Localization of an RNA-binding domain in the nucleocapsid protein of the coronavirus mouse hepatitis virus

Summary The interaction between the nucleocapsid (N) protein of mouse hepatitis virus (MHV) and RNA was studied in an effort to define portions of the N molecule that participate in binding to RNA. N mRNAs transcribed from SP6 and T7 vectors were translated in a rabbit reticulocyte lysate. Analysis of synthesized N protein in a nondenaturing gel system showed that it bound in vitro to an endogenous RNA in the reticulocyte lysate but not to its own mRNA. A set of deletion mutants was constructed in order to localize the RNA-binding activity of the N protein. It was found that removal of as much as 135 amino-terminal or 57 carboxy-terminal amino acids from the molecule had little or no effect on RNA binding. Moreover, deletion mutants lacking both termini still retained RNA-binding ability. By contrast, internal deletions or truncations extending beyond these two limits effectively abolished RNA binding by N protein. Thus, the RNA-binding region of N has been mapped to the second (central) of the three structural domains of the molecule.     

Sequence analysis of the bovine coronavirus nucleocapsid and matrix protein genes

The 3' end of the 20-kb genome of the Mebus strain of bovine enteric coronavirus (BCV) was copied into cDNA and cloned into the PstI site of the pUC9 vector. Four clones from the 3' end of the genome were sequenced either completely or in part to determine the sequence of the first 2451 bases. Within this sequence were identified, in order, a 3'-noncoding region of 291 bases, the gene for a 448-amino acid nucleocapsid protein (N) having a molecular weight of 49,379, and the gene for a 230-amino acid matrix protein (M) having a molecular weight of 26,376. A third large open reading frame is contained entirely within the N gene sequence but is positioned in a different reading frame; it potentially encodes a polypeptide of 207 amino acids having a molecular weight of 23,057. A higher degree of amino acid sequence homology was found between the M proteins of BCV and MHV (87%) than between the N proteins (70%). For the M proteins of BCV and MHV, notable differences were found at the amino terminus, the most probable site of O-glycosylation, where the sequence is N-Met-Ser-Ser-Val-Thr-Thr for BCV and N-Met-Ser-Ser-Thr-Thr for MHV. BCV apparently uses two of its six potential O-glycosylation sites.     

Sequence analysis of the nucleoprotein genes of three enterotropic strains of murine coronavirus

Summary The nucleotide sequences of the nucleoprotein genes of three enterotropic strains of the murine coronavirus mouse hepatitis virus (MHV-Y, MHV-RI and DVIM) were determined and compared with previously reported sequences of three polytropic (respiratory) strains (MHV-A59, MHV-JHM and MHV-S). Greater than 92% homology was found among the six strains by pair-wise comparison at the nucleotide level. The genes encoded proteins of 451 to 455 residues and the deduced amino acid sequences were more than 91% homologous. A unique deletion of twelve nucleotides was found at the carboxy terminus of MHV-Y and a three nucleotide deletion was found in MHV-RI, which corresponded to the one previously reported in MHV-A59 and MHV-S. Two internal open reading frames were found within the coding region of the nucleoprotein, the smaller one was specific for the enterotropic strains. It could potentially encode a truncated version of the hypothetical protein described for MHV-A59 and MHV-S. Sequence relationship of the N gene showed no correlation with tissue tropism and no sequence or even single amino acid change unique to either tropism group was found. This indicates that the nucleoprotein of MHV probably has no part in the determination of the primary tissue tropism of an MHV strain. The role of the potential internal protein warrants further investigation.     

Sequence analysis of the porcine transmissible gastroenteritis coronavirus nucleocapsid protein gene

The 3' end of the 20-kb genome of the Purdue strain of porcine transmissible gastroenteritis coronavirus (TGEV) was copied into cDNA after priming with oligo(dT) and the double-stranded product was cloned into the PstI site of the pUC9 vector. One clone of 2.0-kb contained part of the poly(A) tail and was sequenced in its entirety using the chemical method of Maxam and Gilbert. Another clone of 0.7 kb also contained part of the poly(A) tail and was sequenced in part to confirm the primary structure of the most 3' end of the genome. Two potential, nonoverlapping genes were identified within the 3'-terminal 1663-base sequence from an examination of open reading frames. The first gene encodes a 382-amino acid protein of 43,426 mol wt, that is the apparent nucleocapsid protein on the basis of size, chemical properties, and amino acid sequence homology with other coronavirus nucleocapsid proteins. It is flanked on its 5' side by at least part of the matrix protein gene. The second encodes a hypothetical 78-amino acid protein of 9101 mol wt that is hydrophobic at both ends. A 3'-proximal noncoding sequence of 276 bases was also determined and a conserved stretch of 9 nucleotides near the poly(A) tail was found to be common among TGEV, the mouse hepatitis coronavirus, and the avian infectious bronchitis coronavirus.     

The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.     

Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus

The 3' end of the turkey coronavirus (TCV) genome (1740 bases) including the nucleocapsid (N) gene and 3' untranslated region (UTR) were sequenced and compared with published sequences of other avian and mammalian coronaviruses. The deduced sequence of the TCV N protein was determined to be 409 amino acids with a molecular mass of approximately 45 kDa. The TCV N protein was identical in size and had greater than 90% amino acid identity with published N protein sequences of infectious bronchitis virus (IBV); less than 21% identity was observed with N proteins of bovine coronavirus and transmissible gastroenteritis virus. The 3' UTR showed some variation among the three TCV strains examined, with two TCV strains, Minnesota and Indiana, containing 153 base segments which are not present in the NC95 strain. Nucleotide sequence identity between the 3' UTRs of TCV and IBV was greater than 78%. Similarities in both size and sequence of TCV and IBV N proteins and 3' UTRs provide additional evidence that these avian coronaviruses are closely related.     

Sequence and phylogenetic analysis of nucleocapsid genes of porcine epidemic diarrhea virus (PEDV) strains in China

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration with high mortality rates in swine. It has become increasingly problematic in China. Since the nucleocapsid (N) protein is highly conserved, it is a candidate protein for early diagnosis and vaccine development. In this study, the N genes of 15 PEDV strains were amplified by RT-PCR and cloned into the pMT-19T vector, sequenced, and compared to each other as well as to PEDV reference strains. The nucleotide sequences of the N gene of the Chinese PEDV strains consist of 1326 nucleotides and encode a 441-aa-long peptide. The nucleotide sequences of the fifteen PEDV strains in our study were 96.1-100 % identical to each other, and the deduced amino acid sequences were 94.8-100 % identical. Sequence comparison with other PEDV strains selected from GenBank revealed that their nucleotide sequences were 94.2-99.7 % identical to those of the Chinese PEDV strains, and their deduced amino acid sequences were 94.1-99.5 % identical. In addition, the fifteen strains showed a high degree of nucleotide sequence identity to the early domestic strains (98.4-99.7 %) except the LZC strain, but less sequence identity to the vaccine strain (CV777) used in China (94.7-97.7 %). Phylogenetic analysis showed that the Chinese PEDV strains are composed of a separate cluster including three early domestic strains (JS-2004-02, LJB/03 and DX) but differ genetically from the vaccine strain (CV777) and the early Korean strains (Chinju99 and SM98).     

Identification of antigenic peptides derived from B-cell epitopes of nucleocapsid protein of mouse hepatitis virus for serological diagnosis

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.     

Role of phosphorylation clusters in the biology of the coronavirus infectious bronchitis virus nucleocapsid protein

The coronavirus infectious bronchitis virus (IBV) nucleocapsid (N) protein is an RNA binding protein which is phosphorylated at two conserved clusters. Kinetic analysis of RNA binding indicated that the C-terminal phosphorylation cluster was involved in the recognition of viral RNA from non-viral RNA. The IBV N protein has been found to be essential for the successful recovery of IBV using reverse genetics systems. Rescue experiments indicated that phosphorylated N protein recovered infectious IBV more efficiently when compared to modified N proteins either partially or non-phosphorylated. Our data indicate that the phosphorylated form of the IBV N protein plays a role in virus biology.     

Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable

Partial genomic sequences representing 420 nucleotides of a nonstructional region, 480 nucleotides of the putative RNA polymerase region, and 540 nucleotides of the structural region of epidemic-associated Chinese strains of hepatitis E virus (HEV) were obtained by direct sequencing of PCR-amplified DNA. Comparison with previously published HEV sequences showed a clear relatedness of all Chinese strains to each other and to a Pakistani strain (Sar-55). All eight Chinese strains examined had very similar sequences (98.5-99.8% homology) in the regions examined and were much closer to the Pakistani strain (Sar-55) (97.9-98.4% homology) than to the Burmese strain (92.5-93.3% homology). Sequence comparisons of the three genomic regions in the Chinese strains indicated that the RNA polymerase region was much more conserved than the other nonstructural region or the structural region. HEV isolates from three remote geographic regions of China had sequences closely related to each other.     

Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus.

In a previous study, two murine T-cell hybridomas generated after immunization with infectious bronchitis virus (IBV) were shown to be responsive to the internally localized viral nucleocapsid protein. In the present study, the antigenic determinants were mapped using recombinant expression products and synthetic peptides. Both hybridomas recognized the region spanning amino acid residues 71 to 78 of the nucleocapsid protein. The experimentally determined epitope corresponded with predicted motifs. Both an I-Ed binding motif and a predicted cleavage site for the aspartyl protease cathepsin D were contained within the sequence. The epitope was shown to prime cellular immune responses to IBV in the chicken.     

Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein

Bacterially expressed nucleocapsid (N) protein, from respiratory syncytial virus (RSV), was used to investigate RNA binding in a modified North-Western blotting protocol. The recombinant protein demonstrated no sequence specificity in binding RNA representing either the antigenomic leader sequence or the nonspecific sequence derived from a plasmid vector. When recombinant N was purified on CsCl gradients, two types of structure, both with densities indicating that they contained RNA, could be visualised by negative-stain electron microscopy. Structures similar to nucleocapsids (NC) from RSV-infected cells were observed, as were ring structures. A small fragment of the N (amino acids 1-92) was all that was required for the production of NC-like structures. Another mutant with an internal deletion could form rings but not NC-like structures. This suggests that this domain (amino acids 121-160) may be important for maintaining helical stability. Further analysis has also identified a potential site in the amino-terminus that may be involved in an interaction with the phosphoprotein. A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly.     

小鼠肝炎病毒N蛋白单克隆抗体的制备及鉴定

[目的]制备并鉴定小鼠肝炎病毒(MHV)N蛋白的单克隆抗体(mAb).[方法]以Bac-to-Bac杆状病毒表达系统表达的重组N蛋白为免疫原,免疫BALB/c小鼠,应用常规杂交瘤技术将免疫小鼠脾细胞与Sp2/0骨髓瘤进行融合,用间接ELISA方法筛选分泌N蛋白mAb的杂交瘤细胞系.并用Western blot和IFA方法对所获得的mAb进行鉴定.[结果]共获得4株稳定分泌抗N蛋白mAb的杂交瘤细胞系,其亚类鉴定2株为IgG2a,1株为IgG2b,1株为IgG1,轻链均为k型.经鉴定4株mAb均能与重组N蛋白发生特异性反应.[结论]成功获得了特异性抗MHV N蛋白的mAb,为进一步研究N蛋白的结构功能及建立诊断学方法奠定了基础.     

HBc二聚体组装核衣壳相关功能域突变对HBV复制的影响

目的 探讨乙型肝炎病毒核心蛋白(hepatitis B virus core protein,HBc)二聚体组装相关功能域突变对核心颗粒组装及HBV复制的影响.方法 基于HBc空间结构,PCR定点诱变二聚体组装核衣壳相关功能域重要氨基酸位点,以pcDNA3.1为载体构建4个突变体表达质粒pHBc14-18M、pHBc120-135M、pHBc23-39M和pHBc122-139M.将突变质粒与HBc缺失的含1.2拷贝HBV基因质粒pHBV1.2-core-共转染HepG2细胞,通过Northern blot检测HBV前基因组(pgRNA),Southern blot检测复制中间体,非变性琼脂糖凝胶电泳(native agarose gel electrophoresis)及Western blot检测细胞核心颗粒观察突变质粒自身形成核衣壳情况.将突变质粒与含有1.2拷贝HBV基因质粒pHBV1.2共转染HepG2细胞观测突变质粒对野生型HBc组装及HBV复制的影响.结果 突变质粒与pHBV1.2-core-质粒共转染HepG2细胞,pHBc14-18M、pHBc120-135M和pHBc122-139M突变能够组成核衣壳样结构.pHBc23-39M不能形成核衣壳样结构.Northern blot结果显示所有突变质粒组均未见pgRNA条带,Southern blot检测复制中间体也未见条带.将突变质粒与pHBV1.2共转染HepG2细胞,pHBc14-18M、pHBc120-135M和pHBc122-139M组HBV复制中间体及上清中病毒颗粒明显减少,而pHBc23-39M组无减少.结论 HBc23-39位氨基酸突变可阻止二聚体多聚化,不能形成核衣壳样结构,且不能与野生HBc二聚体相互作用.HBc14-18、120-135、122-139区域的氨基酸突变,能够形成核衣壳样结构但不支持HBV DNA复制,并且能与野生HBc二聚体相互作用,形成杂合体,干扰野生型HBV DNA的复制.     

The cloning and sequencing of the virion protein genes from a British isolate of porcine respiratory coronavirus: comparison with transmissible gastroenteritis virus genes.

Previous analysis of porcine respiratory coronavirus (PRCV) mRNA species showed that mRNAs 2 and 3 were smaller than the corresponding transmissible gastroenteritis virus (TGEV) mRNA species (Page et al. (1991) J. Gen. Virol. 72, 579-587). Sequence analysis showed that mRNA 3 was smaller due to the presence of a new putative RNA-leader binding site upstream of the PRCV ORF-3 gene. However, this observation did not explain the deletion observed in PRCV mRNA 2. Polymerase chain reaction (PCR) was used to generate cDNA from the 3' coding region of the putative polymerase gene to the poly (A) tail of PRCV for comparison to the equivalent region from TGEV. The PRCV S protein was found to consist of 1225 amino acids, which had 98% similarity to the TGEV S protein. However, the PRCV S gene contained a 672 nucleotide deletion, corresponding to 224 amino acids (residues 21 to 245 in TGEV S protein), 59 nucleotides downstream of the S gene initiation codon. The PRCV genome from the ORF-3 gene to the poly (A) tail was sequenced for comparison to TGEV in order to identify other potential differences between the two viruses. Four ORFs were identified that showed 98% similarity to the TGEV ORF-4, M, N and ORF-7 genes. No other deletions or any PRCV specific sequences were identified.