Gene Expression Regulation, Viral ; Proteomics ; methods ; trends ; Viral Proteins ; analysis ; genetics ; Viruses ; genetics ; growth & development ; metabolism

Bombyx mori nucleopolyhedrovirus (BmNPV) bm47 gene is found in all sequenced lepidopteran nucleopolyhedroviruses (NPVs). It is one of the core genes of NPVs. However, the role of bm47 in the biological cycle of NPV remains unknown. In this study, the Red recombination system was used to knock out bm47 from BmNPV to construct bm47-ko-Bacmid in E. coli BW25113 system. Then bm47 gene was introduced back to the viral genome using the Bac-to-Bac system to create the repair virus bm47-re-Bacmid. TCID50 assay and real-time PCR (qPCR) were used to evaluate the effects of bm47 deletion on viral DNA replication, gene transcription, and protein expression. qPCR results showed that bm47 knock-out had no significant effect on viral DNA replication. However, the qPCR results showed that bm47-ko-Bacmid significantly decreased the transcription levels of early gene lef-3, late gene vp39, and very late gene p10 at 48 h and 72 h after viral transfection of BmN cells (P < 0.05). This work will provide a foundation for further studies on the biological function of BmNPV bm47 in viral replication and transcription.
Animals ; Bombyx ; virology ; Gene Deletion ; Gene Expression Regulation, Viral ; Nucleopolyhedrovirus ; genetics ; physiology ; Transcription, Genetic ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo learn the effect of hepatitis B virus (HBV) sequence nt250-453 on the HBV X promoter.

METHODSA plasmid pNRE-XP which contains the NRE and the HBV X promoter was constructed to co-transfect HepG2 cell line with plasmid RL-TK. The firefly luciferase activity and mRNA expression of the firefly luciferase gene were both detected. Then, nt250-453 of HBV was removed from LJ196, which contained HBV full genes. The mutated plasmid LJ196 and plasmid LJ96 which provided core protein and the viral DNA polymerase were used to co-transfect HepG2 cell line. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect the X gene mRNA level.

RESULTSThe activity of firefly luciferase and the expression of firefly luciferase gene mRNA were both down-regulated in the presence of the NRE, while the HBV X gene mRNA expression increased as it was removed from the HBV genes.

CONCLUSIONThis study demonstrates that nt250-453 of HBV acts as a novel negative regulatory element which could suppress the HBV X promoter activity.

Enhancer Elements, Genetic ; Gene Expression ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; Promoter Regions, Genetic ; Trans-Activators ; genetics

OBJECTIVETo investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene.

METHODSHCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level.

RESULTSHCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells.

CONCLUSIONSHCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.

Antimicrobial Cationic Peptides ; genetics ; Cell Line ; Gene Expression Regulation, Viral ; Hepacivirus ; genetics ; Hepcidins ; Humans ; Plasmids ; Transfection ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo establish an experimental model of HCV C-HBV X co-expression protein and explore its effect on the expression of VEGF.

METHODSThe HBV X gene was recovered by enzyme excision and inserted into PBK-CMV and PBK-HCVC, and recombinant plasmids PBK-X and PBK-X-C were constructed. The plasmids PBK-CMV, PBK-X, PBK-HCVC and PBK-X-C were transfected into HepG2 cells with liposomes. After being selected by G418, resistant colonies were obtained. Reverse transcription PCR and Western blot were used to show HBV X and HCV core protein expression. VEGF was analyzed using immunohistochemical methods and Western blot.

RESULTSThe recombinant plasmid PBK-X-C expressed HBV X and HCV core protein efficiently under the control of the vectors promoter. VEGF and VEGF mRNA of the cells co-expressing HCV C-HBV X proteins were higher than those cells expressing HBV X, HCV C and vector alone.

CONCLUSIONHBV X-HCV C co-expression protein can increase the expression of VEGF of HepG2 cells. It suggests that HBV and HCV have a synergic action in the carcinogenesis.

Gene Expression ; Gene Expression Regulation, Viral ; Genetic Vectors ; Hep G2 Cells ; Hepacivirus ; genetics ; Humans ; Trans-Activators ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism ; Viral Core Proteins ; genetics

Contagious ecthyma (also known as orf) is an acute skin zoonosis caused by orf virus (ORFV), which affects sheep, goats and humans. As one of the typical species of the Parapoxvirus genus of the Poxviridae family, orf virus has distinctive and unique characteristics of these species. A range of immuno-modulatory/pathogenesis -related genes acquired by virus that function is to limit (at least transiently) the effectiveness of host immunity during its evolution. This review is aimed to describe the latest progress on the molecular characteristics of ORFV, and upon which we analyzed molecular mechanism of the immune escape designed and a set of strategies developed for ORFV to effective against immune clearance of the host. Known as an essential component in evolutionary system, host is regulated by ORFV for using in population evolution. By the ORFV evolutional immune regulation components and its effect approach, we can understand the viral biological characteristics of ORFV, and it is helpful for us to further study the counter-measures of this disease.
Animals ; Ecthyma, Contagious ; immunology ; virology ; Gene Expression Regulation, Viral ; Immune Evasion ; Orf virus ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

OBJECTIVESTo establish an animal model of HCV transgenic mice to elucidate the pathogenesis of hepatitis C virus infection and function of the viral structural proteins.

