A self-cleaving hammerhead ribozyme gene containing a 14nt target sequence of ASSVd at the 3' end of hammerhead ribozyme was synthesized, amplified and cloned at the Xho I-Hind III site of pGEM7Zf(+). The ends produced by Xho I or Sal I can link together, thus the recognition sites of both enzymes vanish and can't be cut by either one. We used this property to get the recombinant plasmid bearing 2, 4, 6, 8, 10 and 12 copies of self-cleavable ribozyme respectively after successively sub-cloning five times. Linearized recombinat plasmid model catalyzed by T7 RNA polymerase was transcribed in vitro. The multimeric ribozyme molecules efficiently self-cleaved via cis-acting to release many ribozyme molecules It indicates that the concentration of ribozyme transcripts has been enhanced during transcription. Trans-cleavage reaction was carried out by incubating monomeric and multimeric ribozymes with same mol concentration and 32P labeled target ASSVd. Both ribozymes and target transcripts were mixed in 1:1 ratio. Autoradiograms showed the transcripts of multimeric ribozyme were substantially more effective against the ASSVd target RNA than the monomeric ribozymes. We confer that the multimeric self-clevable ribozyme is likely to provide more valuable application in vivo.
Malus ; virology ; RNA, Catalytic ; chemistry ; genetics ; metabolism ; RNA, Viral ; metabolism ; Viroids ; metabolism

OBJECTIVETo screen cellular proteins binding to the core region of hepatitis C virus (HCV) from human hepatoma cells.

METHODSUnlabeled and labeled RNA transcripts were prepared by in vitro transcription. Cytoplasmic extracts were prepared from human hepatoma cells HepG2. Ultraviolet (UV) cross-linking was used to screen the cellular proteins that would bind to the core region of HCV. Competition experiment was performed to confirm the specificity of the binding in which excess unlabeled RNA of HCV core region and plasmid RNA were used as competitors.

RESULTSTwo cellular proteins of 6.6 x 10(4) and 5.5 x 10(4) were found binding to the core region of HCV RNA by UV cross-linking assay. The unlabeled core region of HCV RNA could compete out this binding whereas the unlabeled plasmid RNA could not.

CONCLUSIONThe cellular proteins from HepG2 cells could bind to the core region of HCV RNA.

Binding Sites ; Cross-Linking Reagents ; chemistry ; Hepacivirus ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Ultraviolet Rays ; Viral Core Proteins ; genetics ; metabolism

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryo-eletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451-739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451-739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.
Ebolavirus ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Nucleocapsid ; chemistry ; genetics ; metabolism ; RNA, Viral ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Virus Assembly

Both sides of the picornavirus genome have 5'-untranslated region (5'UTR) and 3'- untranslated region (3'UTR). This study demontrated that both the 5'-and 3'-UTR can form complex structures, such as stem-loop, clover and pseudoknot structure, These structures play an important role in the regulaton of the replication and translation of the viruses. This article reviewed the progress of research on the structure and function of picornavirus' 3'-UTR over recent years.
3' Untranslated Regions ; Animals ; Humans ; Nucleic Acid Conformation ; Picornaviridae ; chemistry ; genetics ; metabolism ; Picornaviridae Infections ; virology ; RNA, Viral ; chemistry ; genetics ; metabolism

Several virus families have been shown to encode microRNAs (miRNAs), which have roles in the infection and replication of viruses in host cells. These virus-encoded miRNAs are identified in double-stranded DNA virus (dsDNA virus) and in several RNA virus families, but not in single-stranded DNA virus (ssDNA virus). We used a bioinformatics approach based on VMir, miRNAFold and MaturePred software to predict virus-encoded miRNA-like small RNAs from the genome of a ssDNA virus: Aedes aegypti densovirus (AaeDV). Northern blotting and stem-loop reverse transcription-polymerase chain reaction (RT-PCR) were used to detect predicted small RNAs. A miRNA-like small RNA termed "AaeDVMD" was identified by stem-loop RT-PCR from predicted candidates. This is the first report demonstrating that a ssDNA virus can encode miRNA-like small RNAs. These data will aid further exploration of the interaction between the AaeDV and its mosquito host.
Aedes ; virology ; Animals ; Base Sequence ; Computational Biology ; Densovirinae ; chemistry ; genetics ; metabolism ; MicroRNAs ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; RNA, Viral ; chemistry ; genetics ; metabolism

OBJECTIVETo study the mechanism of the cellular proteins involved in the process of replication of hepatitis C virus (HCV) negative-strand RNA.

