Hepatitis B virus (HBV) genome P-encoded protein HBV DNA polymerase (Pol) has long been known as a reverse transcriptase during HBV replication. In this study, we investigated the impact of HBV Pol on host cellular processes, mainly apoptosis, and the underlying mechanisms. We showed a marked reduction in apoptotic rates in the HBV Pol-expressed HepG2 cells compared to controls. Moreover, a series of assays, i.e., yeast two-hybrid, GST pull-down, co-immunoprecipitation, and confocal laser scanning microscopy, identified the host factor eEF1A2 to be associated with HBV Pol. Furthermore, knockdown of eEF1A2 gene by siRNA abrogated the HBV Pol-mediated anti-apoptotic effect with apoptosis induced by endoplasmatic reticulum (ER) stress-inducer thapsigargin (TG), thus suggesting that the host factor eEF1A2 is essential for HBV Pol's anti-apoptosis properties. Our findings have revealed a novel role for HBV Pol in its modulation of apoptosis through integrating with eEF1A2.

BACKGROUND:Liver fibrosis is the primary cause of liver cirrhosis and hepatocellular carcinoma and leads to considerable morbidity and mortality. Recent studies have shown that microRNAs are associated with fibrotic processes in liver disorders, but the exact role of miR-202 is still unclear, and its related mechanisms are not fully understood.AIMS:The aim of this research is to analyze the regarded regulation of miR-202 on HGF and its role in the pathological progress of liver fibrosis.METHODS:In the present study, qRT-PCR was used to detect the expression level of miR-202 in serum of patients with liver fibrosis and to compare its expression in patients with different pathological stages. HGF was predicted to be the target gene of miR-202 by TargetScan and was verified by Dual-luciferase reporter gene assay. qRT-PCR and western blot were used to detect the regulatory effect of mir-202 on the mRNA and protein of HGF; effect of miR-202 on the expression of fibrosis factors α-smooth muscle actin (α-SMA), FSP1, and collagen was detected; effect of miR-202 on liver fibrosis in mice was detected by establishing CCL4-induced mouse model.RESULTS:We found that the expression level of miR-202 in serum of patients with liver fibrosis was significantly higher than that of healthy people, and increased with the increase of fibrosis; miR-202 inhibited the expression level of mRNA and protein of HGF by combining with the 3'-UTR of HGF; the expression level of miR-202 significantly increased after hepatic stellate cells (HSC) were stimulated by AngII; the overexpression of miR-202 could up-regulate the expression of fibrotic factors α-SMA, FSP1, and collagen I. In addition, miR-202 up-regulated the expression of collagen I and collagen III in liver tissue of mice with liver fibrosis and promoted the progress of liver fibrosis.CONCLUSIONS:miR-202 could negatively regulate the expression of target gene HGF, activated HSC, and increased the expression levels of various fibrosis factors, and the pathological process of liver fibrosis injury was promoted.

Largemouth bass virus (LMBV) is the causative agent of a disease causing high mortality rates in largemouth bass during summer. However, there is little information available about the development of vaccines for LMBV disease. Hence, a DNA vaccine, named pCDNA3.1(+)-MCP-Flag, was constructed by inserting the cloned LMBV major capsid protein (MCP) gene into the pCDNA3.1(+)-Flag plasmid. The expression of the recombinant plasmid was confirmed by Western blot (WB) and RT-PCR. The WB result revealed that the MCP protein produced a band of approximately 53 kDa, consistent with the expected result. The RT-PCR results also confirmed that MCP was transcribed in the EPC cells transfected with the recombinant plasmid. The largemouth bass in the DNA vaccine group were immunized with the pCDNA3.1(+)-MCP-Flag plasmid by pectoral fin base injection, and the relative percent survival (RPS) of fish challenged with LMBV was 63%. The relative immunological analyses were as follows. Compared with the PBS and pCDNA3.1(+) groups, the DNA vaccine group showed significantly upregulated expression of IL-1β, IL-8, TNF-α and Mx in the spleen, head kidney and liver. All largemouth bass immunized with the DNA vaccine produced a high titre of LMBV-specific neutralizing antibody during the immunization period. The titre was 1:375 ± 40 and peaked at 14 days post-vaccination. The expression of the recombinant plasmid was analysed in the tissues of the DNA vaccine group by RT-PCR. The recombinant plasmid was expressed in the spleen, head kidney and liver, and MCP protein was successfully expressed after vaccination. In conclusion, the recombinant plasmid expressing LMBV MCP induced significant immune responses in largemouth bass, and might represent a potential LMBV vaccine candidate for largemouth bass.