METHODSStructural gene of HCV were amplified and recombined into eukaryotic expression vectors, pcDNA4HisMax and pMT/BiP/V5-His A, after their expressive activity was confirmed to detect the structural protein in the transfected COS7 and S2 cells by Western blot. The fertilized expression element, which contained CMV or pMT promoter, structural gene of HCV and polyadenylation signal sequence, was microinjected into 1736 C57BL/6 mouse fertilized ova. The ova were then replanted into the oviducts of 69 pseudopregnant recipient mice.

RESULTSTwenty-five recipient mice were impregnated and later produced 105 newborns; 49 of them died from unknown causes and 57 survived. After the specific HCV structural genes were identified by PCR and Southern blot hybridization, 26 founders were obtained; among them 10 were stable expression mice and 16 were the inducible ones. The rate of founders developed from implanted embryos was only 1.50%. Through hybridization with normal mice, 58 hybrid mice have been obtained at present.

CONCLUSIONTwo kinds of different transgenic mice of HCV were developed; one is of stable expression, and the other is inducible. This transgenic mice model may create an opportunity for studying the function of the structural gene of HCV and elucidate its pathogenicity.

Animals ; Disease Models, Animal ; Gene Expression Regulation, Viral ; Hepacivirus ; genetics ; Hepatitis C ; Mice ; Mice, Transgenic ; Viral Structural Proteins ; genetics

Gene Expression Regulation ; immunology ; Gene Expression Regulation, Viral ; HIV Infections ; genetics ; immunology ; metabolism ; HIV-1 ; genetics ; immunology ; Humans ; Immunity, Innate ; genetics ; immunology

To investigate the genomic properties of HIV-1, we collected 3,081 sequences from the HIV Sequence Database. The sequences were categorized according to sampling region, country, year, subtype, gene name, and sequence and were saved in a database constructed for this study. The relative synonymous codon usage (RSCU) values of matrix, capsid, and gp120 and gp41 genes were calculated using correspondence analysis. The synonymous codon usage patterns based on the geographical regions of African countries showed broad distributions; when all the other regions, including Asia, Europe, and the Americas, were taken into account, the Asian countries tended to be divided into two groups. The sequences were clustered into nine non-CRF subtypes. Among these, subtype C showed the most distinct codon usage pattern. To determine why the codon usage patterns in Asian countries were divided into two groups for four target genes, the sequences of the isolates from the Asian countries were analyzed. As a result, the synonymous codon usage patterns among Asian countries were divided into two groups, the southern Asian countries and the other Asian countries, with subtype 01_AE being the most dominant subtype in southern Asia. In summary, the synonymous codon usage patterns among the individual HIV-1 subtypes reflect genetic variations, and this bioinformatics technique may be useful in conjunction with phylogenetic methods for predicting the evolutionary patterns of pandemic viruses.
HIV-1/*genetics/*isolation & purification ; Gene Expression Regulation, Viral/*genetics ; Europe/epidemiology ; Codon/*genetics ; Asia/epidemiology ; Americas/epidemiology ; Africa/epidemiology

OBJECTIVESAdeno-associated virus (AAV) Rep78 is known for its inhibitory effects on replication of several viruses and oncogenes transformations. The study was to investigate the effect of Rep78 on hepatitis B virus C (HBV-C) gene and the mechanism of it.

METHODSHBV-C promoter and HBV-C gene with its promoter were amplified by PCR and labeled with 32P-ATP. Electrophoretic mobility shift assay (EMSA) and in vitro transcription were utilized to detect the binding of MBP-Rep78 with HBV-C promoter and the transcription of HBV-C gene.

RESULTSEMSA showed that by increasing the amount of Rep78 protein from 0.1 microg to 1.0 microg, the binding bands got stronger in a dose-dependent manner. In addition, Rep78 antibody was used to certify the specificity of this binding. The compound of Rep78, Rep78 antibody and HBV-C promoter were seen as super shift bands in EMSA. Meanwhile, HBV-C gene transcription was significantly inhibited by in vitro transcription which meant that Rep78 could not only bind with HBV-C promoter, but also could inhibit the transcription of HBV-C gene.

CONCLUSIONAAV Rep78 could inhibit the transcription of HBV-C gene through its binding with HBV-C promoter.

DNA-Binding Proteins ; genetics ; Dependovirus ; genetics ; Gene Expression Regulation, Viral ; Hepatitis B virus ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; Transcription, Genetic ; Viral Proteins ; genetics