Structural proteins of Western equine encephalitis virus: amino acid compositions and N-terminal sequences

The structural proteins of Western equine encephalitis virus, a member of the alphavirus group, have been characterized by the determination of their amino acid compositions and by N-terminal sequence analysis. More than 60 residues of the N-terminal sequences of each of the envelope glycoproteins have been determined. A comparison of these sequences with the previously determined sequences of two related alphaviruses. Sindbis virus and Semliki Forest virus, strongly supports the view that all three viruses have evolved from a common ancestor and provides information on the pattern of this evolution. The analysis of the capsid proteins of Western equine encephalitis virus shows that the nucleocapsid of this virus can accommodate a considerable degree of variability in its protein component and that at least some regions of alphavirus capsid proteins show more extensive differences between different viruses than do the envelope glycoproteins.     

Characteristics of incorporation of carbohydrate radioactivity into structural proteins of the tick-borne encephalitis virus

The propagation of the tick-borne encephalitis virus in the culture of the porcine embryo kidney cells containing the radioactive mannose and glucosamine results in incorporation of radioactive label into hemagglutinin V3(E) as well as into other structural proteins, the nucleocapsid protein V2(C) and membrane protein V1(M). The possible reasons for carbohydrates borne radioactivity incorporation into the proteins V2 and V1 are discussed.     

A tentative model of formation of structural proteins of tick-borne encephalitis virus (flavivirus)

The time course of tick-borne encephalitis virus cell-free protein synthesis was studied by using either [³⁵S]-methionine or formyl[³⁵S]methionyl-tRNA_f^Met as substrates, and the [³⁵S]methionine-labelled products were compared by fingerprinting tryptic peptides. An intermediate in the protein processing, the polypeptide doublet p36/33, was characterized and a tentative model for flavivirus structural protein synthesis and processing was proposed.     

Nucleotide sequence of genes and complete amino acid sequence of tick-borne encephalitis virus strain 205

The 10466 nucleotide long sequence of the cDNA copy of the tick-borne encephalitis strain 205 viral genome has been determined. It includes the 5'-nontranslating region, the genes for structural as well as nonstructural proteins and the first 93 nucleotides of 3'-nontranslating region. The difference in amino acid sequences of structural and nonstructural proteins of strains 205. Sofjin and Neudoerfl of the tick-borne encephalitis virus and the nucleotide changes in 5'- and 3'-nontranslating of these strains are discussed.     

Nucleotide sequence of the genome and complete amino acid sequence of a polyprotein of the tick-borne encephalitis virus

We have cloned and sequenced RNA encoding all virion and nonstructural proteins of tick-borne encephalitis virus (TBEV). Its length is 10,477 bases with a single open reading frame (nucleotides 127-10,363) encoding 3412 amino acids. The 5'- and 3'-noncoding regions have stem- and- loop structure. The polyprotein precursor is proteolytically cleaved, apparently, by a mechanism resembling that proposed for the expression of polyproteins of other flaviviruses, such as yellow fever, West Nile and Kunjin viruses. The deduced TBEV gene order is 5'-C-preM (M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5++ +-3'. The genome and the polyprotein of TBEV and other flaviviruses appears to be structurally similar, although these flaviviruses are transmitted to and from their vertebrate hosts by different carriers, such as ticks or mosquitoes. Analysis of sequence homologies of polyproteins of flaviviruses suggests that TBEV is more closely related to yellow fever virus than to other serological subgroups of flaviviruses (West Nile or Dengue viruses). The hydrophobic profiles of the flaviviruses are highly conservative. Nonstructural proteins NS2A, NS2B, NS4A and NS4B are extremely hydrophobic, suggesting that they are likely to be associated with cellular membranes. Proteins E, NS1, NS3 and NS5 are the most conservative and may be involved in general enzymatic activities related to viral replication and virion assembly.     

Prediction of local structural stabilities of proteins from their amino acid sequences

Hydrogen exchange experiments provide detailed information about the local stability and the solvent accessibility of different regions of the structures of folded proteins, protein complexes, and amyloid fibrils. We introduce an approach to predict protection factors from hydrogen exchange in proteins based on the knowledge of their amino acid sequences without the inclusion of any additional structural information. These results suggest that the propensity of different regions of the structures of globular proteins to undergo local unfolding events can be predicted from their amino acid sequences with an accuracy of 80% or better.     

Purification and N-terminal amino acid sequence comparisons of structural proteins from retrovirus-D/Washington and Mason-Pfizer monkey virus.

