in concert with Y388 and/or F400 seems to play a more critical role in the PUUV N–N formation. This study provides evidence that the amino-terminal regions involved in the N–N interaction of Puumala virus are similar to those reported for Seoul virus (SEOV) and to some extent Hantaan virus (HTNV), even though the identity between PUUV N and SEOV/HTNV N is markedly lower than between PUUV N and Tula virus (TULV) N or Sin Nombre virus (SNV) N.  相似文献   

20.
目的 探讨汉滩病毒(HTNV)部分核蛋白(NP)基因(37-294bp)原核表达产物NP AA1-86多肽的免疫原性及其免疫保护作用。方法 大肠杆菌表达的HTNV部分核蛋白多肽NP AA1-86混合福氏佐剂,腹腔注射免疫BALB/c小鼠,IFA检测抗体应答;^3H-TdR掺入及4h ^51Cr释放试验检测免疫小鼠脾细胞对HTNV的特异淋巴细胞增殖转化指数及细胞毒T淋巴细胞(CTL)杀伤功能;免疫小鼠脾细胞转输感染HTNV A9株的乳鼠,观察对乳鼠致死性的免疫保护作用。结果 免疫后1周小鼠即出现抗体应答反应,抗体滴度最高达1:12800,与重组完整NP免疫组差异无显著性(P>0.05);免疫小鼠的淋巴细胞增殖转化指数、CTL杀伤率较对照组明显增高(P<0.01),与完整NP免疫组差异无显著性(P>0.05),CTL杀伤率随效:靶比例的增高呈逐渐上升趋势;免疫小鼠脾细胞转输可部分保护病毒致死性感染的乳鼠,保护率约25%。结论 大肠杆菌表达的汉滩病毒部分核蛋白多肽NP AA1-86不仅包含NP几乎所有的体液免疫表位,同时含有部分细胞免疫表位,对汉滩病毒感染可产生部分免疫保护作用。  相似文献   

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