The amino acid substitution at position 181 of the Hepatitis B virus (HBV) polymerase is a multi-drug resistance affecting both the L-nucleoside and acyclic phosphonate nucleotide groups. Data is limited on the efficacy of entecavir (ETV) rescuing chronic hepatitis B (CHB) patients with rtA181T/V mutation.

Despite recent results of deletion experiments showing that open reading frame (ORF) UL49 of human cytomegalovirus (HCMV) is essential, the expression, function and functional location of its encoded protein remain unknown. We generated an antibody specific for pUL49 to investigate the protein product encoded by the UL49 ORF and identified its function in HCMV-infected host foreskin fibroblasts. A bacterial artificial chromosome (BAC) of HCMV strain Towne (pRV-Towne) and the UL49-deleted mutant pRV- del UL49Towne were used to observe virus growth by plaque assay. Using a UL49-protein-binding antibody, we located pUL49 in the fibroblast cytoplasm. pUL49 exhibited expression kinetics resembling those of the class β-2 proteins and was detected in the virion tegument. Following deletion of UL49 ORF, the virus failed to replicate, but it could be recovered by addition of pUL49 from pCDNA3.1 (+)-UL49. Our findings indicate that UL49 ORF is essential for HCMV replication in host foreskin fibroblasts.

The UL49 ORF of human cytomegalovirus (HCMV) is essential for viral replication; conserved among all herpes viruses; however, the function is unclear. Once the UL49 ORF was precisely deleted from the start to stop codon, the mutant did not yield infectious progeny. In this study, we find out many alternatively processed ESTs in UL49 locus in HCMV-infected cells, in which there are two novel transcription termination sites in UL49 locus. Most of these ESTs are rare transcripts that contain directed repeat sequences in the intron splicing regions. There is a typical GU-AG intron splicing site in UL49Y transcripts. The 1847 bp UL49Y cDNA spans an ORF from 335 to 1618 and encodes a putative protein of 427 amino acids with a predicted molecular mass of 47.1 kDa. All the new EST sequences and UL49Y cDNA sequence have been deposited in the GenBank database (GenBank Accession nos. GW314860-GW314900 and GU376796). This study provides us with very important clues for revealing the importance of the UL49 locus alternative splicing.

Chinese giant salamander iridovirus (CGSIV) is the emerging causative agent to farmed Chinese giant salamanders in nationwide China. CGSIV is a member of the common midwife toad ranavirus (CMTV) subset of the amphibian-like ranavirus (ALRV) in the genus Ranavirus of Iridoviridae family. However, viral protein information on ALRV is lacking. In this first proteomic analysis of ALRV, 40 CGSIV viral proteins were detected from purified virus particles by liquid chromatography-tandem mass spectrometry analysis. The transcription products of all 40 identified virion proteins were confirmed by reverse transcription polymerase chain reaction analysis. Temporal expression pattern analysis combined with drug inhibition assay indicated that 37 transcripts of the 40 virion protein genes could be classified into three temporal kinetic classes, namely, 5 immediate early, 12 delayed early, and 20 late genes. The presence of major capsid proteins (MCP, ORF019L) and a proliferating cell nuclear antigen (ORF025L) was further confirmed by Western blot analysis. The functions of MCP were also determined by small interfering RNA (siRNA)-based knockdown assay and anti-recombinant MCP serum-based neutralization testing. At low dosages of CGSIV, siRNA-based knockdown of the MCP gene effectively inhibited CGSIV replication in fathead minnow cells. The antiviral effect observed in the anti-MCP serum-based neutralization test confirms the crucial function of the MCP gene in CGSIV replication. Taken together, detailed information on the virion-associated viral proteins of ALRV is presented for the first time. Our results also provide evidence that MCP is essential for CGSIV replication in vitro.

