The nucleotide sequence of cDNA coding for the structural proteins of foot-and-mouth disease virus

The complete nucleotide sequence of cDNA coding for the structural capsid polypeptides of foot-and-mouth disease virus (FMDV) (strain A(10)61) has been determined. Portions of the flanking sequence coding for the nonstructural proteins p20a and p52 are also provided. The three larger structural polypeptides VP1, VP2 and VP3 have unmodified Mrs of 23248, 24649 and 24213, respectively. The size of the smaller polypeptide, VP4, can only be estimated at 7360 because the 5'-limit of its coding region is not yet known with certainty. The sequence data for VP1 (the major immunising antigen) and the amino-terminal quarter of p52 are compared with the data of Kurz et al. (Nucl. Acids Res. 9 (1981) 1919-1931) for a different serotype (O1K). This shows that variation is much greater in the region coding for VP1 than in that coding for p52. This is reflected in the level of amino acid sequence variation predicted for the two proteins. Analysis of relative codon usage reveals a strong bias in favour of C and G over U and A in the third base position. The dinucleotide frequencies show a bias against A-U and U-A, and for A-C and C-A.     

Complete nucleotide sequence of RNA 3 from alfalfa mosaic virus, strain S

We report the sequence of RNA 3 from strain S of Alfalfa mosaic virus (2,055 nucleotides). This RNA codes for a 32.4 kd protein (P3) and for the 24 kd coat protein (P4). The largest part of the sequence was established using RNA sequencing methods. The completion of the sequence in the region coding for P3 was achieved with cloned cDNA synthesized after priming at internal sites of RNA 3. Comparison of the RNA sequences coding P3 and P4 proteins in strain S with those reported in the literature for strain 425 revealed a higher amino acid substitution rate (3%) for P3 than for P4 (congruent to 1%) despite a similar average base substitution of 3-4% in these regions. In P3, two out of nine amino acid changes occur in hydrophilic regions. The amino acid changes in P4 do not modify the local hydrophilicity distribution. The intercistronic region displays a low degree of base substitution (2%) when compared with the untranslated 3'-end region (3.6%) or the 5'-end leader region (8%), the average substitution rate being 3.2%.     

H3N2亚型猪流感病毒的拯救及HA与NA基因的替换

摘要：【目的】为研究流感病毒突破种间屏障分子机制,筛选流感基因工程疫苗株。【方法】本实验以猪流感病毒A/Swine/Henan/S4/01(H3N2)为亲本株,利用反向遗传学操作技术,采用RT-PCR技术对该病毒的8个基因片段分段进行扩增,通过与双向转录载体pHW2000连接, 重组质粒转染293T和MDCK共培养细胞,拯救出全部基因均来自于亲本株的猪流感病毒rgH3N2,并分别以人流感病毒A/ PR/8/34(H1N1)、禽流感病毒A/Duck/Nanchang /4-165/2000 (H4N6 )、马流感病毒A/Equine/Fuyun/2008/(H3N8)的HA和NA基因替换A/Swine/Henan/S4/01的相应基因,【结果】生物学实验结果表明rgH3N2在鸡胚半数感染量、组织培养半数感染量、稳定性试验等方面都与亲本株保持一致。rgH3N2经鸡胚多次传代后血凝价最高可达到1:256,接种MDCK细胞60 h后,血凝价可以达到1:64。基因替换后成功拯救出的重组病毒rgH1N1、rgH4N6和rgH3N8在鸡胚和细胞上均具有较高的增殖能力。【结论】病毒的成功拯救为流感病毒突破种间屏障分子机制,HA、NA基因在流感病毒跨种属传播中所扮演角色的研究和流感基因工程疫苗株的筛选奠定了基础。     

H3N2亚型猪流感病毒M1基因克隆及表达特性分析

从H3N2亚型猪流感病毒感染的鸡胚尿囊液中提取病毒基因组RNA,采用RT-PCR方法克隆M1全长基因,将M1基因亚克隆至pET-28a(+)表达载体中,构建重组表达质粒pET-28a-M1,将该重组质粒转化大肠杆菌BL21并经IPTG诱导表达。诱导产物经SDS-PAGE电泳分析显示重组蛋白M1获得大量表达,表达蛋白纯化后免疫Wistar大鼠制备多克隆抗体。Westernblotting检测结果表明制备的抗M1蛋白多克隆抗体可以识别大肠杆菌表达的M1蛋白和病毒感染细胞的病毒M1蛋白。构建M1基因真核重组质粒p3xFLAG-CMV-7.1-M1并转染Vero细胞,Western blotting检测表明抗M1蛋白多克隆抗体可以识别在Vero细胞中得到表达的M1蛋白,成功建立M1基因的真核表达系统。分析了病毒感染过程中M1蛋白的变化及其作为病毒复制指示分子的可能性。鼠源M1蛋白多克隆抗体的制备和M1基因真核重组表达质粒的构建,为进一步研究猪流感病毒的复制机理以及M1蛋白在病毒复制过程中的生物学功能奠定了基础。     

Comparison of the nucleotide sequence of cloned DNA coding for an apolipoprotein (apoVLDL-II) from avian blood and the amino acid sequence of an egg-yolk protein (apovitellenin I): equivalence of the two sequences

We have compared the amino acid sequences of two low-molecular-weight avian apoproteins: apoVLDL-II from very low-density lipoproteins of hen plasma and apovitellenin I from hen egg yolk. The sequence of White Leghorn apoVLDL-II was derived from the nucleotide sequence of cloned apoVLDL-II DNA (Chan et al., 1980). The sequenator was used to determine the amino acid sequence of apovitellinin I from two breeds of hen (White Leghorn and Australorp). The sequences from the two breeds were not only identical, but they also completely matched the predicted sequence derived from the apoVLDL-II DNA sequence. The identity reported here establishes that this protein is transported intact from the blood to the egg yolk.     

一株H3N2亚型猪流感病毒广东分离株的基因组序列分析

从广东省疑似流感发病猪分离到1株H3N2亚型猪流感病毒(A/Swine/Guangdong/01/2005(H3N2)),对其各个基因进行克隆与测序,并与GenBank中收录的其它猪流感、禽流感和人流感的相关基因进行比较,结果表明,HA全基因与广东2003~2004年分离的H3N2猪流感毒株的核苷酸序列同源性在99%以上,与纽约90年代末分离的H3N2人流感毒株同源性在98.5%以上;NA基因与纽约1998~2000年分离的H3N2人流感毒株的核苷酸序列同源性在99%以上;NS基因、M基因的核苷酸序列与H1N1亚型猪流感毒株A/swine/HongKong/273/1994(H1N1)的核苷酸序列同源性较高,分别为97.9%、98.4%,与美洲A/swine/Iowa/17672/1988(H1N1)的核苷酸序列同源性分别为96.7%、97.1%;其他基因的核苷酸序列与H3N2人流感毒株具有很高的同源性。因此,推测其M和NS基因来源于H1N1亚型猪流感病毒,HA、NA及其他基因均来源于H3N2亚型人流感病毒。表明此H3N2亚型猪流感病毒为H3N2亚型人流感病毒和H1N1亚型猪流感病毒经基因重排而得到的重组病毒。     

The sequence of foot-and-mouth disease virus RNA to the 5′ side of the poly(C) tract

The nucleotide sequence of foot-and-mouth disease virus (FMDV) RNA to the 5' side of the poly(C) tract (S fragment) has been determined for representatives of the A and O serotypes of the virus. The two S fragments differ in length by five nucleotides (nt), with 367 nt for O₁ compared with 362 nt for A₁₀, due to a number of insertions/deletions. However, the two sequences show 86% homology. There are no conserved open reading frames (ORFs). Secondary structure predictions reveal a high degree of potential base-pairing, such that the entire S fragment sequence can be folded into a hairpin structure.     

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage λ Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.     

Construction and characterization of a bacterial clone containing the hemagglutinin gene of the WSN strain (HON1) of influenza virus

A synthetic dodecadeoxynucleotide primer has been used to prepare a double-stranded DNA form of the hemagglutinin (HA) gene of a human influenza virus (WSN strain, HON1). This DNA has been inserted in plasmid pBR322 and cloned in bacterial cells. The insert contains nearly the complete hemagglutinin gene. A restriction map of this insert has been determined and structurally important areas of the HA gene have been sequenced. Amino acid sequences of several regions of the HA protein were deduced from the DNA sequences and compared to the known amino acid sequences of other influenza A viruses. WSN HA shows extensive homology to all influenza A viruses in a few regions, namely the first 17 amino acids of the N-terminus of HA1 (N-terminal polypeptide of HA) and the first 24 amino acids of the N-terminus of HA2 (C-terminal polypeptide of HA). The sequence diverges extensively from other influenza A viruses in most other areas. The sequence of WSN virus HA is similar to that of other HON1 viruses with the exception of the C-terminus of the HA1 peptide. The change in this area may contribute to some of the unique properties of WSN virus among the HON1 viruses. In addition, WSN HA contains a 17-amino-acid precursor before the N-terminus of HA1 and a single amino acid, arginine, connecting HA1 and HA2.     

H5N2亚型禽流感病毒血凝素裂解位点碱性氨基酸对应mRNA核苷酸的二级结构分析

以H5N2亚型禽流感病毒毒株血凝素蛋白裂解位点碱性氨基酸为研究对象,对其密码子偏好性和对应mRNA序列的折叠二级结构特点进行研究和分析。旨在探讨裂解位点氨基酸对应mRNA核苷酸片段的二级结构与病毒致病力的关系,希望能对禽流感病毒的研究提供一些基础性信息。将mRNA样本按照序列等步长递增的方法,用RNAstructure 4．1程序预测这些样本的动态延伸折叠二级结构。序列和结构的分析结果：裂解位点的碱性氨基酸对富含腺嘌呤的密码子有强烈偏好;与碱性氨基酸对应的mRNA片段上的核苷酸主要位于折叠二级结构的单链环区,少数位于配对螺旋区。结果表明：裂解位点氨基酸对应的mRNA核苷酸形成发夹端环的大小与其碱性氨基酸的多少具有正相关性。     

Molecular cloning of AKR xenotropic murine leukemia virus unintegrated proviral DNA

Suprahelical proviral DNA of AKR xenotropic murine leukemia virus was purified from agarose gels and cloned in lambda Charon 28 DNA (BamHI sites). Nine viral DNA recombinants were identified and mapped with 12 restriction endonucleases. Three calsses of cloned viral DNA inserts were found: (1) Six inserts were apparently full-length 9.0-kb DNA with tandem long terminal repeat (LTR) elements; (2) two inserts contained DNAs with deletions in or adjacent to the LTR regions; (3) a single isolate contained an inversion of 2.3 kb around the LTR in the envelope gene.     

Molecular cloning of cDNA from foot-and-mouth disease virus C1-Santa Pau (C-S8). Sequence of protein-VP1-coding segment

cDNA segments copied from the RNA of foot-and-mouth disease virus (FMDV) C₁-Santa Pau (isolate C-S8) have been cloned in plasmid pBR322. A 998-bp DNA fragment, that includes the region coding for capsid protein VP1, the carboxy terminus of VP3, and the amino terminus of precursor protein p52 has been sequenced. Comparison of the nucleotide sequence with those from FMDV O₁K, A₁₀61, a₁₂ and C₃ Indaial (Kurz et al., Nucl. Acids Res. 9 (1981) 1919–1931; Kleid et al., Science 214 (1981) 1125–1129; Boothroyd et al., Gene 17 (1982) 153–161; Makoff et al., Nucl. Acids Res. 10 (1982) 8285–8295) indicates extensive variability between the corresponding gene segments, including short insertions and deletions. Base transversions are more frequent than transitions within the VP1 coding segment, but not in the sequence coding for the amino-terminal end of p52. The nucleotide sequence divergence is reflected in variability in both the primary and the predicted higher-order structures of the encoded VP1s.     

HNHC: a triple resonance experiment for correlating the H2, N1(N3) and C2 resonances in adenine nucleobases of <Superscript>13</Superscript>C-, <Superscript>15</Superscript>N-labeled RNA oligonucleotides

A novel NMR pulse sequence has been developed that correlates the H2 resonances with the C2 and the N1 (N3) resonances in adenine nucleobases of ¹³C, ¹⁵N labeled oligonucleotides. The pulse scheme of the new 3D-HNHC experiment is composed of a ²J-¹⁵N-HSQC and a ¹J-¹³C-HSQC and utilizes large ²J(H2, N1(N3)) and ¹J(H2, C2) couplings. The experiment was applied to a medium-size ¹³C, ¹⁵N-labeled 36mer RNA. It is useful to resolve assignment ambiguities occurring especially in larger RNA molecules due to resonance overlap in the ¹H-dimension. Therefore, the missing link in correlating the imino H3 resonances of the uracils across the AU base pair to the H8 resonances of the adenines via the novel pulse sequence and the TROSY relayed HCCH-COSY (Simon et al. in J Biomol NMR 20:173–176 2001) is provided. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Replication of the cauliflower mosaic virus: role and stability of the cloned delta 3 discontinuity sequence

A fragment of cauliflower mosaic virus (CaMV) DNA, containing delta 3, one of the three discontinuity sequences, was cloned in various ways into CaMV DNA deleted for the delta 3 sequence. The series of constructions was monitored for the appearance of the typical single-strand (ss) discontinuity after hybrid CaMV replication in plants. The delta 3 discontinuity was observed only if the orientation of inserted DNA sequence was the same as in the wild-type virus. Long polylinker sequences used for insertion of the fragment into cloned viral DNA, affected the stability of the insert in progeny viral DNA in plants by acting as recombination targets.     

RNA-Mediated Dimerization of the Human Deoxycytidine Deaminase APOBEC3H Influences Enzyme Activity and Interaction with Nucleic Acids

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4 + T cells. When aberrantly expressed in lung or breast tissue, APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12-nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, and deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single-molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA-mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.