Human astrovirus (HAstV) is one of the leading causes of actue virual diarrhea in infants. HAstV-induced epithdlial cell apoptosis plays an important role in the pathogenesis of HAstV infection. Our previous study indicated that HAstV non-structural protein nsPla C-terminal protein nsPla/4 was the major apoptosis functional protein and probably contained the main apoptosis domains. In order to screen for astrovirus encoded apoptotic protien, nsPla/4 and six turncated proteins, which possessed nsPla/4 protein different function domain ,were cloned into green fluorescent protein (GFP) vector pEG-FP-N3. After 24-72 h transfection, the fusion protein expression in BHK21 cells, was analysis by fluorescence microscope and Western blot. The results indicated seven fusion proteins were observed successfully in BHK21 cell after transfected for 24 h. Western blot analysis showed that the level of fusion protein expressed in BHK21 cells was increased significantly at 72h compared to 48h in transfected cells. The successful expression of deletion mutants of nsPla/4 protein was an important foundation to gain further insights into the function of apoptosis domains of nsPla/4 protein and it would also provide research platform to further confirm the molecule pathogenic mechanism of human astrovirus.
Amino Acid Motifs ; Astroviridae Infections ; virology ; Humans ; Mamastrovirus ; genetics ; metabolism ; Mutation ; Sequence Deletion ; Transfection ; Viral Nonstructural Proteins ; chemistry ; genetics ; metabolism

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

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Encephalitis Virus, Japanese ; enzymology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; Protease Inhibitors ; chemistry ; RNA Helicases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Serine Endopeptidases ; metabolism ; Viral Nonstructural Proteins ; metabolism

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 ; Amino Acid Substitution ; Antigens, Viral/chemistry/*genetics/metabolism ; Capsid Proteins/chemistry/*genetics/metabolism ; Cloning, Molecular ; Foot-and-Mouth Disease Virus/classification/*genetics/*metabolism ; Gene Expression Regulation, Viral ; Molecular Sequence Data ; Phylogeny ; Viral Nonstructural Proteins/chemistry/*genetics/metabolism

The present study was aimed to isolate the active compounds from the fermentation products of Fusarium oxysporum, which had hepatitis C virus (HCV) NS3 protease inhibitory activity. A bioactive compound was isolated by reverse-phase silica-gel column chromatography, silica-gel column chromatography, semi-preparative reverse-phase High Performance Liquid Chromatography (HPLC), and then its molecular structure was elucidated based on the spectrosopic analysis. As a result, the compound (H1-A, 1) Ergosta-5, 8 (14), 22-trien-7-one, 3-hydroxy-,(3β, 22E) was isolated and identified. To the best of our knowledge, this was the first report on the isolation of H1-A from microorganisms with the inhibitory activity of NS3 protease.
Enzyme Inhibitors ; chemistry ; isolation & purification ; metabolism ; Fusarium ; chemistry ; metabolism ; Hepacivirus ; drug effects ; enzymology ; genetics ; Hepatitis C ; virology ; Humans ; Magnetic Resonance Spectroscopy ; Viral Nonstructural Proteins ; antagonists & inhibitors ; metabolism

Treatments for chronic hepatitis C has evolved significantly in the past 15 years. The standard of care (SOC) is peginterferon alfa-2a/-2b with ribavirin for 48 weeks or 24 weeks in patients infected with HCV genotype 1 or 2/3, respectively. The treatment duration can be individualized based on the baseline viral load and the speed of the virologic response during treatment. However, current therapies are associated with side effects, complications, and poor patient tolerability. Therefore, there is an urgent need to identify better strategies for treating this disease. An improved sustained virologic response (SVR) can be achieved with new HCV-specific inhibitors against NS3/4A and NS5B polymerases. Recent trials have found SVR rates in patients with HCV genotype 1 infection of 61~68% and 67~75% for combining the SOC with the protease inhibitors telaprevir and boceprevir, respectively. Several new HCV-specific inhibitors such as protease inhibitors and nucleoside and non-nucleoside polymerase inhibitors as well as non-HCV-specific compounds with anti-HCV activity are currently in clinical evaluation. In this review we discuss these new treatments for chronic hepatitis C.
Antiviral Agents/*therapeutic use ; DNA-Directed RNA Polymerases/antagonists & inhibitors/metabolism ; Hepatitis C, Chronic/*drug therapy ; Humans ; Interferons/therapeutic use ; Nucleotides/chemistry/therapeutic use ; Protease Inhibitors/*therapeutic use ; Viral Nonstructural Proteins/antagonists & inhibitors/metabolism ; Virus Internalization/drug effects

OBJECTIVETo construct point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal, and express the constructs in Huh 7 cells.

