Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.     

Structure, sequence and expression of the hepatitis delta (delta) viral genome

Biochemical and electron microscopic data indicate that the human hepatitis delta viral agent contains a covalently closed circular and single-stranded RNA genome that has certain similarities with viroid-like agents from plants. The sequence of the viral genome (1,678 nucleotides) has been determined and an open reading frame within the complementary strand has been shown to encode an antigen that binds specifically to antisera from patients with chronic hepatitis delta viral infections.     

A conformational switch controls hepatitis delta virus ribozyme catalysis

Ribozymes enhance chemical reaction rates using many of the same catalytic strategies as protein enzymes. In the hepatitis delta virus (HDV) ribozyme, site-specific self-cleavage of the viral RNA phosphodiester backbone requires both divalent cations and a cytidine nucleotide. General acid-base catalysis, substrate destabilization and global and local conformational changes have all been proposed to contribute to the ribozyme catalytic mechanism. Here we report ten crystal structures of the HDV ribozyme in its pre-cleaved state, showing that cytidine is positioned to activate the 2'-OH nucleophile in the precursor structure. This observation supports its proposed role as a general base in the reaction mechanism. Comparison of crystal structures of the ribozyme in the pre- and post-cleavage states reveals a significant conformational change in the RNA after cleavage and that a catalytically critical divalent metal ion from the active site is ejected. The HDV ribozyme has remarkable chemical similarity to protein ribonucleases and to zymogens for which conformational dynamics are integral to biological activity. This finding implies that RNA structural rearrangements control the reactivity of ribozymes and ribonucleoprotein enzymes.     

Folding Kinetics of HDV Ribozyme with C13A:G82U and A16U:U79A Mutations

Gene mutations influence the folding kinetics of hepatitis delta virus(HDV) ribozyme. In this work, we study the effect of the double mutation on the folding kinetics of HDV ribozyme. By using the master equation method combined with RNA folding free energy landscape, we predict the folding kinetics of C13A:G82U and A16U:U79A mutated HDV sequences. Their folding pathways are identified by recursively searching the states with high net flux-in(out) population starting from the native state. The results indicate that the folding kinetics of C13A:G82U mutation sequence is bi-phasic, which is similar to the wild type(wt HDV) sequence. While the folding kinetics of A16U:U79A mutation sequence is mono-phasic, it quickly folds to the native state in 30 s. Thus, the folding kinetics of double mutated HDV ribozyme depends on the mutation sites.     

High diversity of unknown picorna-like viruses in the sea

Picorna-like viruses are a loosely defined group of positive-sense single-stranded RNA viruses that are major pathogens of animals, plants and insects. They include viruses that are of enormous economic and public-health concern and are responsible for animal diseases (such as poliomyelitis), plant diseases (such as sharka) and insect diseases (such as sacbrood). Viruses from the six divergent families (the Picornaviridae, Caliciviridae, Comoviridae, Sequiviridae, Dicistroviridae and Potyviridae) that comprise the picorna-like virus superfamily have the following features in common: a genome with a protein attached to the 5' end and no overlapping open reading frames, all the RNAs are translated into a polyprotein before processing, and a conserved RNA-dependent RNA polymerase (RdRp) protein. Analyses of RdRp sequences from these viruses produce phylogenies that are congruent with established picorna-like virus family assignments; hence, this gene is an excellent molecular marker for examining the diversity of picorna-like viruses in nature. Here we report, on the basis of analysis of RdRp sequences amplified from marine virus communities, that a diverse array of picorna-like viruses exists in the ocean. All of the sequences amplified were divergent from known picorna-like viruses, and fell within four monophyletic groups that probably belong to at least two new families. Moreover, we show that an isolate belonging to one of these groups is a lytic pathogen of Heterosigma akashiwo, a toxic-bloom-forming alga responsible for severe economic losses to the finfish aquaculture industry, suggesting that picorna-like viruses are important pathogens of marine phytoplankton.     

Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus.

