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

OBJECTIVETo secreted express envelope glycoprotein (E) of dengue virus type 2 extracellularly.

METHODSThe entire prM/E gene was amplified by RT-PCR. An optimized signal sequence gene from Japanese encephalits virus (JEV, SA14-14-2 strain) was introduced using fusion PCR. The impact of E protein transmembrane and cytoplasmatic domains was compared by amplifying prM and E with full length of E gene, with 20% truncation of the E gene at 3' terminus and one chimeric gene, which was generated by replacing the 3' terminal 20% region of E gene with the corresponding sequence of JEV (SA14-14-2 strain). The PCR segments were inserted into the NheI and NotI sites of pcDNA5/FRT vector or into the NheI and XhoI sites of pAcUW51-M. Then they were transfected into 293T cells or Sf9 cells respectively. The expression and secretion of E protein were detected by immunofluorescence assay (IFA) and Western Blot.

RESULTSAfter transected into 293T cells or Sf9 cells, all constructs expressed E protein intracellularly indentified by IFA while only two plasmids could secret detectable E protein into tissue culture using Western Blot analysis.

CONCLUSIONSignal peptide as well as the transmembrane and cytoplasmatic domains is crucial for the secretion of dengue E protein.

Animals ; Cell Line ; Dengue ; metabolism ; virology ; Dengue Virus ; genetics ; metabolism ; Gene Expression ; Humans ; Protein Structure, Tertiary ; Protein Transport ; Spodoptera ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
Animals ; Coronavirus Infections ; metabolism ; virology ; Coronavirus NL63, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Mice ; Mice, Inbred BALB C ; Protein Engineering ; Protein Structure, Tertiary ; Receptors, Virus ; metabolism ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

The highly virulent PRRSV isolate strain HN-1/06 was cultivated on Marc-145. To study the viral entry mechanisms, the GP5 gene of PRRSV isolate was amplified by RT-PCR and cloned into pcDNA3.0 to generate the expressing plasmid pcDNA-GP5. pcDNA-GP5 was transfected into 293T by the calcium phosphate precipitation method. Analysis of flow cytometry confirmed that the GP5 proteins were expressed in surface of the 293T cells. Then 293T cells were transfected with pcDNA-GP5, pHIT60 and pHIT111 plasmids to generate pseudotyping virus. The pseudotyping virus supernatant was harvested 48 hours post-transfection and was detected by Western blotting and infection assay. Western blotting indicated that the GP5 glycoproteins were incorporated into the retroviral pseudotyped virus. Infection assay showed that the pseudotyped virus infected 293T and Mark-145 cell. The pseudotyped virus could be used to further study infectious mechanism of PRRSV.
Animals ; Cell Line ; Cloning, Molecular ; Endothelial Cells ; cytology ; metabolism ; virology ; Leukemia Virus, Murine ; genetics ; metabolism ; Mice ; Porcine respiratory and reproductive syndrome virus ; chemistry ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Swine ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; Virion ; genetics ; metabolism

In this study, Recombinant baculoviruses rBac-NF and rBac-NG were generated for expressing F and G proteins Nipah virus (NiV) . The expression of recommbinant G (rNG) and F (rNF) protein in rBac-NF and rBac-NG infected cells were confirmed by western-blot. Both rNG and rNF showed sensitive and specific antigenic reaction to rabbit serum anti-Nipah virus in indirect immunofluorescence detection and indirect ELISA. Immunization with rBac-NF and rBac-NG infected insect cells elicited G ad F protein specific antibody responses in mice. Furthermore, the G ad F specific antibodies could neutralize the infectivity of the VSVdeltaG* F/G, the NiV F and G envelope glycoproteins psudotyped recombinant Vesicular Stomatitis Virus expressing green fluorescence protein. The results demonstrated F and G protein expressed by the recombinant baculoviruses could be safe economic diagnostic antigens for the surveillance and monitoring of NiV and promising subunit vaccines for the prevention of NiV.
Animals ; Antigens, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Nipah Virus ; chemistry ; genetics ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Recombination, Genetic ; Viral Envelope Proteins ; biosynthesis ; genetics ; immunology

The glycoprotein 3 (GP3) of type II porcine reproductive and respiratory syndrome virus has the characteristic domains of a membrane protein. However, this protein has been reported to be retained in the endoplasmic reticulum (ER) rather than transported to the plasma membrane of the cell. In this study, we performed confocal laser scanning microscopy analysis of variants of GP3 and foundthat the signal sequence of the GP3 led to confinement of GP3 in the ER, while the functional ortransmembrane domain did not affect its localization. Based on these results, we concludedthat the signal sequence of GP3 contains the ER retention signal, which might play an important role in assembly of viral proteins.
Animals ; Cell Line ; Cell Membrane/*metabolism/virology ; Cricetinae ; Endoplasmic Reticulum/*metabolism/virology ; Microscopy, Confocal/veterinary ; Plasmids/genetics/metabolism ; Porcine respiratory and reproductive syndrome virus/*genetics/metabolism ; *Protein Sorting Signals ; Sequence Analysis, Protein/veterinary ; Viral Envelope Proteins/chemistry/*genetics/metabolism

OBJECTIVETo construct sub-unit vaccines of dengue virus type 1 to 4 and to analyze its immunogenicity.

METHODSEnvelope domain III s of dengue serotypes 1 and 2, as well as 3 and 4, were spliced by a linker (Gly-Gly-Ser-Gly-Ser)3 and cloned into vector pET-30a, then transformed into E. coli to express recombinant fusion proteins. The recombinant proteins were purified by high-performance liquid chromatography and mixed to immunize BALB/c mice. The neutralizing antibodies were tested by neutralizing assay, as well as in newborn mice challenged intracranially with dengue virus type 1 to 4.

