mRNA的序列、结构以及翻译速率与蛋白质结构的关系

mRNA所包含的核苷酸序列通过三联体密码子决定了蛋白质的氨基酸序列。但是, 由于对氨基酸同义密码使用频率上的差异, 密码子与反密码子相互作用效率上的不同, 以及密码子上下文关系和mRNA 不同区域二级结构上的差异, 造成了核糖体对mRNA 不同区域翻译速度上的差异, 加之共翻译折叠的作用, 使得mRNA 的序列和结构影响着蛋白质空间结构的形成。     

枯草芽孢杆菌同义密码子使用偏性对蛋白质折叠速率的影响

目前,有关同义密码子使用偏性对蛋白质折叠的影响研究中,样本蛋白均来源于不同的物种。考虑到同义密码子使用偏性的物种差异性,选取枯草杆菌的核蛋白为研究对象。首先,将每条核蛋白按二级结构截取为α螺旋片段、β折叠片段和无规卷曲（α-β混合）片段,并计算其蛋白质折叠速率。然后,整理每个片段相应的核酸序列信息,计算其同义密码子使用度。在此基础上,分析枯草芽孢杆菌核蛋白的同义密码子使用偏性与蛋白质折叠速率的相关性。发现对于不同二级结构的肽链片段,都有部分密码子的使用偏性与其对应的肽链折叠速率显著相关。进一步分析发现,与肽链片段折叠速率显著相关的密码子绝大部分为枯草杆菌全序列或核蛋白序列的每一组同义密码子中使用度最高的密码子。结果表明,在蛋白质的折叠过程中,枯草芽孢杆菌的同义密码子使用偏性起着重要作用。     

一株H5N2亚型鹦鹉源禽流感病毒分子进化分析

2005年在广东进行流行病学调查时分离到一株鹦鹉源禽流感病毒,经鉴定为H5N2亚型禽流感病毒(A/Parrot/Guangdong/268/2005)。该毒株的HA裂解位点附近的氨基酸序列为RETRGLF,只含有一个碱性氨基酸,符合低致病性禽流感病毒的HA裂解位点附近氨基酸序列的分子特征;与H5N2亚型禽流感代表毒株相比,该毒株HA和NA基因的糖基化位点、HA基因的受体结合位点编码区、NA基因的耐药性位点均未发生变异。将该毒株全基因组序列与GenBank已公布的19株H5N2亚型禽流感病毒株的相应序列进行比较分析并绘制系统进化树后发现:其与低致病性禽流感毒株A/Pheasant/NJ/1355/1998(H5N2)-like的亲缘关系最近,位于以A/Chicken/Pennsylvania/1/1983(H5N2)为代表的美洲进化分支。     

同义密码子用语与蛋白质结构的关系

对大肠杆菌的５４种三维结构已知的蛋白质及其对应的ｍＲＮＡ序列的集合的分析结果表明,绝大部分密码子与其编码的氨基酸在蛋白质α螺旋、β折叠和卷曲等三种二级结构中的偏好性没有显著的统计性差异。同时,又对Ａｄｚｈｕｂｅｉ提供的相应哺乳类数据集作了同样的偏好性检验,表明９种氨基酸的同义密码子携带蛋白质二级结构信息,与他们的结论一致。推测,这种差异可能由翻译机制的进化趋异造成的。这些结果对于真核基因在原核生物中实现高表达可能有重要的实际意义。     

mRNA的序列,结构以及翻译速率与蛋白质结构的关系

ｍＲＮＡ所包含的核苷酸序列通过三联体密码子决定了蛋白质的氨基酸序列,但是,由于对氨基酸同义密码使用频率上的差异,密码子与反密码子相互作用效率上的不同,以及密码子上下文关系和ｍＲＮＡ不同区域二级结构上的差异,造成了核糖体对ｍＲＮＡ不同区域翻译速度上的差异,加之共翻译折叠的作用,使得ｍＲＮＡ的序列和结构影响着蛋白质空间结构的形成。     

