狂犬/丙型肝炎嵌合病毒的构建和拯救

目的 以狂犬病病毒为载体,构建表达丙型肝炎病毒包膜糖蛋白E1E2的狂犬/丙型肝炎嵌合病毒,为发展新型丙肝载体疫苗奠定基础.方法 在狂犬病病毒反向遗传系统CTN-GFP的基础上,通过传统分子克隆方法,将HCV E1E2基因分别克隆人复制型和复制缺陷型狂犬病病毒载体,构建狂犬/丙肝嵌合病毒CTN-HCV E1E2和CTNΔG-HCV E1E2.结果 免疫荧光（DFA）和反转录聚合酶链式反应（RT-PCR）结果 显示嵌合病毒拯救成功,嵌合病毒能够再次感染正常细胞并且能够在mRNA水平检测到HCV E1E2基因的表达.结论 本研究成功构建了表达丙型肝炎病毒包膜糖蛋白E1E2的狂犬/丙肝嵌合病毒,提示以狂犬病病毒为载体发展新型丙肝载体疫苗在理论和技术上都是可行的.     

Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

L-ficolin, one of lectin families, is a recently identified complement factor that initiates lectin pathway of complement. Little is known about its role in viral hepatitis. In the present study, we found that L-ficolin in serum from 103 patients with hepatitis C virus （HCV）, were significantly higher than that in 150 healthy controls. We further found that L-ficolin expressions were significantly increased in vitro study by HCV JFH-1 infected human hepatocyte cell line Huh7.5.1. Investigation of the mechanisms of the L-ficolin action on HCV demonstrated that L-ficolin protein could recognize and bind to envelope glycoproteins E1 and E2 of HCV, activating the lectin complement pathway-mediated cytolytic activity in HCV-infected hepatocyte. This interaction between L-ficolin and HCV E1 and E2 glycoproteins was attributed to the N-glycans of E1 and E2. These findings provide new insights into the biological functions of L-ficolin in clinically important hepatic viral diseases.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

人类免疫缺陷病毒-1包膜糖蛋白进化对抗原表位及病毒亲嗜性的影响

Objective To investigate the evolution of HIV-1 envelope (env) gene from the individuals infected by the virus from one donor, the entry mediated by the envelope glycoprotein and the variation in the main neutralizing epitopes of envelope. Methods The genetic distances of the HIV-1 envelope genes derived from previous studies were analyzed. A series of envelope-pseudotyped viruses were constructed by co-transfecting HEK293T cells with a HIV-1 plasmid bearing the firefly luciferase reporter gene and an envelope expression plasmid. The entry ability of the envelope-pseudotyped viruses into U87. CD4. CCR5 or U87. CD4. CXCR4 cell lines was examined. The ami-no acid sequences representing the epitopes to the broad-neutralizing antibodies within the envelope glycoproteins were also investigated. Results It was found that the genetic distance of the 24 env genes with complete open reading frame was (7.91 ±0.78)% towards HIV-1 CNHN24, and (6.90 ±0.79)% towards RL42. Among the variable regions, the genetic distance of V1/V2 showed the biggest distance, and that of V3 showed the smallest distance. There were CCR5-tropic, CXCR4-tropic and CCR5/CXCR4-dual-tropic Env-pseudoviruses. Furthermore, in these envelopes, the epitopes to IgG1 b12 2F5 and 4E10 antibody were conserved, while the epitope to 447-52D was variable. Conclusion There is definite env gene variation among the viruses derived from the same donor. The variation influences the entry ability and tropism of emelope pseudoviruses. The epitopes to the main broad-neutralizing antibodies are conserved.     

Death-associated protein kinase 1 is an IRF3/7-interacting protein that is involved in the cellular antiviral immune response

Interferon regulatory factor （IRF） 7 has been demonstrated to be a master regulator of virus-induced type I interferon production （IFN）, and it plays a central role in the innate immune response against viruses. Here, we identified death-associated protein kinase 1 （DAPK1） as an IRF7-interacting protein by tandem affinity purification （TAP）. Viral infection induced DAPKI-IRF7 and DAPKI-IRF3 interactions and overexpression of DAPK1 enhanced virus-induced activation of the interferon-stimulated response element （ISRE） and IFN-p promoters and the expression of the IFNB1 gene. Knockdown of DAPK1 attenuated the induction of IFNB1 and RIG.lexpression triggered by viral infection or I FN-p, and they were enhanced by viral replication. In addition, viral infection or IFN-p treatment induced the expression of DAPK1. IFN-p treatment also activated DAPK1 by decreasing its phosphorylation level at serine 308. Interestingly, the involvement of DAPK1 in virus-induced signaling was independent of its kinase activity. Therefore, our study identified DAPK1 as an important regulator of the cellular antiviral response.     

Research progress of gene therapy for vascularization of tissue engineering北大核心

BACKGROUND: The rapid formation of functional vascular system in large-scale engineered graft is the basic prerequisite for its successful survival in the host. The vascularization of tissue engineering by genetic engineering technology has the advantages of good therapeutic effect, low cost and high safety. It is of great significance to carry out the research on gene therapy for vascularization of tissue engineering for long-term effective tissue repair. OBJECTIVE: To summarize the current research status and main problems of seed cells, target genes and gene vectors of gene therapy in tissue engineering vascularization so as to further explore the application prospect of gene therapy in tissue engineering vascularization,. METHODS: The literature retrieval was conducted on PubMed, Web of Science, and CNKI with the key words of “tissue engineering; vascularization; gene therapy; seed cells; target genes; vectors” in Chinese and English, and 61 articles closely related to this study were selected for the review. RESULTS AND CONCLUSION: Mesenchymal stem cells, vascular endothelial cells and endothelial progenitor cells are the most potential seed cells for vascularization of tissue engineering using gene therapy. They not only have good vascular induction, but also benefit the introduction of many virus and non-viral vectors and the expression of vascularization target genes. Vascular endothelial growth factor, angiopoietin-1, basic fibroblast growth factor, bone morphogenetic protein-2, hypoxia inducible factor-1 α and other vascularization target genes are mainly used to construct efficient and stable vascular network in engineering grafts by means of combination of double/multiple gene, coupling of osteogenesis and angiogenesis, and regulation of upstream gene. Since different viral and non-viral vectors have their own advantages and disadvantages, suitable vectors should be selected according to the efficiency of gene transfection, biological safety, and cost in application. At present, although great progress has been made in the application of gene therapy in tissue engineering vascularization, there are still many key technologies to be broken through for clinical application, such as how to improve the targeted release of target growth factors and reduce the safety risk, which is also the research direction and hot spot of tissue engineering vascularization based on gene therapy in the future. © 2022, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Apoptosis and proinflammatory cytokine responses of primary mouse microglia and astrocytes induced by human H1N1 and avian H5N1 influenza viruses

Patients with an influenza virus infection can be complicated by acute encephalopathy and encephalitis. To investigate the immune reactions involved in the neurocomplication, mouse microglia and astrocytes were isolated, infected with human H1N1 and avian H5N1 influenza viruses, and examined for their immune responses. We observed homogeneously distributed viral receptors, sialic acid （SA）-a2,3-Galactose （Gal） and SA-a2,6-Gal, on microglia and astrocytes. Both viruses were replicative and productive in microglia and astrocytes. Virus-induced apoptosis and cytopathy in infected cells were observed at 24 h post-infection （p.i.）. Expression of IL-1β, IL-6 and TNF-a mRNA examined at 6 h and 24 h p.i. was up-regulated, and their expression levels were considerably higher in H5N1 infection. The amounts of secreted proinflammatory IL-1β, IL-6 and TNF-a at 6 h and 24 h p.i. were also induced, with greater induction by H5N1 infection. This study is the first demonstration that both human H1N1 and avian H5N1 influenza viruses can infect mouse microglia and astrocytes and induce apoptosis, cytopathy, and proinflammatory cytokine production in them in vitro. Our results suggest that the direct cellular damage and the consequences of immunopathological injury in the CNS contribute to the influenza viral pathogenesis. Cellular ＆ Molecular Immunology.     

Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus

Small interfering RNA (siRNA) is currently being evaluated not only as a powerful tool for functional genomics, but also as a potentially promising therapeutic agent for cancer and infectious diseases. Inhibitory effect of siRNA on viral replication has been demonstrated in multiple pathogenic viruses. However, because of the high sequence specificity of siRNA-mediated RNA degradation, antiviral efficacy of siRNA directed to viral genome will be largely limited by emergence of escape variants resistant to siRNA due to high mutation rates of virus, especially RNA viruses such as poliovirus and hepatitis C virus (HCV). To investigate the therapeutic feasibility of siRNAs specific for the putative cellular cofactors for HCV, we constructed adenovirus vectors expressing siRNAs against La, polypyrimidine tract-binding protein (PTB), subunit gamma of human eukaryotic initiation factors 2B (eIF2Bgamma), and human VAMP-associated protein of 33 kDa (hVAP-33). Adenoviral-mediated expression of siRNAs markedly diminished expression of the endogenous genes, and silencing of La, PTB, and hVAP-33 by siRNAs substantially blocked HCV replication in Huh-7 cells. Thus, our studies demonstrate the feasibility and potential of adenoviral-delivered siRNAs specific for cellular cofactors in combating HCV infection, which can be used either alone or in combination with siRNA against viral genome to prevent the escape of mutant variants and provide additive or synergistic anti-HCV effects.     

Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the context of JFH1 are more robust in the release of viral particles. To understand the contributions of structural and nonstructural genes to HCV replication potential and infectivity, we have characterized intragenotypic recombinant genotype 2a viruses with different portions of the J6 isolate engineered into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone. Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility of the chimeric junction. Moreover, NTRNS2 a chimeric virus equivalent to the previously reported FL-J6/JFH chimera, showed a 10-fold enhancement of virus titers compared to CNS2. NTRNS2 differs from CNS2 by three nucleotide differences residing in the 5' NTR and core coding sequence and all three nucleotide changes were necessary for increased virion production. Importantly, cells producing Cp7 virus showed increased apoptosis compared with JFH1, an effect correlating with virion production. These studies begin to unravel requirements for robust virus replication and the relationship between increased virion production and host cell viability.     

Immunogenic epitopes on the surface of the hepatitis A virus capsid: Impact of secondary structure and/or isoelectric point on chimeric virus assembly

Hepatitis A virus (HAV) protein 2A has the capacity to harbor and expose a short foreign epitope. The chimeric virus, HAV-gp41, bearing seven amino acids of the 2F5 epitope of the HIV glycoprotein gp41, was shown to replicate in cell culture and laboratory animals and to induce a humoral immune response. As an extension of this work, we now investigated the possibility to insert longer epitopes, their impact on genetic stability, and the production of chimeric HAV. Twenty-seven amino acid residues of either HIV gp41, comprising the 2F5 epitope, or of a mimotope (F78) of the hypervariable region 1 of the hepatitis C virus (HCV) envelope protein E2 were inserted near the C-terminus of HAV 2A and viral capsid formation and replication were studied. The genome of the chimeric virus (HAV-F78) had reduced replication ability, yet the sedimentation profile of the chimeric particles was unchanged and the HCV sequence was maintained over serial viral passages. In contrast, no capsids were formed when an extended HIV epitope of 27 residues was inserted, precluding the rescue of infectious chimeric virus. Based on structural analyses, the data suggest that the isoelectric point (pI) and/or the secondary structure of the chimeric proteins are essential determinants that affect HAV particle formation for which protein 2A serves as an assembly signal.     

High-level expression of hepatitis C virus (HCV) structural proteins by a chimeric HCV/BVDV genome propagated as a BVDV pseudotype

A chimeric cDNA genome was constructed in which the core, E1 and E2 genes of hepatitis C virus (HCV) replaced the core, E(rns), E1 and E2 genes of bovine viral diarrhea virus (BVDV). High levels of HCV structural proteins were expressed in a small number of human or bovine cells following transfection with chimeric RNA. However, in one cell line, bovine embryonic trachea cells [EBTr(A)], the number of cells expressing HCV proteins increased to greater than 70% following serial passage of culture medium. These cells were persistently infected with a non-cytopathogenic BVDV helper virus. In these cells, the chimeric genome was packaged into infectious particles that accumulated in the culture medium at a titer as high as 10(7)-10(9) genome equivalents per ml. The virus particles were pseudotypes, because they were neutralized by anti-BVDV but not by anti-HCV.     

CD81 engineered with endocytotic signals mediates HCV cell entry: implications for receptor usage by HCV in vivo

Although CD81 has been shown to bind HCV E2 protein, its role as a receptor for HCV remains controversial. In this study, we constructed two CD81 chimeras by linking the cytoplasmic domains of recycling surface receptors, low-density lipoprotein receptor (LDLR), and transferrin receptor (TfR), respectively, to CD81 and compared their internalization properties to wild-type CD81. Binding experiments with anti-hCD81 antibody showed that cell-surface CD81 chimeric receptors were internalized much more efficiently than wild-type CD81. In addition, CD81 chimeras, but not wild-type CD81, could internalize recombinant E2 protein and E2-enveloped viral particles from the serum of HCV-infected patients into Huh7 liver cells. The latter resulted in persistent positive-strand viral RNA and accumulation of replication intermediates, negative-strand viral RNA, in the infected cells, suggesting that the internalized viruses have undergone replication. Therefore, it appeared that CD81, possibly in association with a liver-specific endocytotic protein(s), represents one of the pathways by which HCV can infect hepatocytes.     