RESULTSMice immunized with proteins could produce neutralizing antibodies, with titers of 1:34. 9, 1: 45.3, 1: 24.7 and 1:38.4 for DEN-1 to 4 respectively. 100% newborn mice challenged with DEN-1 or 2 in combination with sera from mice immunized with recombinant proteins were protected, whereas 83% protection was obtained when challenged with DEN-3 or 4.

CONCLUSIONThe recombinant proteins possess excellent immunogenicity to induce neutralizing antibodies and would be valuable for development of a tetravalent sub-unit vaccine.

Animals ; Antibodies, Neutralizing ; immunology ; Dengue Vaccines ; chemistry ; genetics ; immunology ; Dengue Virus ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology

BACKGROUNDTo investigate the interaction between the host cell and the truncated S fragments to identify the receptor-binding domain of the spike (S) protein of SARS-associated coronavirus (SARS-CoV).

METHODSTwo different fragments S260-600 and S397-796 of the SARS-CoV S protein were expressed in Escherichia coli (E.coli) using a pET expression vector, respectively. The two recombinant proteins were separately verified by Western blot, purified by nickel-affinity chromatography, and incubated with Vero cells, a susceptible cell line of SARS-CoV infection, for cell binding assay. After the sequential probing with sera from convalescent SARS-patients and FITC-labeled anti-human IgG, the cells were analyzed by flow cytometry. The NIH 3T3 cell, a non-permissive cell line of SARS-CoV infection, was used as controls.

RESULTSThe recombinant proteins S260-600 and S397-796 were efficiently expressed in an insoluble form in E.coli. The appropriate expression of the proteins was confirmed by Western blotting using both SARS patients' sera and anti-6 x histidine antibody. The flow cytometry results showed that the both proteins were able to bind Vero cells, but the binding ability of S260-600 was somewhat stronger than that of S397-796. In contrast, the S260-600 protein did not bind NIH3T3 cells.

CONCLUSIONBoth S260-600 and S397-796 exhibited different receptor binding activity. The S260-600 fragment probably contains the important receptor binding domain and could be a potential candidate for the development of SARS vaccine and anti-SARS therapeutics.

Animals ; Binding, Competitive ; Blotting, Western ; Cercopithecus aethiops ; Escherichia coli ; genetics ; metabolism ; Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Mice ; NIH 3T3 Cells ; Peptide Fragments ; chemistry ; genetics ; metabolism ; Protein Binding ; Receptors, Cell Surface ; metabolism ; Recombinant Proteins ; isolation & purification ; metabolism ; SARS Virus ; genetics ; metabolism ; Spike Glycoprotein, Coronavirus ; Vero Cells ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo observe the ability of dengue virus recombinant envelope protein domain expressed in E. coli to inhibit virus infection and induce the neutralizing antibody.

METHODSE III protein of Dengue virus serotypes 1-4 were expressed in E. coli BL21(DE3) then purified. Recombinant proteins were tested to inhibit DV2 from infecting BHK-21 cell. Rabbits were immunized with recombinant proteins to produce anti-E III serum. Antibody titers were determined by neutralizing assay.

RESULTSThe recombinant E III proteins of Dengue virus serotypes 1-4 were expressed in E. coli. They effectively protected BHK cells in culture against DV2 infection. All four type anti-E III sera can neutralize DV2 but their efficacies are different.

CONCLUSIONproteins of dengue virus expressed in E. coli can directly inhibit DV2 infection. Neutralizing antibodies were induced by E III proteins. Both E III protein of dengue virus and the neutralizing antibodies they induced are more efficient in inhibiting homologous dengue serotypes infection than heterologous serotypes.

Animals ; Antibodies, Viral ; immunology ; Cell Line ; Cricetinae ; Dengue ; immunology ; prevention & control ; virology ; Dengue Virus ; chemistry ; genetics ; immunology ; physiology ; Down-Regulation ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunization ; Mesocricetus ; Protein Structure, Tertiary ; Rabbits ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Envelope Proteins ; chemistry ; genetics ; immunology ; Virus Replication

During July to November in 2007, several outbreaks of Hemangiomas in Hy-line Brown laying hens were observed in China. The virus that infected these flocks was identified in cultured DF-1 cells by PCR and indirect fluorescent assay (IFA) with ALV-J specific monoclonal antibody JE-9. The gp85 gene of one strain named WS0705 of ALV-J was cloned and expressed. Phylogenetic analysis showed that gp85 amino sequences of WS0705 strain had the highest homology with that of the prototype HPRS-103. The gp85 gene from a constructed plasmid pMD18-T-WS0705gp85 was cloned into baculovirus transfer vector. rBac-WS0705gp85 was obtained by the Bac-to-Bac baculovirus expression system. The rBac-WS0705gp85 protein was analyzed by indirect immunofluor escence assay and Western blot. The results showed that positive green fluorescent was present in Sf9 cells infected with the recombinant virus and a 35 kD band was present in western blot. It is concluded that WS0705 gp85 gene was expressed in Sf9 cells infected with the recombinant virus and the SU protein of WS0705 can bind specifically to JE9 MAb of ALV-J. The expressed protein can be used to detect hemangiomas induced by ALV-J.
Amino Acid Sequence ; Animals ; Avian Leukosis Virus ; classification ; genetics ; Blotting, Western ; Cell Line ; Cloning, Molecular ; Electrophoresis, Polyacrylamide Gel ; Gene Expression ; Hemangioma ; virology ; Phylogeny ; Polymerase Chain Reaction ; Sequence Alignment ; Viral Envelope Proteins ; chemistry ; genetics ; isolation & purification ; metabolism