蛋白质折叠速率与其氨基酸序列极性的关系

以大肠杆菌60个蛋白酶以及几种常见病毒（SARS病毒、艾滋病病毒、丙型肝炎病毒及乙型肝炎病毒）各蛋白质序列中的所有α-螺旋和β-折叠片段为研究对象,计算了各片段的折叠速率和平均极性,分别在各物种的α-螺旋和β-折叠两类二级结构片段中分析了二者的相关性。得到结论：不论是大肠杆菌中的蛋白酶还是病毒蛋白,其中的两类氨基酸片段的平均极性与折叠速率都是极显著相关的：对于所有的α片段,二者呈线性正相关,而对于所有的β片段,二者成线性负相关。结果证实了在蛋白质折叠中,氨基酸的极性起着重要的作用。     

我国四株狂犬病毒M和P基因序列分析和所编码蛋白的分析

为了解我国狂犬病毒M、P基因序列和结构特点,用RT-PCR方法获得目的基因片段,测定核苷酸序列后,计算机分析核苷酸和氨基酸序列及其功能区位点结构。结果显示四株病毒M基因核苷酸和氨基酸序列同源性分别为83.9%～99.5%和93.1%～99%,四株狂犬病毒M蛋白上调节病毒RNA转录和复制功能的第58位氨基酸残基均为谷氨酰胺残基(E),与特异性细胞蛋白WW区域作用的PPxY结构序列均为PPEY保守序列;四株病毒P基因核苷酸和氨基酸序列同源性分别为83.6%～99.8%和87.2%～99%,P蛋白与胞浆动力蛋白轻链LC8相互作用的序列位于143～148位氨基酸残基,均为DKSTQT,四株病毒P基因与L蛋白、N蛋白作用位点序列显示未发生影响其生物学功能的变异。研究结果证实了这两种蛋白结构在病毒致病性中起重要作用的推论。     

鸽源H5N1亚型禽流感病毒株的HA和NA基因特征性分析

本研究自行设计合成两对特异性引物,通过RT-PCR扩增出1株鸽源H5N1亚型禽流感病毒血凝素（HA）和神经氨酸酶（NA）两个基因的cDNA片段,将它们成功克隆于pMD18-T载体上,然后进行序列测定。结果表明,HA基因全长1707bp,编码568个氨基酸, HA基因有7个糖基化位点,在裂解位点附近有连续6个碱性氨基酸（R-R-R-K-K-R）的插入,具有高致病性毒株的分子特征。受体结合位点的氨基酸分别为YWIHELY,左侧壁氨基酸为SGVSSA,右侧壁为NGQSGR;NA基因全长1350bp,编码446个氨基酸,NA基因有3个糖基化位点。     

大肠杆菌mRNA编码区长度、形成二级结构倾向与密码子偏好性的关系

从GenBank获得大肠杆菌K-12MG1655株的全基因组序列,计算了与基因密码子偏好性相关的多个参数(Nc、CAI、GC、GC3s),对其mRNA编码区长度、形成二级结构倾向与密码子偏好性之间的关系进行了统计学分析,发现虽然翻译效率(包括翻译速度和翻译精度)是制约大肠杆菌高表达基因的密码子偏好性的主要因素,同时,mRNA编码区长度及其形成二级结构的倾向也是形成这种偏好性的不可忽略的原因,而且对偏好性有一定程度的削弱。另外对mRNA编码区形成二级结构倾向的生物学意义进行了讨论分析。     

青海湖地区5株 H9N2亚型禽流感病毒全基因组序列进化分析

2012年7~9月从来源于青海湖地区活禽市场的环境样本中分离到5株H9N2亚型禽流感病毒,为了了解其基因遗传进化情况,本研究通过RT-PCR技术扩增分离毒株的8个基因片段,并进行全基因序列测定。对其分子特征及全基因序列进行遗传进化分析。结果显示5株病毒的HA基因片段的核苷酸相似度为93.2%~99.1%。NA基因核苷酸的相似度为94.5%~99.8%。A/environment/qinghai/017/2012的裂解位点为PSKSSRGLF,其它4个毒株的HA裂解位点均为PSRSSRGLF。5个病毒的HA基因第226位受体结合位点均为L。M1基因片段中发生了N30D和T215A替换。遗传进化分析表明5株病毒同2005年湖南分离的A/chicken/Hunan/5260/2005(H9N2)毒株类似,为一种重配基因型禽流感病毒。其中HA、NA、NS基因片段属于Y280-like支系,MP基因片段属于G1-like支系,NP、PB1、PB2、PA四个基因片段属于F98-like支系。     

Structural features influencing hemagglutinin cleavability in a human influenza A virus.

