DegU调控单核细胞增生李斯特菌感染宿主细胞及适应高温的机制研究

【目的】本研究旨在探究孤儿调节因子DegU在介导单核细胞增生李斯特菌(Listeria monocytogenes)宿主感染和高温环境适应性方面的调控机制。【方法】本研究以单增李斯特菌参考菌株EGD-e、degU基因缺失菌株ΔdegU和回补菌株CΔdegU为研究材料,通过细胞模型、实时荧光定量聚合酶链式反应和凝胶阻滞试验等方法探究DegU对单增李斯特菌感染宿主细胞和适应高温的调控机制。【结果】研究结果表明:缺失degU后,单增李斯特菌在Caco-2上的黏附和侵袭能力显著降低,在RAW264.7中的增殖能力显著降低,在L929中的空斑形成能力也显著降低;进一步通过实时荧光定量聚合酶链式反应检测degU基因缺失后引起的单增李斯特菌毒力因子转录水平变化,发现多个重要毒力因子转录水平均显著下调;该试验结果还发现与毒力相关的热应激基因clpE(受CtsR抑制的ATP依赖蛋白水解酶编码基因)转录水平显著升高,而在43℃高温条件下,clpE转录水平显著降低;进一步通过凝胶阻滞试验结果表明DegU能够与clpE的启动子直接结合。【结论】综上所述,degU基因缺失能够降低单增李斯特菌在宿主感染过程中的细胞黏附、侵袭、胞内增殖和胞间迁移能力;DegU能够与clpE启动子结合,直接调控clpE基因的转录水平来适应高温环境。该研究为进一步深入解析单增李斯特菌的宿主感染和环境适应性机制奠定了试验基础。

Characterization of the Receptor-binding Domain of Ebola Glycoprotein in Viral Entry

Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

Targeting host factors:A novel rationale for the management of hepatitis C virus

Hepatitis C is recognized as a major threat to global public health. The current treatment of patients with chronic hepatitis C is the addition of ribavirin to interferon-based therapy which has limited efficacy, poor tolerability, and significant expense. New treatment options that are more potent and less toxic are much needed. Moreover, more effective treatment is an urgent priority for those who relapse or do not respond to current regimens. A major obstacle in combating hepatitis C virus （HCV） infection is that the fidelity of the viral replication machinery is notoriously low, thus enabling the virus to quickly develop mutations that resist compounds targeting viral enzymes. Therefore, an approach targeting the host cofactors, which are indispensable for the propagation of viruses, may be an ideal target for the development of antiviral agents because they have a lower rate of mutation than that of the viral genome, as long as they have no side effects to patients. Drugs targeting, for example, receptors of viral entry, host metabolism or nuclear receptors, which are factors required to complete the HCV life cycle, may be more effective in combating the viral infection. Targeting host cofactors of the HCV life cycle is an attractive concept because it imposes a higher genetic barrier for resistance than direct antiviral compounds. However the principle drawback of this strategy is the greater potential for cellular toxicity.

Sensitive semi-quantitative detection of respiratory syncytial virus by dark-field light scattering imaging of the infected host cells

A novel sensitive semi-quantitative virus detection technique was developed using the respiratory syncytial virus(RSV) as an example, through dark-field light scattering imaging of the surface state of the virusinvaded host cells. In this method, anti-RSV-antibody modified gold nanoparticles(Au NPs) could bind with the invading virus on the cell membrane of the infected host cells through the specific antibody-antigen binding. Then,the host cells could be imaged by the localized surface plasmon resonance light scattering properties of Au NPs under a dark-field light scattering microscopy, which could be further used to semi-quantify the invading virus.

Viral proteomics: The emerging cutting-edge of virus research

Viruses replicate and proliferate in host cells while continuously adjusting to and modulating the host environment.They encode a wide spectrum of multifunctional proteins,which interplay with and modify proteins in host cells.Viral genomes were chronologically the first to be sequenced.However,the corresponding viral proteomes,the alterations of host proteomes upon viral infection,and the dynamic nature of proteins,such as post-translational modifications,enzymatic cleavage,and activation or destruction by proteolysis,remain largely unknown.Emerging high-throughput techniques,in particular quantitative or semi-quantitative mass spectrometry-based proteomics analysis of viral and cellular proteomes,have been applied to define viruses and their interactions with their hosts.Here,we review the major areas of viral proteomics,including virion proteomics,structural proteomics,viral protein interactomics,and changes to the host cell proteome upon viral infection.

Kaposi's sarcoma-associated herpesvirus:Towards better understanding of its latency and pathogenesis

Kaposi's sarcoma-associated herpesvirus(KSHV) is a double stranded DNA virus.It was found to be related to Kaposi's sarcoma(KS),primary effusion lymphoma(PEL) and multicentric Castleman's disease(MCD),which cause severe illness in AIDS patients.As a member of human herpesvirus family,KSHV displays two distinct phases in its life cycle,the default latent and lytic replication phase.Following primary infection,the virus can quickly establish latent infection in the host.However,it is still not fully understood up to date how KSHV establishes and maintains viral latency in the host cells.KSHV mainly infects endothelial cells in the host,promoting proliferation and angiogenesis.Abundant angiogenesis is the key feature of KS and is the critical factor for KS progression.The mechanism of KSHV mediated pathogenesis is also largely unknown.In this review,we summarize the recent progress in the mechanisms of KSHV latency and pathogenesis,with particular views from our work.