SiRNA抑制柯萨奇B3病毒的复制和表达

目的 研究观察体外合成siRNA对培养HELA细胞中柯萨奇B3病毒（Coxsackievirus B3,CVB3）的影响。方法根据siRNA靶序列设计原则,针对编码CVB3病毒聚合酶、VP1蛋白和5’非编码区基因组,特异性地体外合成三对siRNA,同时合成一对与CVB基因组序列无关的阴性对照siRNA。利用脂质体转染进入Hela细胞,用CVB3感染培养HELA细胞,观察转染后HELA细胞病变;采用RT-PCR技术检测感染CVB3各组的病毒RNA;用免疫荧光技术检测各组CVB3蛋白的表达;并用培养细胞上清液再感染HELA细胞观察病毒滴度。结果针对CVB3病毒聚合酶的siR-NA能有效的抑制病毒的复制和CVB3蛋白的表达,并能抑制病毒的再感染;而针对VP1蛋白和5’非编码区的siRNA能部分抑制病毒的复制和CVB3蛋白的表达。结论我们设计合成针对编码CVB3病毒聚合酶基因组的siRNA能有效抑制CVB3病毒复制和表达。     

Comparison of the major antigenic determinants of different serotypes of foot-and-mouth disease virus.

Complete nucleotide sequences which code for the capsid protein VP1 of two foot-and-mouth disease virus serotypes, O1Campos/Brazil/58 and C3Indaial/Brazil/71, have been determined. Ten available VP1 sequences (three serotype O, three serotype C, and four serotype A) were aligned and compared. Our evidence suggests that O1BFS/Britain/68 and O1K/Germany/66 are closely related to O1Campos/Brazil/58. Significant variations were observed between the nucleotide sequences of C3Indaial determined by two different laboratories. These differences are probably the result of virus adaptation and propagation in different laboratories. In one of the isolates (C3Biogen), a 13-base-pair stem and 13-base-pair loop structure is located in the 134-158 amino acid variable region. Isolates of different serotypes differ at two highly variable regions, amino acid positions 42-60 and 134-158, but isolates of the same serotype show major differences only in the variable region between amino acids 134 and 158. Since the remaining amino acid sequence of VP1 is highly conserved, we conclude that the 134-158 amino acid variable region is involved in subtype specificity, whereas both variable regions contribute to serotype differences.     

Recombination in circulating enteroviruses

Recombination is a well-known phenomenon for enteroviruses. However, the actual extent of recombination in circulating nonpoliovirus enteroviruses is not known. We have analyzed the phylogenetic relationships in four genome regions, VP1, 2A, 3D, and the 5' nontranslated region (NTR), of 40 enterovirus B strains (coxsackie B viruses and echoviruses) representing 11 serotypes and isolated in 1981 to 2002 in the former Soviet Union states. In the VP1 region, strains of the same serotype expectedly grouped with their prototype strain. However, as early as the 2A region, phylogenetic grouping differed significantly from that in the VP1 region and indicated recombination within the 2A region. Moreover, in the 5' NTR and 3D region, only 1 strain of 40 grouped with its prototype strain. Instead, we observed a major group in both the 5' NTR and the 3D region that united most (in the 5' NTR) or all (in the 3D region) of the strains studied, regardless of the serotype. Subdivision within that major group in the 3D region correlated with the time of virus isolation but not with the serotype. Therefore, we conclude that a majority, if not all, circulating enterovirus B strains are recombinants relative to the prototype strains, isolated mostly in the 1950s. Moreover, the ubiquitous recombination has allowed different regions of the enterovirus genome to evolve independently. Thus, a novel model of enterovirus genetics is proposed: the enterovirus genome is a stable symbiosis of genes, and enterovirus species consist of a finite set of capsid genes responsible for different serotypes and a continuum of nonstructural protein genes that seem to evolve in a relatively independent manner.     

A mutation in the puff region of VP2 attenuates the myocarditic phenotype of an infectious cDNA of the Woodruff variant of coxsackievirus B3.