Recently,Infectious diseases are implicated in the declines and the economic loss in cul-tured and wild Chinese giant salamander.One of pathogens has been recognized as Chinese giant sala-mander iridovirus (CGSIV).The putative ORF22R of CGSIV was amplified and sequenced.The ORF consists of 1 818 bp,which codes for a protein of 605 aa with a predicted molecular mass of 65.185 kD. Comparative studies of the identity of the amino acid sequence of ORF22R were carried out with different iridovirus species.Alignment of the identity of the amino acid sequence of this ORF of CGSIV and amphib-ian-like ranaviruses(ALRV)showed 95.9%-98.2% identity,but significantly higher than that of GIV-like viruses (31.7%)and the Lymphocystis disease virus 1 and C in other genus of Ranavirus (~16%). ORF22R was cloned into the pET22b vector,and expressed in E.coli Rosetta.The protein ORF22R was purified with nickel column chromatography after inclusion body lysis,and isolated by SDS-PAGE.

Human cytomegalovirus (HCMV) is a ubiquitous virus. Although the infection in healthy children and adults is usually asymptomatic, in immunocompromised individuals and newborns it is a significant cause of morbidity and mortality. UL49, an essential gene of HCMV, is highly conserved among various HCMV strains. The expression of UL49 is correlated with the production of virions. When UL49 is inhibited in the HCMV, the production of virions is reduced severely. In this study, RNA interference was applied to further investigate the roles of UL49 in viral replication. Two effective small interfering RNAs against UL49 were selected. Silencing of UL49 in HCMV-infected human foreskin fibroblast cells reduced the transcription levels of early and late genes, but not immediate-early ones. In addition, the viral DNA content was significantly reduced. This is the first time to uncover the role of UL49 in viral DNA synthesis, which indicates that UL49 might play an important role in this period. So the down-regulation of UL49 mRNA using RNAi might be a potential clinical therapy against the virus.

Objective To construct a targeting ribozyme(M1GS) which is specific to HCV genome.MethodsAccording to the sequence of conservative region(5′UTR) of HCV genome,a guide sequence was designed and synthesized.And then by the PCR method,a kind of targeting ribozyme can be constructed by covalently linking the guide sequence to the 3′terminus of M1 RNA,the catalytic subunit of RNase P from Escherichia coli.ResultsThe engineered ribozyme(M1GS-HCV/C67) is targeted to the 5′UTR of HCV genome,and can effectively cleave the substrate RNA segment in vitro.The cleavage is specific and the cleavage site is between 67 nt and 68 nt of the target region.Conclusion The M1GS-HCV/C67 would be a useful experimental material to further study its cleavage activity in vivo,and can be even used for evaluating its anti-viral effect in the animal model.It was believed that this study would markedly facilitate the research of a general gene targeting agent for anti-HCV applications,and layed the foundation for developing a new nucleic acid drug of anti-HCV therapy.

Objective To construct the mammalian expression plasmid of the pro-survival molecule prosaposin and establish the stable transfected cell line.Methods The open reading frame of prosaposin was amplified by PCR.Annealed oligonucleotides containing a c-Myc antibody recognition sequence and an in-frame stop codon were cloned downstream of the prosaposin cDNA sequence and then subcloned into the pcDNA3.1(+) expression vector to construct the mammalian expression plasmid pcDNA-Psap-Myc.The recombined plasmid was sequenced and then transfected into NIH3T3 cell by lipofectamineTM 2000.After screening by G418,stable transfected cell line was established,and the expression of prosaposin-Myc was identified by RT-PCR and western blotting assays.Results The mammalian expression plasmid pcDNA-Psap-Myc was constructed successfully and the stable transfected cell line Psap-Myc in NIH3T3 was established.RT-PCR and western blotting results indicated that prosaposin-Myc fusion protein was expressed successfully in NIH3T3 cell.Conclusion The construction of the recombinant eukaryotic expression pcDNA-Psap-Myc and the establishment of the stable transfected cell line Psap-Myc in NIH3T3 will facilitate further functional study on prosaposin and its interaction with other proteins.