Double-stranded RNA viruses have an RNA-dependent RNA polymerase activity associated with the viral particles which is indispensable for their replication cycle. Using the yeast L-A double-stranded RNA virus we have investigated the mechanism by which the virus encapsidates its genomic RNA and RNA polymerase. The L-A gag gene encodes the principal viral coat protein and the overlapping pol gene is expressed as a gag-pol fusion protein which is formed by a -1 ribosomal frameshift. Here we show that Gag alone is sufficient for virus particle formation, but that it fails to package the viral single-stranded RNA genome. Encapsidation of the viral RNA requires only a part of the Pol region (the N-terminal quarter), which is presumably distinct from the RNA polymerase domain. Given that the Pol region has single-stranded RNA-binding activity, these results are consistent with our L-A virus encapsidation model: the Pol region of the fusion protein binds specifically to the viral genome (+) strand, and the N-terminal gag-encoded region primes polymerization of Gag to form the capsid, thus ensuring the packaging of both the viral genome and the RNA polymerase.     

Genetic recombination between RNA components of a multipartite plant virus

Genetic recombination of DNA is one of the fundamental mechanisms underlying the evolution of DNA-based organisms and results in their diversity and adaptability. The importance of the role of recombination is far less evident for the RNA-based genomes that occur in most plant viruses and in many animal viruses. RNA recombination has been shown to promote the evolutionary variation of picornaviruses, it is involved in the creation of defective interfering (DI) RNAs of positive- and negative-strand viruses and is implicated in the synthesis of the messenger RNAs of influenza virus and coronavirus. However, RNA recombination has not been found to date in viruses that infect plants. In fact, the lack of DI RNAs and the inability to demonstrate recombination in mixedly infected plants has been regarded as evidence that plants do not support recombination of viral RNAs. Here we provide the first molecular evidence for recombination of plant viral RNA. For brome mosaic virus (BMV), a plus-stranded, tripartite-genome virus of monocots, we show that a deletion in the 3' end region of a single BMV RNA genomic component can be repaired during the development of infection by recombination with the homologous region of either of the two remaining wild-type BMV RNA components. This result clearly shows that plant viruses have available powerful recombinatory mechanisms that previously were thought to exist only in animal hosts, thus they are able to adapt and diversify in a manner comparable to animal viruses. Moreover, our observation suggests an increased versatility of viruses for use as vectors in introducing new genes into plants.     

吉林地区病毒性肝炎分型分析

目的 了解吉林地区病毒性肝炎的型别分布及感染状况．方法 采用ELlSA法检测．结果 男性检出39例，女性检出21例，检出HAV23例，占38％；HBV25例，占42％，NCV6例，占10％；HVD未检出，HEV4例，占7％，HGV2例，占3％．乙型肝炎检出率最高，其次为甲型肝炎、丙型肝炎、戊型肝炎、庚型肝炎．丁型肝炎检出率为0．单纯感染56例；1例甲、丙、庚三重感染，1例为甲、乙二重感染．结论 甲、乙型肝炎仍为吉林地区人群感染的主要病原．     

A complete sequence and comparative analysis of a SARS-associated virus(Isolate BJ01)

Severe Acute Respiratory Syndrome (SARS) is a newly identified infectious disease[1—5]. The global outbreak of SARS has been threatening the health of people worldwide and has killed 353 people and infected more than 5462 in 27 countries, as reported by WHO on April 29, 2003 (http://www.who.int/csr/sarscountry/en). Although it has been recognized that a variant of virus from the family of coronavirus might be the candidate pathogen of SARS[1—5], its identity as the unique pathogen sti…     

A complete sequence and comparative analysis of a SARS-associated virus （Isolate BJO1）

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-assoclated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs.We report the complete genome sequence and comparative analysis of an isolate (B J01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 20RFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host.Two amino acid changes have been detected in the M protein,which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).     

正链RNA病毒基因组的复制

许多正链RNA病毒是严重危害人类健康的病原体,是造成经济植物动物死亡的致病因子.正链RNA病毒的基因组为正链RNA,其复制酶是依赖RNA的RNA聚合酶,非编码区是病毒基因组复制的主要调控位点,3’非编码区是复制酶的第一结合位点,正链RNA病毒基因组大多可能按copy-back模型进行复制.瘟病毒基因组的复制过程出现正链复制本的数量大于负链复制本的数量,这可能是以RF中间体的负链RNA为模板、正链RNA被置换的形式进行复制的结果.本文概述了HCV细胞培养系统的研究进展.     