The cleavability of the hemagglutinin (HA) molecule is related to the virulence of avian influenza A viruses, but its influence on human influenza virus strains is unknown. Two structural features are involved in the cleavage of avian influenza A virus HAs: a series of basic amino acids at the cleavage site and an oligosaccharide side chain in the near vicinity. The importance of these properties in the cleavability of a human influenza A virus (A/Aichi/2/68) HA was investigated by using mutants that contained or lacked an oligosaccharide side chain and had either four or six basic amino acids. All mutants except the one that contains a single mutation at the glycosylation site were cleaved, although not completely, demonstrating that a series of basic amino acids confers susceptibility to cellular cleavage enzymes among human influenza virus HAs. The mutants containing six basic amino acids at the cleavage site showed limited polykaryon formation upon exposure to low pH, indicating that cleavage was adequate to impart fusion activity to the HA. Deletion of the potential glycosylation site had no effect on the cleavability of these mutants; hence, the oligosaccharide side chain appears to have no role in human influenza virus HA cleavage. The inability to induce high cleavability in a human influenza A virus HA by insertion of a series of basic amino acids at the cleavage site indicates that other, as yet unidentified structural features are needed to enhance the susceptibility of these HAs to cellular proteases.     

Molecular determinants within the surface proteins involved in the pathogenicity of H5N1 influenza viruses in chickens

Although it is established that the cleavage site and glycosylation patterns in the hemagglutinin (HA) play important roles in determining the pathogenicity of H5 avian influenza viruses, some viruses exist that are not highly pathogenic despite possessing the known characteristics of high pathogenicity (i.e., their HA contains multiple basic amino acids at the cleavage site and has glycosylation patterns similar to that of the highly pathogenic H5 viruses). Currently little is known about the H5N1 viruses that fall into this intermediate category of pathogenicity. We have identified strains of H5N1 avian influenza viruses that have markers typical of high pathogenicity but distinctly differ in their ability to cause disease and death in chickens. By analyzing viruses constructed by reverse-genetic methods and containing recombinant HAs, we established that amino acids 97, 108, 126, 138, 212, and 217 of HA, in addition to those within the cleavage site, affect pathogenicity. Further investigation revealed that an additional glycosylation site within the neuraminidase (NA) protein globular head contributed to the high virulence of the H5N1 virus. Our findings are in agreement with previous observations that suggest that the activities of the HA and NA proteins are functionally linked.     

Generation of seal influenza virus variants pathogenic for chickens, because of hemagglutinin cleavage site changes.

Influenza virus A/seal/Mass/1/80 (H7N7) was adapted to grow in MDCK cells and chicken embryo cells (CEC) in the absence of exogenous protease. The biological properties of the virus variants obtained coincided with intracellular activation of the hemagglutinin (HA) by posttranslational proteolytic cleavage and depended on the cell type used for adaptation. MDCK cell-adapted variants contained point mutations in regions of the HA more distant from the cleavage site. It is proposed that these mutations are probably responsible, through an unknown mechanism, for enhanced cleavability of HA in MDCK cells. Such virus variants were apathogenic in chickens. CEC-adapted variants, on the other hand, contained an insertion of basic amino acids at the HA cleavage site, in addition to scattered point mutations. The insertions converted the cleavage sites in the variant virus HAs so that they came to resemble the cleavage site found in highly pathogenic avian influenza viruses. CEC variants with such cleavage site modifications were highly pathogenic for chickens. The lethal outcome of the infection in chickens demonstrated for the first time that an influenza virus derived from a mammalian species can be modified during adaptation to a new cell type to such an extent that the resulting virus variant becomes pathogenic for an avian species.     