BACKGROUND:Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that typically causes asymptomatic infections in healthy individuals but may lead to serious complications in newborns and immunodeficient individuals. The emergence of drug-resistant strains of HCMV has posed a need for the development of new drugs and treatment strategies. Antisense molecules are promising gene-targeting agents for specific regulation of gene expression. External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. The UL49-deletion BAC of HCMV was significantly defective in growth in human foreskin fibroblasts. Therefore, UL49 gene may serve as a potential target for novel drug development to combat HCMV infection. In this study, DNA-based EGS molecules were synthesized to target the UL49 mRNA of human cytomegalovirus (HCMV).RESULTS:By cleavage activity assessing in vitro, the EGS aimed to the cleavage site 324 nt downstream from the translational initiation codon of UL49 mRNA (i.e. EGS324) was confirmed be efficient to direct human RNase P to cleave the target mRNA sequence. When EGS324 was exogenously administered into HCMV-infected human foreskin fibroblasts (HFFs), a significant reduction of ~76% in the mRNA and ~80% in the protein expression of UL49 gene, comparing with the cells transfected with control EGSs. Furthermore, a reduction of about 330-fold in HCMV growth were observed in HCMV-infected HFFs treated with the EGS.CONCLUSIONS:These results indicated that UL49 gene was essential for replication of HCMV. Moreover, our study provides evidence that exogenous administration of a DNA-based EGS can be used as a potential therapeutic approach for inhibiting gene expression and replication of a human virus.

AIM:pUL23 is a protein encoded by human cytomegalovirus,which inhibits self propagation of the virus.The study was to investigate the function of pUL23 protein encoded by human cytomegalovirus and reveal the mechanism which inhibits propagation of the virus itself.METHODS: We screened a host proteins interacting with pUL23 from human embryonic kidney cDNA library using the yeast two-hybrid assay.GST-pull-down and immunoprecipitation techniques were applied to study the physical interaction between them in vitro and in vivo,respectively.RESULTS: We confirmed the interaction between pUL23 and IGFBP4 by pull-down and immunoprecipitation technology.CONCLUSION: These results might provide important information to the function of pUL23 in regulating self-reproducting.

Objective:To construct the recombinant eukaryotic expression plasmid of the homeobox gene Rhox5 and establish the stable transfected cell line.Methods:The full length cDNA sequence of Rhox5 was amplified by PCR.Annealed oligonucleotides containing a HA antibody recognition sequence and an in-frame stop codon were cloned downstream of the Rhox5 cDNA sequence and then subcloned into the pcDNA3.1(-) expression vector to construct the recombinant eukaryotic expression plasmid pcDNA-Rhox5-HA.The recombinant plasmid and the pcDNA3.1 vector were transfected into NIH3T3 cell by lipofectamineTM 2000 respectively.After screening by hygromycin B,stable transfected cell line was established,and the expression of Rhox5-HA was identified by RT-PCR and western blotting assays.Results:The eukaryotic expression plasmid pcDNA-Rhox5-HA was constructed successfully and the stable transfected cell line Rhox5-HA in NIH3T3 was established.RT-PCR and western blotting results indicated that Rhox5-HA fusion protein was expressed successfully in NIH3T3 cell.Conclusion:The construction of the recombinant eukaryotic expression pcDNA-Rhox5-HA and its stable expression in NIH3T3 cell will facilitate further functional study on Rhox5 and its interaction with other proteins.

Incubated HCMV Towne to the well growing HFF cells; Collected the cells while the HFF cells appearing cytopathic effects; Extracted the HCMV RNA from the infected cells. Amplified the cDNA of the gene by RT-PCR. Finally, the production of RT -PCR were cloned into pMD18 -T vectors and sequenced subsequently. And the sequence of UL49X was analyzed by bioinformatics. Our research show that, there is a new gene in the UL49 region of HCMV which we called it UL49X. The sequence of UL49X gene was from 68 783 bp to 78 608 bp, and we found that there was a intron between 69 194 bp and 71 530 bp.

Human cytomegalovirus (HCMV) is extremely species specific and does not replicate in experimental animal tissues.To overcome the problem and establish suitable animal models for studying antiviral strategies,the expression of HCMV UL49 gene was explored in mice.UL49-GFP gene was subcloned into the adenovirus shuttle plasmid pDC316,the products(pDC316-UL49-GFP)were co-transfected with helper plasmid pBHGloxE1,3Cre into HEK293 cell lines by liposome reagent,recombinant adenovirus(Ad-UL49-GFP) was generated and confirmed by PCR and Western blot.Ad-UL49-GFP was propagated in 293 cells and purified.The titer of viral stocks was determined by end-point dilution assay.The purified adenoviruses were delivered into mice via the tail vein injection.Fluorescence quantitative PCR and Western blot experiments were used to examine the tissue distribution and duration of UL49 gene expression.The results showed that the recombinant adenovirus were present in vivo.The expression level in tissues arranged in descending order was liver,spleen,kidney,heart and lung.3 days after injection,the liver,spleen,kidney,heart and lung expressed protein UL49 in high lever and then declined gradually.14 days after injection,UL49 protein expression was disappear in some organs except liver and spleen.In conclusion,transgene animal model carrying UL49 gene was successfully established.Therefore,the system may be suitable for selecting anti-HCMV drugs targeting UL49 gene.