METHODSUsing pSG5/M-H05-5/4A as the template (A1-1) and primers designed according to the typing criteria, 4 single point mutation plasmids, namely pSG5/M-H05-5(A1-2)/4A(A1-2) (Y56F), pSG5/M-H05-5(B1-1)/4A(B1-1) (L80Q), pSG5/M-H05-5(B2-1)/4A(B2-1) (V51A), and pSG5/M-H05-5(B2-2)/4A(B2-2) (S61A), were constructed. With A1-2, B2-1, and B2-2 as the templates, the leucine to glutamine mutation at position 80 (L80Q) was induced to construct another 3 double point mutation plasmids pSG5/M-H05-5(B1-2)/4A(B1-2), pSG5/M-H05-5(A2-1)/4A(A2-1), and pSG5/M-H05-5(A2-2)/4A(A2-2), respectively. DNA sequencing was performed for confirmation of the mutations. Huh 7 cells were transfected with the constructs using FuGene 6 transfection reagents. Indirect immunofluorescence staining and Western blotting were used to detect the expression of the constructs.

RESULTSIndirect immunofluorescence assay revealed 4 subcellular localization patterns of NS3 protein, including dot-like staining, diffuse staining, doughnut-like staining, and rod-shape staining. Western blotting also demonstrated successful expression of the constructs and weak in cis and in trans NS3 serine protease activities of subtypes A2-1 and B2-1 in comparison with other subtypes.

CONCLUSIONThe point mutation plasmids expressing HCV NS3/4A with different secondary structures at amino-terminal are constructed successfully, which provides the basis for further study of different subtypes of HCV.

Amino Acid Sequence ; Cell Line ; Gene Expression ; Genetic Engineering ; methods ; Hepacivirus ; enzymology ; Immunohistochemistry ; Intracellular Space ; metabolism ; Molecular Sequence Data ; Plasmids ; genetics ; Point Mutation ; Protein Structure, Secondary ; Protein Transport ; Viral Nonstructural Proteins ; chemistry ; genetics ; metabolism

The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5'- and 3'-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3'-end region of HCV minus-strand RNA and the X-RNA at the 3'-end of HCV RNA genome was also initiated de novo. No formation of dimersize self-primed RNA products resulting from extension of the 3'-end hydroxyl group was observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polymerase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genome.
3' Untranslated Regions/genetics ; 5' Untranslated Regions/genetics ; Animals ; Cell Line ; Gene Expression ; Genome ; Genome, Viral ; Hepacivirus/*enzymology/genetics ; RNA/biosynthesis/genetics ; RNA, Viral/genetics/metabolism ; Recombinant Proteins/genetics/isolation & purification/metabolism ; Spodoptera ; Templates, Genetic ; Uridine Monophosphate/metabolism ; Viral Nonstructural Proteins/chemistry/*genetics/isolation & purification/*metabolism

To develop a sensitive and specific ELISA for detection of antibodies to the nonstructural protein of FMDV. We cloned and expressed FMDV nonstructural protein 3AB in Escherichia coli expression system. The recombinant protein 3AB was purified with Ni-NTA HisBind Resins and characterized by Western blotting. An indirect ELISA based on purified protein 3AB as a coating antigen was established. The specificity and sensitivity of this assay were evaluated by comparison with a commercial 3ABC-ELISA kit in detecion of serum samples. The results showed that the recombinant protein 3AB was expressed as a formation of inclusion bodies in Escherichia coli. The purified protein could specificially react with FMDV infection antibodies in Western blotting assay, but no reaction with the immune antibodies induced with vaccine. Two assays were no significant differences in specificity and sensitivity for detection of field samples (P>0.05). Therefore, we speculated that the recombinant protein 3AB is a promising molecular marker, which may effectively differentiate FMD-infected from vaccinated animals in a herd.
Animals ; Antibodies, Viral ; analysis ; Antigens, Viral ; biosynthesis ; genetics ; immunology ; Cattle ; Cattle Diseases ; diagnosis ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Foot-and-Mouth Disease ; diagnosis ; immunology ; Foot-and-Mouth Disease Virus ; chemistry ; genetics ; isolation & purification ; Genetic Vectors ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

Human enterovirus 71 (EV71) is one of the major etiological agents for the hand, foot, and month disease (HFMD) and is causing frequent, widespread occurrence in the mainland of China. The single positive-stranded RNA genome of EV71 is translated into a single polyprotein which is autocleavaged into structural and nonstructural proteins. The functions of many nonstructural proteins characterized in the life cycle of virus are potential targets for blocking viral replication. This article reviews the studies of the structures and functions of nonstructural proteins of EV71 and the anti-enterovirus 71 drugs targeting on these nonstructural proteins.
Antiviral Agents ; pharmacology ; Enterovirus A, Human ; enzymology ; genetics ; isolation & purification ; Hand, Foot and Mouth Disease ; drug therapy ; virology ; Humans ; Molecular Targeted Therapy ; Peptide Hydrolases ; chemistry ; metabolism ; physiology ; Protein Kinase Inhibitors ; pharmacology ; RNA, Viral ; genetics ; Viral Nonstructural Proteins ; chemistry ; metabolism ; physiology ; Virus Replication ; drug effects