Coxsackievirus B3 (CVB3) infections induce myocarditis in humans and mice. Little is known about the molecular characteristics of CVB3 that activate the cellular immunity responsible for cardiac inflammation. Previous experiments have identified an antibody escape mutant (H310A1) of a myocarditic variant of CVB3 (H3) that attenuates the myocarditic potential of the virus in mice in spite of ongoing viral replication in the heart. We have cloned full-length infectious cDNA copies of the viral genome of both the wild-type myocarditic H3 variant of CVB3 and the antibody escape mutant H310A1. Progeny viruses maintained the myocarditic and attenuated myocarditic potential of the parent viruses, H3 and H310A1. The full sequence of the H3 viral cDNA is reported and compared with those of previously published CVB3 variants. Comparison of the full sequences of H3 and H310A1 viruses identified a single nonconserved mutation (A to G) in the P1 polyprotein region at nucleotide 1442 resulting in an asparagine-to-aspartate mutation in amino acid 165 of VP2. This mutation is in a region that corresponds to the puff region of VP2. Nucleotide 1442 of the H3 and H310A1 cDNA copies of the viral genome was mutated to change amino acid 165 of VP2 to aspartate and asparagine, respectively. The presence of asparagine at amino acid 165 of VP2 is associated with the myocarditic phenotype, while an aspartate at the same site reduces the myocarditic potential of the virus. In addition, high-level production of tumor necrosis factor alpha by infected BALB/c monocytes is associated with asparagine at amino acid 165 of VP2 as has been previously demonstrated for the H3 virus. These findings identify potentially important differences between the H3 variant of CVB3 and other previously published CVB3 variants. In addition, the data demonstrate that a point mutation in the puff region of VP2 can markedly alter the ability of CVB3 to induce myocarditis in mice and tumor necrosis factor alpha secretion from infected BALB/c monocytes.     

The complete nucleotide sequence of RNa1 of barley mild mosaic virus (asl1) and attempts at bacterial expression of the P3 protein

The complete nucleotide sequence of RNA1 of an Aschersleben isolate of barley mild mosaic virus (BaMMV) was determined. It consists of 7263 nucleotides (nt) excluding the 3' poly (A) tail. The 5' and 3' nontranslated regions (NTR) are 148 and 338 nt in length, respectively, and flank a single large open reading frame coding for a precursor polypeptide with a calculated molecular mass of 256 kDa. Sequence comparison revealed a 96% amino acid (aa) identity to RNA1 translation products of Japanese and French BaMMV isolates. Conserved nucleotide motifs in the 3' sense and 5' complementary sense NTR of the two genomic RNAs were identified that may represent the polymerase recognition sites. A range of constructs containing various parts of the coding region of the P3 nonstructural protein was prepared for expression in Escherichia coli . A short stretch of 35 aa in the C-proximal region of P3 appeared to be highly toxic to the bacterium.     

Nucleotide sequence of an attenuated mutant of coxsackievirus B3 compared with the cardiovirulent wildtype: assessment of candidate mutations by analysis of a revertant to cardiovirulence

Background: Coxsackievirus B3 (CVB3) causes myocarditis in the SWR (H^2q) mouse model and persistence of CVB3 in myocardium disposes to the development of dilated cardiomyopathy. An attenuated strain of CVB3 has been isolated, sequenced and several candidate mutations for attenuation identified. Derivation of a revertant to cardiovirulence allows the significance of these mutations to be assessed.Objectives: To ascertain which candidate mutation(s) determine(s) the attenuated phenotype.Study design: A revertant to cardiovirulence was isolated following passage through severe combined immunodeficient disease (SCID) mouse heart. The 5′-non-translated region (NTR) and region coding for capsid proteins were sequenced and compared to the wildtype and attenuant.Results: There are five candidates for attenuation: (1) A–G at base 580 in the 5′-NTR; (2) A–T at base 690 in the 5′-NTR; (3) CG–GC at bases 1401/2 (Thr to Ser at amino acid 151 in VP2); (4) AA–GT at bases 2691/2 (Lys to Ser at amino acid 80 in VP1); (5) A–G at base 2916 (Asp to Gly at amino acid 155 in VP1). It was shown previously that mutations at 580, 690 and 2691/2 are not important in attenuation. Additionally, there are three novel mutations in the coding region of the revertant and one in the 5′-NTR which are unlikely to be relevant for attenuation as they are not present in the attenuant. Of nucleotide changes seen at 1401/2 and 2916 in the attenuant, only 2916 reverts to the wildtype sequence and so is a strong candidate for a determinant of attenuation.Conclusions: The A–G mutation at 2916 (Asp to Gly at amino acid 155 in VP1) is a strong candidate for attenuation. It is located at the top of the receptor binding cleft and mutation of the Asp to a Gly may destabilise the receptor binding site.     

The primary sequence of the late region of polyoma virus DNA II. The expression of the late genes and comparison with DNA sequences of SV40 and BKV.