A new D-type retrovirus originally designated SAIDS-D/Washington and here referred to as retrovirus-D/Washington (R-D/W) was recently isolated at the University of Washington Primate Center, Seattle, Wash., from a rhesus monkey with an acquired immunodeficiency syndrome and retroperitoneal fibromatosis. To better establish the relationship of this new D-type virus to the prototype D-type virus, Mason-Pfizer monkey virus (MPMV), we have purified and compared six structural proteins from each virus. The proteins purified from each D-type retrovirus include p4, p10, p12, p14, p27, and a phosphoprotein designated pp18 for MPMV and pp20 for R-D/W. Amino acid analysis and N-terminal amino acid sequence analysis show that the p4, p12, p14, and p27 proteins of R-D/W are distinct from the homologous proteins of MPMV but that these proteins from the two different viruses share a high degree of amino acid sequence homology. The p10 proteins from the two viruses have similar amino acid compositions, and both are blocked to N-terminal Edman degradation. The phosphoproteins from the two viruses each contain phosphoserine but are different from each other in amino acid composition, molecular weight, and N-terminal amino acid sequence. The data thus show that each of the R-D/W proteins examined is distinguishable from its MPMV homolog and that a major difference between these two D-type retroviruses is found in the viral phosphoproteins. The N-terminal amino acid sequences of D-type retroviral proteins were used to search for sequence homologies between D-type and other retroviral amino acid sequences. An unexpected amino acid sequence homology was found between R-D/W pp20 (a gag protein) and a 28-residue segment of the env precursor polyprotein of Rous sarcoma virus. The N-terminal amino acid sequences of the D-type major gag protein (p27) and the nucleic acid-binding protein (p14) show only limited amino acid sequence homology to functionally homologous proteins of C-type retroviruses.     

N-terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O, and C.

Sequences of amino acids at the N-termini of virus proteins VP1, VP2, and VP3 were determined for foot-and-mouth disease virus types A12 strain 119, O1Brugge and C3Resende. In the polyacrylamide gel electrophoresis system used to purify the proteins, VP3 migrated faster than VP1 or VP2; and in the virion, VP3 could be cleaved by trypsin into VP3a and VP3b. The N-terminal amino acids for each of the virus types were glycine in VP1, aspartic acid in VP2, and threonine in VP3. No divergences in sequence across the virus types were indicated until at least the fourth position in VP1, and the third in VP3. For virus types A12, O1 and C3, the sequences were, respectively: for VP1 (Gly-ile-phe,pro,val---), (Gly,ile,phe---) and Gly-ile-phe,ala---); for VP2 (Asp,X,met---), (Asp---) and Asp-leu---); and for VP3 (Thr-thr-ala-thr---), (Thr-thr-ser---) and (Thr-thr---). Unresolved mixtures of VP3a and VP3b, from either A12 or O1 viruses, appeared to have the N-terminal amino acids threonine, which is presumed to be the same threonine as in uncleaved VP3 and serine, which is generated by the tryptic cleavage.     

N-terminal amino acid sequences and amino acid compositions of the Spirochaeta aurantia flagellar filament polypeptides.

The amino-terminal sequences and amino acid compositions of the three major and two minor polypeptides constituting the filaments of Spirochaeta aurantia periplasmic flagella were determined. The amino-terminal sequence of the major 37.5-kDa outer layer polypeptide is identical to the sequence downstream of the proposed signal peptide of the protein encoded by the S. aurantia flaA gene. However, the amino acid composition of the 37.5-kDa polypeptide is not in agreement with that inferred from the sequence of flaA. The 34- and 31.5-kDa major filament core polypeptides and the 33- and 32-kDa minor core polypeptides show a striking similarity to each other, and the amino-terminal sequences of these core polypeptides show extensive identity with homologous proteins from members of other genera of spirochetes. An additional 36-kDa minor polypeptide that occurs occasionally in preparations of S. aurantia periplasmic flagella appears to be mixed with the 37.5-kDa outer layer polypeptide or a degradation product of this polypeptide.     

Synthesis and membrane-dependent processing of a precursor of tick-borne encephalitis virus (flavivirus) structural proteins in cell-free systems

Conditions that permitted cell-free synthesis of at least one of the non-structural, in addition to two-structural, polypeptides of tick-borne encephalitis virus have been found. The time course of accumulation of virus-specific polypeptides in extracts of Krebs-2 cells and reticulocyte lysates as well as the peptide maps of the products synthesised were studied. A model of generation of viral structural polypeptides has been proposed, according to which a common precursor of these proteins while in a nascent form, is processed in a membrane-dependent reaction into a C-terminal segment, corresponding to the polypeptide moiety of envelope glycoprotein E, and an N-terminal segment, doublet p36/33. Subsequently, an N-terminal segment, corresponding to the core polypeptide C, is cleaved off from p36/33. The remaining C-terminal segment of p36/33 is possibly a precursor of the membrane polypeptide M. The translational strategy of flaviviruses is compared to that of other positive-stranded RNA viruses.     

