生物谷报道：利用低温电子显微镜技术和计算机图像分析方法，美国Scripps研究所(全球最著名的免疫研究所之一)的两个实验室将两个存在于被感染细胞中的乙肝病毒的两种中间体形式显像出来。此外，他们还通过分析从患者血液中分离获得的传染性乙肝病毒，确定出了病毒的三维图。研究的结果刊登在6月23日的Molecular Cell学术期刊上。这些结果将帮助研究人员更清楚地了解乙肝病毒在细胞中如何复制，并为乙肝新疗法的研究开辟新路。
据统计，全世界约有3.5亿人被乙肝病毒所感染，而且每年因急性和慢性感染以及肝细胞癌死亡的人数高达100多万人。这种攻击肝脏的病毒通过输入被病毒感染的血液、进行静脉注射的药物滥用者共用针头和性接触等途径传播。
在乙肝病毒中，病毒的遗传物质被蛋白质外壳所保护。乙肝病毒颗粒即Dane颗粒的尺寸大约为40纳米，而且蛋白质外壳还被一层膜包被。虽然乙肝病毒蛋白外壳在离体状况下已经进行过大量的研究，但是人们对活体的被感染细胞中存在的病毒外壳的结构和装配却几无所知，甚至对成熟的病毒颗粒的结构也是知之甚少。
在新的研究中，Mark Yeager教授和Francis V. Chisari教授的领导的两个实验室使用低温电子显微镜和图像分析方法检测了转基因小鼠的乙肝病毒蛋白外壳和人类患者血样中分离的病毒颗粒的天然结构。通过迅速冷冻样本，研究人员能够利用低温电子显微镜成像这种病毒颗粒。成像过程获得了三维图像，该图像首次揭示出蛋白质壳如何与外层液体膜封套反应。
三维图像在分子水平上显示出乙肝病毒比血红蛋白分子大将近10倍。与人类的基因组类似，乙肝病毒的基因组也是由双链DNA构成，并被蛋白质壳包被。这个蛋白质壳是二十面体对称，类似多面穹顶几何结构。蛋白质壳本身被由液体双分子层形成的外膜包围住——就像围住人类细胞的细胞膜一样。乙肝病毒的这种膜镶嵌着许多糖蛋白长刺，这种长刺能与肝脏细胞上的受体结合，进而感染细胞。
在被乙肝病毒感染的肝细胞中，病毒DNA的转录过程产生一种类型的RNA——它被包裹进蛋白质外壳中。在这种蛋白质壳中，逆转录过程制作出乙肝单链DNA拷贝，它充当第二条DNA链合成所需的模板。最终产生的颗粒通过细胞的内质网膜萌芽出来，并被运送到病毒的外膜封套。
在转基因小鼠中，研究人员发现了两种不同密度的病毒蛋白质壳。虽然两种类型的蛋白质壳都是二十面体结构，但较低密度的蛋白质壳含有病毒DNA的中间体。这些低密度的蛋白质壳可能由细胞核释放。这些发现为人们了解活体状态下的乙肝病毒复制提供新的线索。
原始出处：
Native Hepatitis B Virions and Capsids Visualized by Electron Cryomicroscopy
Kelly A. Dryden, Stefan F. Wieland, Christina Whitten-Bauer, John L. Gerin, Francis V. Chisari, and Mark Yeager
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New findings may aid in understanding how virus infects cells
LA JOLLA, CA, June 22, 2006 -- Using electron cryomicroscopy and computer image analysis, the scientists visualized two intermediate forms of the virus that exist within infected cells. In addition, they were able to determine a three-dimensional map by analysis of infectious hepatitis B virus isolated from patient blood samples. These results could help future researchers understand more clearly how hepatitis B virus replicates in the cell and point the way toward new therapeutic approaches that could disrupt the hepatitis B virus infection pathway.
The study, a collaborative effort between the laboratories of Scripps Research scientists Professor Mark Yeager, M.D., Ph.D., and Professor Francis V. Chisari, M.D., was published in the June 23, 2006 (Volume 22, Issue 6) edition of the journal Molecular Cell.