The nucleotide sequence of the late region of the polyoma virus genome has been deduced, which codes for the major capsid protein VP1 and the C-terminal region of the minor proteins VP2 and VP3. The amino acid sequence of VP1 predicted from the nucleotide sequence is in good agreement with the partial N-terminal sequence 1 and amino acid composition of VP1 2,3. When both nucleotide and amono acid sequences are compared with their counterparts in the related viruses, SV40 4,5 and BKV (R. Young, personal communication), extensive homologies are found along the entire regions of the viral genes. Maximum homologies appear to occur in the regions which code for the C-terminal of VP1, on the contrary of the result of heteroduplex analysis 6 with 6 with SV40 and polyoma virus DNAs.     

Rearrangement of the VP6 gene of a group A rotavirus in combination with a point mutation affecting trimer stability.

A group A rotavirus isolated from a lamb with diarrhea in Qinhai province, China, was serially passaged in fetal calf kidney cells. In passage 96, rearrangements of RNA segments 5 and 6 of the viral genome were found. Here we report the nucleotide and predicted amino acid sequences of normal and rearranged RNA 6, coding for the major inner capsid protein VP6. In comparison with the normal gene (N6), the rearranged RNA 6 (R6) contained the normal open reading frame followed by a 473-nucleotide (nt) duplication of the gene beginning 23 nt after the termination codon. The duplicated region starts at nt 768 and runs through to the 3' end of the gene. In accordance with the nucleotide sequence of the rearranged RNA 6, a normal-length VP6 product was found in cells infected with the mutant. However, a single-amino-acid change from proline to glutamine at position 309 slightly affected the electrophoretic mobility of the VP6 monomer of the R6 mutant and reduced the stability of VP6 trimers on gels and at low pH values compared with the normal gene product. The degree of relatedness of VP6 of the Chinese lamb rotavirus Lp14 to those of other group A rotaviruses was determined.     

Internal initiation of translation in retroviral vectors carrying picornavirus 5' nontranslated regions.

Previous work has shown that picornavirus 5' nontranslated regions (NTRs) can initiate internal translation of downstream coding regions both in vitro and in transient in vivo assays. We have used 5' NTR sequences from encephalomyocarditis virus and poliovirus to construct retroviral vectors that are designed to express two proteins from a single mRNA. Inclusion of 5' NTR sequences did not adversely affect vector titer. Protein expression was studied with stable cell lines generated by vector infection of mouse NIH 3T3 cells and human and canine skin fibroblasts. Expression of a coding region in the downstream position was at levels from 25 to 100% of the same coding region in the upstream position. Expression of downstream coding regions in control vectors that did not contain the 5' NTR sequences was very low, in agreement with the predictions of the scanning model for eukaryotic translation. These experiments demonstrate coordinate expression of two coding regions from a single mRNA in stable cell lines and provide further support for the model of internal translation initiation by sequences in the 5' NTRs of picornaviruses.     

Cleavage sites within the poliovirus capsid protein precursors.

Partial amino-terminal sequence analysis was performed on radiolabeled polio-virus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein.     

RNA recombination plays a major role in genomic change during circulation of coxsackie B viruses

RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5'-nontranslated region (5'-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, approximately 320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5'-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.     

Complete genome sequence of a recombinant coxsackievirus b4 from a patient with a fatal case of hand, foot, and mouth disease in guangxi, china

The coxsackievirus B4 (CVB4) belongs to human enterovirus B species within the family Picornaviridae. Here we report a novel complete genome sequence of a recombinant CVB4 strain, CVB4/GX/10, which was isolated from a patient with a fatal case of hand, foot, and mouth disease in China. The complete genome consists of 7,293 nucleotides, excluding the 3' poly(A) tail, and has an open reading frame that maps between nucleotide positions 742 and 7293 and encodes a 2,183-amino-acid polyprotein. Phylogenetic analysis based on different genome regions reveals that CVB4/GX/10 is closest to a CVB4 strain, EPIHFMD-CLOSE CONTACT-16, in the 5' half (VP4～2B) of the genome, although it is closer to a Chinese CVB5 strain, CVB5/Henan/2010, in the 3' half (2C～3D) of the genome. Furthermore, similar bootscan analysis based on the whole genomes demonstrates that recombination has possibly occurred within the 2C domain and that CVB4/GX/10 is a possible progeny of intertypic recombination of the CVB4 strain EPIHFMD-CLOSE CONTACT-16 and CVB5/Henan/2010 that occurred during their cocirculation and evolution, which is a relatively common phenomenon in enteroviruses.     