Transformation by murine and feline sarcoma viruses specifically blocks binding of epidermal growth factor to cells.

Normal cells in culture have membrane receptors for epidermal growth factor (EGF); EGF stimulates cells to divide by binding to these receptors. Cells transformed by murine and feline sarcoma viruses rapidly lose the ability to bind EGF, whereas cells transformed by the DNA tumour viruses, polyoma and SV40, or infected with non-transforming RNA tumour viruses have normal levels of functional EGF receptors. The results suggest that a product of the sarcoma virus genome specifically changes cell EGF receptors; the sarcoma gene product may, then, be functionally related to EGF.     

农作物病毒病害绿色防控技术创新

作物病毒病害是限制我国农作物优质稳产的主要因素之一,其主要由媒介生物传播,作物普遍缺乏抗病毒病的种质资源,病毒病害防治难度大,主要依赖化学农药通过防控媒介昆虫进行控制。近年来,随着纳米生物学、分子生物学和组学研究的飞速发展,以及微观生物学技术和理念对宏观生物学的快速渗透与交叉,基因组编辑技术的快速发展,病毒—昆虫—植物三者互作机制等各方面研究取得了诸多重要进展,在寻找病毒病害流行爆发的宏观生态学现象背后的分子生物学与生物化学机制上取得了长足进步,从而促进新型病毒病害的持久防控技术的发展,提供了科学依据和潜在新方案。文章简要回顾了近年来这些新技术的主要机理,并抛砖引玉提出了对未来发展的建议和思考。建议国家和社会组织研究力量,大力加强1)探索利用微纳米递送化学药物或者核酸干扰等防治病毒病害的新方法研究;2)病害发生的多元生物体系互作机制及科学防控科技支撑能力建设;3)基因组编辑创建病毒病害绿色防控的技术等方向的探索与突破,力求在病毒病害绿色生态防控方面为农业提供强有力的科技创新和支撑能力。     

Antigen chimaeras of poliovirus as potential new vaccines

Polioviruses occur as three distinct serotypes, 1, 2 and 3, and are composed of a single-stranded positive-sense RNA genome of approximately 7,450 nucleotides enclosed in an icosahedral particle of diameter 27 nm. The three-dimensional crystallographic structure of poliovirus type 1 has been determined at 2.9 A resolution, providing a detailed knowledge of the folding and arrangement of the individual virus proteins, VP1-VP4. From this and the characterization of monoclonal antibody-resistant mutants, the amino acids contributing to antigenic sites have been identified and located on the surface of the virus particle. Here we describe the construction and characterization of a poliovirus chimaera having a defined region of type 3 inserted into type 1. This virus has composite antigenicity and the substitute site is immunogenic in small animals and primates. The ability to construct such viruses has implications for the design of improved poliovirus vaccine strains or vaccines against other picornaviruses, such as hepatitis A.     

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.     

植物病毒编码的RNA沉默抑制因子

RNA沉默广泛存在于真核生物中,是一种发生于RNA水平的基因表达调节机制。寄主植物利用RNA沉默防御病毒的入侵,而病毒编码抑制因子以抵御这种防卫反应,从而达到在植物体内扩增、形成侵染的目的。近几年越来越多的沉默抑制因子被鉴定出来。本文就已鉴定出的沉默抑制因子的特点进行了系统介绍。     

Glycyrrhizic acid inhibits virus growth and inactivates virus particles.

Screening investigations in antiviral action of plant extracts have revealed that a component of Glycyrrhiza glabra roots, found to be glycyrrhizie acid, is active against viruses. We report here that this drug inhibits growth and cytopathology of several unrelated DNA and RNA viruses, while not affecting cell activity and ability to replicate. In addition, glycyrrhizic acid inactivates herpes simplex virus particles irreversibly.