蜱传脑炎病毒对人单核细胞的致病性北大核心CSCD

【目的】确定蜱传脑炎病毒(Tick-born encephalitis virus,TBEV)对人单核细胞的感染性及对其复制增殖的影响。【方法】用蜱传脑炎病毒感染单核细胞THP-1,观察细胞病变情况。取不同时间点的细胞培养上清,测定病毒滴度,并用Real Time RT-PCR方法检测病毒核酸;用流式细胞法检测细胞感染率,以确定TBEV在THP-1细胞中的复制增殖情况;同时进行细胞活力检测,以确定TBEV感染后THP-1细胞的变化。【结果】TBEV病毒感染THP-1细胞后,可进行复制增殖,流式细胞法可检测到细胞内的病毒,感染病毒后的单核细胞活力显著降低。【结论】TBEV可在单核细胞THP-1中复制增殖,并可造成细胞活力的显著降低,提示单核细胞可能在TBEV感染机体并扩散至各组织器官过程中发挥了重要作用。     

Diol dehydratase: N-terminal amino acid sequences and subunit stoichiometry

N-terminal sequence analysis of diol dehydratase and its constituent subunits shows that the ratio of the 60K:51K:29K:15K subunits in the native enzyme is 2:1:2:2. From the amino acid compositions of the individual subunits diol dehydratase appears to be a peripheral membrane protein.     

森林脑炎病毒分子生物学研究进展

森林脑炎(TBE)病毒属黄病毒科,基因姐RNA含有单个开放阅读框架,5′端编码病毒的结构蛋白,3′端编码非结构蛋白。翻译成聚蛋白后,通过细胞和病毒编码的蛋白酶裂解产生单个的病毒蛋白。成熟的病毒是由两个相关的E和M膜蛋白脂质包膜所包围的立体对称的核衣壳组成。包膜E蛋白在病毒的感染周期中对细胞的识别和穿入细胞具有极其重要的功能,同时E蛋白诱导保护性的免疫反应,E蛋白内某一位点单个氨基酸的改变可引起病毒毒力的改变。因此,对TBE病毒分子生物学的研究有助于了解病毒与宿主细胞相互作用的机理,为病毒感染的特异性诊断、疫苗的研制和抗病毒药物的设计提供理论依据。     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

Availability of short amino acid sequences in proteins

Much attention is being paid to protein databases as an important information source for proteome research. Although used extensively for similarity searches, protein databases themselves have not fully been characterized. In a systematic attempt to reveal protein-database characters that could contribute to revealing how protein chains are constructed, frequency distributions of all possible combinatorial sets of three, four, and five amino acids ("triplets," "quartets," and "pentats"; collectively called constituent sequences) have been examined in the nonredundant (nr) protein database, demonstrating the existence of nonrandom bias in their "availability" at the population level. Nonexistent short sequences of pentats were found that showed low availability in biological proteins against their expected probabilities of occurrence. Among them, six representative ones were successfully synthesized as peptides with reasonably high yields in a conventional Fmoc method, excluding the possibility that a putative physicochemical energy barrier in forming them could be a direct cause for the low availability. They were also expressed as soluble fusion proteins in a conventional Escherichia coli BL21Star(DE3) system with reasonably high yield, again excluding a possible difficulty in their biological synthesis. Together, these results suggest that information on three-dimensional structures and functions of proteins exists in the context of connections of short constituent sequences, and that proteins are composed of evolutionarily selected constituent sequences, which are reflected in their availability differences in the database. These results may have biological implications for protein structural studies.     

A single amino acid substitution in envelope protein E of tick-borne encephalitis virus leads to attenuation in the mouse model.

We have determined the virulence characteristics of seven monoclonal antibody escape mutants of tick-borne encephalitis virus in the mouse model. One of the mutants with an amino acid substitution from tyrosine to histidine at residue 384 revealed strongly reduced pathogenicity after peripheral inoculation of adult mice but retained its capacity to replicate in the mice and to induce a high-titered antibody response. Infection with the attenuated mutant resulted in resistance to challenge with virulent virus. Assessment of nonconservative amino acid substitutions in other attenuated flaviviruses suggests that a structural element including residue 384 may represent an important determinant of flavivirus virulence in general.