Attenuating mutations in coxsackievirus B3 map to a conformational epitope that comprises the puff region of VP2 and the knob of VP3

Ten antibody escape mutants of coxsackievirus B3 (CVB3) were used to identify nucleotide substitutions that determine viral virulence for the heart and pancreas. The P1 region, encoding the structural genes of each mutant, was sequenced to identify mutations associated with the lack of neutralization. Eight mutants were found to have a lysine-to arginine mutation in the puff region of VP2, while two had a glutamate-to-glycine substitution in the knob of VP3. Two mutants, EM1 and EM10, representing each of these mutations, were further analyzed, initially by determining their entire sequence. In addition to the mutations in P1, EM1 was found to have two mutations in the 3D polymerase, while EM10 had a mutation in stem-loop II of the 5' nontranslated region (5'NTR). The pathogenesis of the mutants relative to that of CVB3 strain RK [CVB3(RK)] then was examined in A/J mice. Both mutants were found to be less cardiotropic than the parental strain, with a 40-fold (EM1) or a 100- to 1,000-fold (EM10) reduction in viral titers in the heart relative to the titers of CVB3(RK). The mutations in VP2, VP3, and the 5'NTR were introduced independently into the RK infectious clone, and the phenotypes of the progeny viruses were determined. The results substantiated that the VP2 and VP3 mutations reduced cardiovirulence, while the 5'NTR mutation in EM10 was associated with a more virulent phenotype when expressed on its own. Stereographic imaging of the two mutations in the capsomer showed that they lie in close proximity on either side of a narrow cleft between the puff and the knob, forming a conformational epitope that is part of the putative binding site for coreceptor DAF.     

Molecular cloning and nucleotide sequences of the complementary DNAs to chicken skeletal muscle myosin two alkali light chain mRNAs.

We report here the molecular cloning and sequence analysis of DNAs complementary to mRNAs for myosin alkali light chain of chicken embryo and adult leg skeletal muscle. pSMA2-1 contained an 818 base-pair insert that includes the entire coding region and 5' and 3' untranslated regions of A2 mRNA. pSMA1-1 contained a 848 base-pair insert that included the 3' untranslated region and almost all of the coding region except for the N-terminal 13 amino acid residues of the A1 light chain. The 741 nucleotide sequences of A1 and A2 mRNAs corresponding to C-terminal 141 amino acid residues and 3' untranslated regions were identical. The 5' terminal nucleotide sequences corresponding to N-terminal 35 amino acid residues of A1 chain were quite different from the sequences corresponding to N-terminal 8 amino acid residues and of the 5' untranslated region of A2 mRNA. These findings are discussed in relation to the structures of the genes for A1 and A2 mRNA.     

Nucleotide sequence and genetic organization of the polyoma late region: features common to the polyoma early region and SV40.

The nucleotide sequence of the late region of the polyoma genome has been determined. It consists of 2366 bp and encodes the virion capsid proteins VP1, VP2 and VP3. Extensive open reading frames identify the possible coding sequences of VP2 and VP3 toward the 5′ end of the late region, and of the major capsid protein VP1 toward the 3′ end of the late region. The 5′ end of the sequence encoding VP1 overlaps the 3′ VP2/VP3 region by 29 nucleotides and is in a different reading frame. The predicted amino acid sequences for all three known capsid proteins show extensive homology with the analogous capsid proteins of SV40 throughout most of their length. The VP2/VP3 amino acid homology between the two viruses is 34%, while the major capsid protein VP1 is much more highly conserved, showing 54% homology. These homologies together with the extent of open reading frames help to define the extent of the coding sequences. The VP2 initiator begins at position 269 and the coding region extends to the first termination codon beginning at 1226. The predicted size of VP2 is 35,007 daltons. A probable VP3 initiator is within the VP2 coding sequence at position 614 and is in the same frame as VP2. This coding sequence can also utilize the terminator at position 1226, and the predicted size of the VP3 translation product is 22,979 daltons. The VP1 coding region begins at position 1197 and continues in a frame different from that of VP2/ VP3 to a termination point at 2349. The molecular weight of VP1 is predicted to be 42,834 daltons. The 5′ untranslated region contains sequences that resemble a potential ribosomal binding site and a possible mRNA capping sequence similar to those found in other eucaryotic systems. There is also a sequence (5′-TCAAGTAAGTGA-3′) almost identical to one found in two regions containing potential splice sites in the early region of polyoma. The 5′ untranslated region does not show the extensive repeated sequences found in the similar region of SV40. The 3′ untranslated region contains the sequence 5′-AATAAA-3′, thought to represent a polyadenylation signal. As in the early region of polyoma, the extensive nucleotide and deduced amino acid homology with SV40 indicate a close evolutionary relationship between the two viruses, and help to identify regions of common and important structure-function relationships.     