虎源流感病毒HA 基因的系统发生分析及其在杆状病毒系统中的表达

通过对虎源流感病毒A/ Tiger/ Harbin/01/ 2003 (H5N1)的HA 基因进行克隆与序列测定,证明该基因全长为1 731 bp,读码框由1 707个碱基组成,编码568 个氨基酸。对HA 基因的进化分析表明,该基因与H5 亚型流感病毒的HA 基因同源性最高,其HA 裂解位点由6 个碱性氨基酸插入序列(RRRKKR)组成,符合高致病性禽流感病毒的分子特征。将HA 基因克隆入杆状病毒转座载体质粒pFastBacⅠ,构建重组质粒pFastBac-HA;再将该重组质粒转化DH10 Bac 感受态细菌,在体内进行重组,并经三重抗性与蓝白斑筛选,得到杆状病毒重组质粒Bacmid-HA;将Bacmid-HA 转染sf9 细胞,获得重组杆状病毒。经Western-blotting 检测,HA 蛋白在重组杆状病毒中获得表达。用感染重组病毒的sf9 细胞免疫小鼠,2 次免疫后2 周可诱导小鼠产生1∶ 8 ～1∶ 16 的血凝抑制抗体,表明虎源流感病毒的HA 基因在重组杆状病毒系统中得到了正确表达。     

2017–2019年我国南方地区高致病性H5N6亚型禽流感病毒血凝素蛋白分子特征分析

【背景】自2014年以来,H5N6禽流感病毒在我国家禽和活禽市场持续进化,成为人类和动物健康的重大威胁。【目的】对2017–2019年中国南方地区93株高致病性H5N6禽流感病毒的HA基因进行分子进化分析。【方法】接种9–11日龄鸡胚分离核酸检测阳性的H5N6标本,运用下一代测序平台对病毒分离物进行全基因组测序,从NCBI和GISAID数据库下载参考序列,利用BLAST、MEGA6.1及Clustal X等软件进行序列分析。【结果】2017–2019年,从189份江苏省H5亚型禽类/环境标本和1名H5N6患者咽拭子标本中共分离到43株病毒,完成了33株H5N6病毒的全基因组测序。下载网上同时期中国其他地区流行的H5N6毒株序列,对总计93株H5N6病毒的HA基因进行分子进化分析。93株H5N6病毒中有78株属于Clade 2.3.4.4h,9株病毒属于Clade 2.3.4.4e,4株H5N6病毒属于Clade 2.3.4.4b,1株属于Clade 2.3.4.4f,1株属于Clade 2.3.4.4g。所有93株病毒HA蛋白的裂解位点含有多个碱性氨基酸,表明它们都属于高致病性禽流感病...     

Sequence analysis of recent H7 avian influenza viruses associated with three different outbreaks in commercial poultry in the United States

The hemagglutinin (HA) and neuraminidase (NA) genes of H7 avian influenza virus (AIV) isolated between 1994 and 2002 from live-bird markets (LBMs) in the northeastern United States and from three outbreaks in commercial poultry have been characterized. Phylogenetic analysis of the HA and NA genes demonstrates that the isolates from commercial poultry were closely related to the viruses circulating in the LBMs. Also, since 1994, two distinguishing genetic features have appeared in this AIV lineage: a deletion of 17 amino acids in the NA protein stalk region and a deletion of 8 amino acids in the HA1 protein which is putatively in part of the receptor binding site. Furthermore, analysis of the HA cleavage site amino acid sequence, a marker for pathogenicity in chickens and turkeys, shows a progression toward a cleavage site sequence that fulfills the molecular criteria for highly pathogenic AIV.     

Human influenza virus hemagglutinin with high sensitivity to proteolytic activation.

To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.     

Interplay between carbohydrate in the stalk and the length of the connecting peptide determines the cleavability of influenza virus hemagglutinin.

The ability of many viruses to replicate in host cells depends on cleavage of certain viral glycoproteins, including hemagglutinin (HA). By generating site-specific mutant HAs of two highly virulent influenza viruses, we established that the relationship between carbohydrate in the stalk and the length of the connecting peptide is a critical determinant of cleavability. HAs that lacked an oligosaccharide side chain in the stalk were cleaved regardless of the number of basic amino acids at the cleavage site, whereas those with the oligosaccharide side chain resisted cleavage unless additional basic amino acids were inserted. This finding suggests that the oligosaccharide side chain interferes with HA cleavage if the number of basic amino acids at the cleavage site is not adequate to nullify this effect. Similar interplay could influence cleavage of other viral glycoproteins, such as those of human and simian immunodeficiency viruses and paramyxoviruses.