【Objective】 To establish a human embryonic lung fibroblast(HELF) cell line which stably expresses human cytomegalovirus(HCMV) UL49 gene.【Methods】 HCMV UL49 gene was amplified by RT-PCR from HCMV infected HELF cells and then cloned into pLEGFP-N1 vector using standard procedures to develop recombinant plasmid pLEGFP-N1-FLAG-UL49.The packaging cell line,AmphoPackTM-293 cells were transiently transfected by pLEGFP-N1-FLAG-UL49 to generate retrovirus carrying UL49 DNA,which can be used to infect HELF cells.Cells integrated the retrovirus were selected by antibiotic G418.【Results】 Protein encoded by HCMV UL49 gene was detected in the retrovirus-infected HELF cells by IFA,Western blot and RT-PCR assay.【Conclusion】 HELF cell line stably expressing UL49 has been established,which may provide a powerful tool for functional research of UL49.

To study the interaction between prosaposin with exon 8 isform and Rhox5 protein,the prosaposin cDNA including exon 8 spliced forms was amplified using overlap extension PCR and then was subcloned into pGBKT7 and pGADT7 vectors to construct pGBKT7-PsapE8 and pGADT7-PsapE8 plasmids.Yeast two-hybrid assay was used to detect whether PsapE8 could bind Rhox5 protein in yeast.PsapE8 proteins was translated in vitro and labeled with 35S-Met using a TNT T7 Quick Coupled transcription/translation reaction kit.GST pull-down was used to confirm the interaction between PsapE8 and Rhox5 protein in vitro.The recombinant plasmids pGBKT7-PsapE8 and pGADT7-PsapE8 were constructed successfully.Yeast two-hybrid assay indicated PsapE8 could bind RHOX5 in yeast.GST pull-down assay further confirmed this interaction in vitro.The results suggested that Prosaposin with exon 8 isform(PsapE8) could interact with Rhox5 protein.

Objective:To construct the recombinant eukaryotic expression vector pEYFP-C1 /Myf5 carrying encoding gene of mouse Myf5 and test its location in 10T1 /2 cells.Method:The full length of Myf5 cDNA sequence was amplified from mouse cDNA library by PCR.Then Myf5 CDS fragment was ligated into plasmid pEYFP-C1.C3H10T1 /2 cells were transfected with pEYFP-C1 /Myf5 vector.The expression of Myf5 was observed by fluorescence microscope.Result:Myf5 CDS which was about 760bp and obtained by PCR was recombinant into pEYFP-C1 voctor.C3H10T1 /2 cells were transfected with pEYFP-C1 /Myf5 vector and Myf5 protein was found locating in nucleus.Conclusion:The eukaryotic expression plasmid pEYFP-C1 /Myf5 has been successfully constructed and expressed in the C3H10T1 /2 cells.Myf5 protein has been proved locating in nucleus.

Objective:To analyze the sequences of RhoxF1 protein,construct the bait plasmid of RhoxF1 in yeast two-hybrid system and test its self-activation acitivity.Method:Conserved domains were analyzed and the homology between mouse Rhox5 protein and human RhoxF1 protein was compared by bioinformatics method.PCR was used to amplify RhoxF1 gene and the fragment was inserted into the PMD 18-T vector.After verified,the fragment was subcloned into pGBKT7 vector.After that,the 1 plasmid was transformed into the yeast strain Saccharomyces cerevisiae AH109 and its toxicity and transcriptional activation was tested by both the phenotype assay and β galactosidase assays.Result:Several conserved DNA binding sites were found and the homology between Rhox5 and RhoxF1 amounted up to 30%.The bait plasmid pGBKT7-RhoxF1 was constructed correctly.Activity test of β-galactosidase revealed that pGBKT7-RhoxF1 couldn't be self-activated.Conclusion:Human RhoxF1 protein has the high homogeneous characteristics with mouse Rhox5 protein.In addition,pGBKT7-RhoxF1,as a bait plasmid of the yeast two-hybrid system,can be used to screen the protein interacting with RhoxF1.