柯萨奇B3病毒基因在重组痘苗病毒中的表达

将编码柯萨奇Ｂ３病毒（ＣＶＢ３）衣壳蛋白ＶＰ１和ＶＰ２的基因,分别克隆到具有７．５ｋ启动子的痘苗病毒表达载体ｐＧＪＰ５上;将ＣＶＢ３衣壳蛋白全基因克隆到具有Ｔ７启动子的痘苗表达载体ｐＴＭ１上,并筛先到相应的重组痘苗病毒ＶＶＰ１、ＶＶＰ２和ＶＶＰ／４／２／３／１。ＶＶＰ１和ＶＶＰ２稳定表达产物为ＣＶＢ３衣壳蛋白ＶＰ１和ＶＰ２,而ＶＶＰ４／２／３／１为一无分泌性的多聚蛋白,且这三种表达产物均属无分泌性     

Construction of viable deletion and insertion mutants of the Sabin strain of type 1 poliovirus: function of the 5'' noncoding sequence in viral replication.

A number of deletion and insertion sequences were introduced into the 5' noncoding sequence (742 nucleotides long) of the genome of the Sabin strain of type 1 poliovirus by using an infectious cDNA clone of the virus strain. The genomes of all three poliovirus serotypes contained highly homologous sequences (nucleotide positions 509 to 639) as well as highly variable sequences (positions 640 to 742) in the 5' noncoding region. The viability of mutant viruses was tested by transfecting mutant cDNA clones into African green monkey kidney cells and then estimating the plaque sizes displayed on the cells. The results suggested that the highly variable sequence next to the VP4 coding region did not play an important role, at least in the in vitro culture system used, that the loci of highly conserved nucleotide sequences were not always expected to be the genome regions essential for viral replication, that the sequence between positions 564 and 599 carried genetic information to maintain the efficiency of certain steps in viral replication, and that the sequence between positions 551 to 563 might play an essential role in viral replication. Four-base deletion or insertion mutations were introduced into relatively variable sequences in the genome region upstream of position 509. The results suggest that variable sequences do not always indicate that the corresponding genome regions are less important. Apparent revertants (large-plaque variants) were easily generated from one of the viable mutants with the small-plaque phenotype. The determination of nucleotide sequences of the revertant genomes revealed the second mutation site. The results suggested that the different loci at around positions 200 and 500 might specifically interact with each other. This interaction may result in the formation of a functional structure that influences the efficiency of certain steps in the viral replication.     

Genetic analysis of sequences in the 3' nontranslated region of hepatitis C virus that are important for RNA replication

The genome of the hepatitis C virus (HCV) is a plus-strand RNA molecule that carries a single long open reading frame. It is flanked at either end by highly conserved nontranslated regions (NTRs) that mediate crucial steps in the viral life cycle. The 3' NTR of HCV has a tripartite structure composed of an about 40-nucleotide variable region, a poly(U/UC) tract that has a heterogeneous length, and a highly conserved 98-nucleotide 3'-terminal sequence designated the X tail or 3'X. Conflicting data as to the role the sequences in the 3' NTR play in RNA replication have been reported. By using the HCV replicon system, which is based on the self-replication of subgenomic HCV RNAs in human hepatoma cell line Huh-7, we mapped in this study the sequences in the 3' NTR required for RNA replication. We found that a mutant with a complete deletion of the variable region is viable but that replication is reduced significantly. Only replicons in which the poly(U/UC) tract was replaced by a homouridine stretch of at least 26 nucleotides were able to replicate, whereas RNAs with homopolymeric guanine, adenine, or cytosine sequences were inactive. Deletions of individual or all stem-loop structures in 3'X were not tolerated, demonstrating that this region is most crucial for efficient RNA replication. Finally, we found that none of these deletions or substitutions within the 3' NTR affected RNA stability or translation, demonstrating that the primary effect of the mutations was on RNA replication. These data represent the first detailed mapping of sequences in the 3' NTR assumed to act as a promoter for initiation of minus-strand RNA synthesis.     

Disoxaril mutants of Coxsackievirus B1: phenotypic characteristics and analysis of the target VP1 gene

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackievirus B1 (CVB1/RES) were derived from the wild disoxaril-sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril-resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196-258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented.