1.Advances in UL7 gene of herpesvirus.
Jie HUANG ; An-Chun CHENG ; Ming-Shu WANG
Chinese Journal of Virology 2011;27(5):501-504
Animals
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Herpesviridae
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genetics
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metabolism
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Humans
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Viral Proteins
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chemistry
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genetics
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metabolism
2.The baculovirus enhancin.
Xiao-xia ZHANG ; Xiao-hui CHEN ; Zhen-pu LIANG ; Su-mei CAO ; Fen XU ; Guan-hua QIAO ; Xing-ming YIN
Chinese Journal of Virology 2010;26(5):418-423
Baculoviridae
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genetics
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metabolism
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Phylogeny
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Viral Proteins
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chemistry
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classification
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genetics
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metabolism
3.Screening of cellular proteins binding to the core region of hepatitis C virus RNA by ultraviolet cross-linking assay.
Hai-xia SU ; Jing-xia ZHANG ; Xiao-ning ZHAO ; Juan LU ; Yong-ping YAN
Chinese Journal of Hepatology 2005;13(9):656-659
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
4.In vitro assembly of Ebola virus nucleocapsid-like complex expressed in E. coli.
Ruchao PENG ; Tengfei ZHU ; Babayemi Olawale OLADEJO ; Abednego Moki MUSYOKI ; Yingzi CUI ; Yi SHI ; Peiyi WANG ; George Fu GAO
Protein & Cell 2016;7(12):888-898
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
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chemistry
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genetics
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metabolism
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Escherichia coli
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genetics
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metabolism
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Gene Expression
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Nucleocapsid
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chemistry
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genetics
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metabolism
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RNA, Viral
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chemistry
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genetics
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metabolism
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Recombinant Proteins
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chemistry
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genetics
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metabolism
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Virus Assembly
5.Progress about genetic and variant study of rabies virus glycoprotein gene.
Chinese Journal of Virology 2011;27(2):184-188
Animals
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Evolution, Molecular
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Glycoproteins
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chemistry
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genetics
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immunology
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metabolism
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Mutation
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Phylogeny
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Rabies virus
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genetics
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physiology
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Viral Proteins
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chemistry
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genetics
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immunology
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metabolism
6.Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87.
Majid ESMAELIZAD ; Saber JELOKHANI-NIARAKI ; Khadije HASHEMNEJAD ; Morteza KAMALZADEH ; Mohsen LOTFI
Journal of Veterinary Science 2011;12(4):363-371
The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3Dpol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3Dpol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp26-->Glu substitution in a beta sheet located within a small groove of the 3Dpol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.
Amino Acid Sequence
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Amino Acid Substitution
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Antigens, Viral/chemistry/*genetics/metabolism
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Capsid Proteins/chemistry/*genetics/metabolism
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Cloning, Molecular
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Foot-and-Mouth Disease Virus/classification/*genetics/*metabolism
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Gene Expression Regulation, Viral
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Molecular Sequence Data
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Phylogeny
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Viral Nonstructural Proteins/chemistry/*genetics/metabolism
7.Proteomic Analyses of Purified Particles of the Rabies Virus.
Zhongzhong TU ; Wenjie GONG ; Yan ZHANG ; Ye FENG ; Nan LI ; Changchun TU
Chinese Journal of Virology 2015;31(3):209-216
The rabies virus (RABV) is an enveloped RNA virus. It mainly damages the central nervous system and causes anencephaly in mammals and humans. There is now compelling evidence that enveloped virions released from infected cells can carry many host proteins, some of which may play an important part in viral replication. Several host proteins have been reported to be incorporated into RABV particles. However, a systematic study to reveal the proteomics of RABV particles has not been conducted. In the present study, after virus culture and purification by sucrose density gradient ultracentrifugation, a proteomics approach was used to analyze the protein composition of purified RABV particles to understand the molecular mechanisms of virus-cell interactions. Fifty host proteins, along with five virus-encoded structural proteins, were identified in purified RABV particles. These proteins could be classified into ten categories according to function: intracellular trafficking (14%), molecular chaperone (12%), cytoskeletal (24%), signal transduction (8%), transcription regulation (12%), calcium ion-binding (6%), enzyme binding (6%), metabolic process (2%), ubiquitin (2%) and other (14%). Of these, four proteins (beta-actin, p-tubulin, Cofilin, Hsc70) were validated by western blotting to be present in purified RABV particles. This novel study of the composition of host proteins in RABV particles may aid investigation of the mechanism of RABV replication.
Animals
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Humans
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Molecular Sequence Data
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Proteomics
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Rabies
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genetics
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metabolism
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virology
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Rabies virus
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chemistry
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genetics
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metabolism
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Viral Proteins
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analysis
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chemistry
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genetics
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metabolism
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Virion
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chemistry
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genetics
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metabolism
8.Hepatitis C virus: virology and life cycle.
Chang Wook KIM ; Kyong Mi CHANG
Clinical and Molecular Hepatology 2013;19(1):17-25
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
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Genome, Viral
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Hepacivirus/genetics/*physiology
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Humans
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RNA, Viral/metabolism
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Scavenger Receptors, Class B/metabolism
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Viral Envelope Proteins/chemistry/metabolism
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Viral Nonstructural Proteins/chemistry/metabolism
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Virus Assembly
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Virus Internalization
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Virus Replication
9.Efficient soluble expression and purification of influenza A nucleoprotein in Escherichia coli.
Bao-ying HUANG ; Wen-ling WANG ; Xiu-ping WANG ; Tao JIANG ; Wen-jie TAN ; Li RUAN
Chinese Journal of Virology 2011;27(1):50-57
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
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Cloning, Molecular
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Escherichia coli
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genetics
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metabolism
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Gene Expression
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Humans
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RNA-Binding Proteins
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chemistry
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genetics
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isolation & purification
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metabolism
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Solubility
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Viral Core Proteins
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chemistry
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genetics
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isolation & purification
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metabolism
10.Functional analysis of the late expression factor genes of plutella xylostella granulovirus.
Chinese Journal of Virology 2012;28(5):560-566
Plutella xylostella granulovirus (PlxyGV) contains homologs of 15 Autographa californica MNPV (AcMNPV) late expression factor (lef) genes. The prospective products of 14 PlxyGV lef genes (ie-0 is not included) share 13%-53% amino acid similarity with their corresponding homologs of AcMNPV, among which LEF-9, LEF-8 and P47, three subunits of the virus-encoded RNA polymerase, share 49%, 53% and 46% sequence identity, respectively. In this study, an established transient expression system was used to test the ability of the PlxyGV LEFs to activate an AcMNPV vp39 promoter-driven reporter gene in SF9 cells. It was shown that PlxyGV le f-2 replaced the corresponding AcMNPV gene and exhibited partial activity in the context of the remaining set of AcMNPV le fs. PlxyGV LEF-2 was found to contain additional 100aa and 70aa at the C-terminus in comparison with the LEF-2 of other GVs and lepidopteran NPVs respectively.
Amino Acid Sequence
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Animals
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Gene Expression Regulation, Viral
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Granulovirus
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chemistry
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genetics
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metabolism
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Molecular Sequence Data
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Moths
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virology
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Nucleopolyhedrovirus
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genetics
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metabolism
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Sequence Alignment
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Viral Proteins
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genetics
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metabolism