METHODSUltraviolet (UV) cross-linking was used to identify the cellular proteins that would bind to the 3'-end of HCV negative-strand RNA. Competition experiment was used to confirm the specificity of this binding, in which excess nonhomologous protein and RNA transcripts were used as competitors. The required binding sequence was determined by mapping, then the binding site was predicted through secondary structure analysis.

RESULTSA cellular protein of 45 kD (p45) was found to bind specifically to the 3'-end of HCV negative-strand RNA by UV cross-linking. Nonhomologous proteins and RNA transcripts could not compete out this binding, whereas the unlabeled 3'-end of HCV negative-strand RNA could. Mapping of the protein-binding site suggested that the 3'-end 131-278nt of HCV negative-strand RNA was the possible protein-binding region. Analysis of RNA secondary structure presumed that the potential binding site was located at 194-GAAAGAAC-201.

CONCLUSIONThe cellular protein p45 could specifically bind to the secondary structure of the 3'-end of HCV intermediate negative-strand RNA, and may play an important role in HCV RNA replication.

Binding Sites ; Hepacivirus ; genetics ; Nucleic Acid Conformation ; RNA, Viral ; chemistry ; metabolism ; RNA-Binding Proteins ; analysis ; metabolism ; Virus Replication

Hepatitis C virus (HCV) is a positive sense, single-stranded RNA virus in the Flaviviridae family. It causes acute hepatitis with a high propensity for chronic infection. Chronic HCV infection can progress to severe liver disease including cirrhosis and hepatocellular carcinoma. In the last decade, our basic understanding of HCV virology and life cycle has advanced greatly with the development of HCV cell culture and replication systems. Our ability to treat HCV infection has also been improved with the combined use of interferon, ribavirin and small molecule inhibitors of the virally encoded NS3/4A protease, although better therapeutic options are needed with greater antiviral efficacy and less toxicity. In this article, we review various aspects of HCV life cycle including viral attachment, entry, fusion, viral RNA translation, posttranslational processing, HCV replication, viral assembly and release. Each of these steps provides potential targets for novel antiviral therapeutics to cure HCV infection and prevent the adverse consequences of progressive liver disease.
Antigens, CD81/metabolism ; Genome, Viral ; Hepacivirus/genetics/*physiology ; Humans ; RNA, Viral/metabolism ; Scavenger Receptors, Class B/metabolism ; Viral Envelope Proteins/chemistry/metabolism ; Viral Nonstructural Proteins/chemistry/metabolism ; Virus Assembly ; Virus Internalization ; Virus Replication

To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.
Escherichia coli ; genetics ; metabolism ; Influenza A virus ; genetics ; metabolism ; Influenza B virus ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Ribonucleases ; chemistry ; Virion ; genetics ; metabolism

To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; RNA-Binding Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Solubility ; Viral Core Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

Previously, we have established an epsilon library and selected out a series of RNA aptamers with higher affinity to P protein based on the in vitro Systematic Evolution of Ligands by Exponential Enrichment (SELEX) in duck hepatitis B virus (DHBV) system. In order to study the structural elements within the epsilon that is essential for initiating priming of HBV reverse transcriptase (P protein), all selected aptamers were subjected to in vitro priming assay and RNA secondary structure probing. We found that all those aptamers supporting priming had an undamaged bulge, while those lacking of the bulge no more support priming. Our results suggest an undamaged bulge within Depsilon is indispensable for initiating priming of P protein.
Base Sequence ; Hepatitis B Virus, Duck ; chemistry ; enzymology ; genetics ; Molecular Sequence Data ; Nucleic Acid Conformation ; RNA, Viral ; chemistry ; genetics ; RNA-Directed DNA Polymerase ; genetics ; metabolism ; Reverse Transcription ; Sequence Alignment ; Viral Proteins ; genetics ; metabolism