An adaptive mutation in NS2 is essential for efficient production of infectious 1b/2a chimeric hepatitis C virus in cell culture

The development of JFH1 based intergenotypic recombinants which exploit the unique replication characteristics of JFH1 has made it possible to study infectious HCV encoding the structural genes of additional HCV genotypes including genotype 1b. Although, intergenotypic 1b/2a chimeric genomes replicate efficiently in transfected cells they produce very low viral titers, limiting the utility of this system. Here, intergenotypic 1b/2a variants were generated by serially passaging the virus in a novel highly permissive Huh-7 cell clone. The adapted virus was 1000-fold more infectious than the parental unadapted virus and six adapted mutations were identified throughout the genome. Of the mutations identified, L839S in the NS2 gene was the most critical for the adapted phenotype by enhancing the infectivity of assembled viral particles. Overall, the efficient production of infectious 1b/2a virus particles will facilitate the discovery and characterization of inhibitors targeting steps that involve the structural genes of genotype 1b HCV.     

多型别HCV-E1复合表位抗原研究

目的 设计多型别HCV-E1表位复合免疫原,通过免疫小鼠,探讨其在丙型肝炎治疗性疫苗与诊断试剂研究中的应用.方法 分析比较HCV-E1包膜糖蛋白B细胞表位序列,选取几个代表性基因型的中和性优势抗原表位,再结合泛DR辅助性T细胞表位(PADRE),构建含有不同HCV型别E1表位抗原基因和通用T辅助细胞表位基因的原核表达质粒.结果 成功构建了含有HCV 1a、1b、2a、3a、4a和6a等基因型E1中和性表位以及通用T辅助细胞表位的质粒pBVIL1/E1s-PADRE,该质粒转化大肠杆菌后获得的工程菌,通过诱导培养可以高效表达重组多型别HCV-E1表位复合抗原,所获得的多型别HCV-E1表位复合免疫原可在BALB/c小鼠体内诱发强烈的体液免疫反应,ELISA检测抗体水平可达到1:12 800.结论 新构建的HCV-E1表位复合免疫原具有很好的免疫原性,为进一步研究HCV疫苗奠定了基础.     

Hepatitis C virus and the related bovine viral diarrhea virus considerably differ in the functional organization of the 5' non-translated region: implications for the viral life cycle

The 5' non-translated regions (5'NTRs) of hepatitis C virus (HCV) and bovine viral diarrhea virus (BVDV) initiate translation of the viral RNA genome through an internal ribosomal entry site (IRES) and operate as major determinants of the RNA replication cycle. We report on comparative studies with both virus systems demonstrating that the functional organization of the 5'NTRs of HCV and BVDV shows evident differences despite a similar RNA structure. In the BVDV 5'NTR, replication signals are restricted to the 5' terminal domain I. With HCV, we defined specific replication signals in domain I but also in domains II and III that constitute the functional IRES. While the BVDV domain I supports IRES activity, the HCV domain I appears to down-regulate IRES function. These data suggest that HCV and BVDV apply different mechanisms to coordinate viral protein and RNA synthesis, which may explain differences in the replication efficiency of both related viruses.     

Mechanism of the interferon alpha response against hepatitis C virus replicons

Interferon alpha (IFN-alpha) inhibits hepatitis C virus (HCV) replication in vivo and in cell cultures by one or several mechanisms that are not yet understood. We sought to identify the viral targets of the IFN-alpha-induced cellular antiviral program in Huh7 cells expressing HCV subgenomic replicons. Our results revealed a tight linkage between translation, assembly of replication complexes and viral RNA synthesis, and indicated that the stability of amplified plus strand RNA was reduced in the presence of the cytokine. Moreover, HCV internal ribosomal entry site (IRES)-directed translation was inhibited approximately 2-fold in IFN-treated cells. In contrast, the synthesis of viral RNA did not seem to be directly affected by the antiviral program induced by the cytokine. Our results were consistent with a model predicting that the IFN-alpha-induced antiviral program could inhibit multiple steps of the HCV replication cycle, leading to a reduction in viral protein synthesis and eventually inhibition of viral RNA amplification.