More than 350 million people worldwide are infected with hepatitis B virus, which kills more than a million each year due to acute and chronic hepatitis, and hepatocellular carcinoma. The virus, which attacks the liver, is spread through infected blood transfusions, needle sharing by intravenous drug abusers and sexual contact.
Virions are inert virus particles that carry the virus genome from cell to cell. In the hepatitis B virus, this genetic material is protected by a shell of protein molecules called a capsid. Hepatitis B virions, also known as Dane particles, are approximately 40 nanometers in size, and the capsid is surrounded by a membrane envelope. While the structure of the hepatitis B capsid has been studied intensively in vitro, until this study little was known about the structure and assembly of native capsids present in infected cells in vivo, and even less was known about the structure of mature virions.
"We used cryomicroscopy and image analysis to examine the native structure of HBV [hepatitis B virus ] capsids from transgenic mice and virions isolated from patient blood samples," Yeager said. "By rapidly freezing the samples we were able to use cryo-electron microscopy to image the particles while they were maintained at the temperature of liquid nitrogen-around-300?F-which preserves them in a state close to what exists in vivo. Image processing allowed us to derive 3-D maps that revealed for the first time how the outer lipid envelope interacts with the capsid shell. "
The 3-D maps showed that in terms of molecular size, hepatitis B virus is enormous-nearly 10 times larger than a hemoglobin molecule. Like the human genome, the genome of the hepatitis B virus is formed by double-stranded DNA and enclosed by the capsid, which has icosahedral symmetry, resembling the geometric structure of a geodesic dome. The capsid itself is contained within an outer envelope formed by a lipid bilayer, similar to the membranes that enclose all human cells. The membrane of hepatitis B virus is studded with glycoprotein spikes, which bind to receptors on liver cells that mediate infection.
In hepatitis B-infected liver cells, transcription of the viral DNA produces a type of RNA that is packaged into capsids. Within the capsid, reverse transcription produces a single-strand DNA copy that serves as the template for second strand DNA synthesis. The resulting particles bud through membranes of the endoplasmic reticulum-the part of the cell involved with protein folding, assembly, and transport-to acquire the outer membrane envelope of the virus, a step that confers infectivity.
"In the transgenic mice, we found two types of capsids with different densities," Yeager said. "While both types of capsids were assembled as similar icosahedral structures, we found that the lower density capsids did not contain any viral DNA or viral RNA, while the higher density capsids contained viral DNA intermediates. It seems likely that these lower density capsids were released from the cell nucleus. These results may offer new clues how the virus replicates in vivo."
Other researchers of the study, titled "Native Hepatitis B Virions and Capsids Visualized by Electron Cryomicroscopy," were Kelly A. Dryden, Stefan F. Wieland, Christina Whitten-Bauer, and John L. Gerin.
关于The Scripps Research Institute
The Scripps Research Institute, headquartered in La Jolla, California, in 18 buildings on 40 acres overlooking the Pacific Ocean, is one of the world's largest independent, non-profit biomedical research organizations. It stands at the forefront of basic biomedical science that seeks to comprehend the most fundamental processes of life. Scripps Research is internationally recognized for its research into immunology, molecular and cellular biology, chemistry, neurosciences, autoimmune, cardiovascular, and infectious diseases, and synthetic vaccine development. Established in its current configuration in 1961, it employs approximately 3,000 scientists, postdoctoral fellows, scientific and other technicians, doctoral degree graduate students, and administrative and technical support personnel.
Scripps Florida, a 364,000 square-foot, state-of-the-art biomedical research facility, will be built in Palm Beach County. The facility will focus on basic biomedical science, drug discovery, and technology development. Palm Beach County and the State of Florida have provided start-up economic packages for development, building, staffing, and equipping the campus. Scripps Florida now operates with approximately 160 scientists, technicians, and administrative staff at 40,000 square-foot lab facilities on the Florida Atlantic University campus in Jupiter.
