生物谷報道：利用低溫電子顯微鏡技術(shù)和計算機圖像分析方法,，美國Scripps研究所(全球最著名的免疫研究所之一)的兩個實驗室將兩個存在于被感染細胞中的乙肝病毒的兩種中間體形式顯像出來,。此外,，他們還通過分析從患者血液中分離獲得的傳染性乙肝病毒,，確定出了病毒的三維圖,。研究的結(jié)果刊登在6月23日的Molecular Cell學(xué)術(shù)期刊上,。這些結(jié)果將幫助研究人員更清楚地了解乙肝病毒在細胞中如何復(fù)制,，并為乙肝新療法的研究開辟新路,。
據(jù)統(tǒng)計,，全世界約有3.5億人被乙肝病毒所感染,，而且每年因急性和慢性感染以及肝細胞癌死亡的人數(shù)高達100多萬人。這種攻擊肝臟的病毒通過輸入被病毒感染的血液,、進行靜脈注射的藥物濫用者共用針頭和性接觸等途徑傳播,。
在乙肝病毒中，病毒的遺傳物質(zhì)被蛋白質(zhì)外殼所保護,。乙肝病毒顆粒即Dane顆粒的尺寸大約為40納米,，而且蛋白質(zhì)外殼還被一層膜包被。雖然乙肝病毒蛋白外殼在離體狀況下已經(jīng)進行過大量的研究,，但是人們對活體的被感染細胞中存在的病毒外殼的結(jié)構(gòu)和裝配卻幾無所知,，甚至對成熟的病毒顆粒的結(jié)構(gòu)也是知之甚少。
在新的研究中,，Mark Yeager教授和Francis V. Chisari教授的領(lǐng)導(dǎo)的兩個實驗室使用低溫電子顯微鏡和圖像分析方法檢測了轉(zhuǎn)基因小鼠的乙肝病毒蛋白外殼和人類患者血樣中分離的病毒顆粒的天然結(jié)構(gòu),。通過迅速冷凍樣本，研究人員能夠利用低溫電子顯微鏡成像這種病毒顆粒,。成像過程獲得了三維圖像,，該圖像首次揭示出蛋白質(zhì)殼如何與外層液體膜封套反應(yīng)。
三維圖像在分子水平上顯示出乙肝病毒比血紅蛋白分子大將近10倍,。與人類的基因組類似,，乙肝病毒的基因組也是由雙鏈DNA構(gòu)成，并被蛋白質(zhì)殼包被,。這個蛋白質(zhì)殼是二十面體對稱,，類似多面穹頂幾何結(jié)構(gòu)。蛋白質(zhì)殼本身被由液體雙分子層形成的外膜包圍住——就像圍住人類細胞的細胞膜一樣,。乙肝病毒的這種膜鑲嵌著許多糖蛋白長刺,，這種長刺能與肝臟細胞上的受體結(jié)合，進而感染細胞,。
在被乙肝病毒感染的肝細胞中,，病毒DNA的轉(zhuǎn)錄過程產(chǎn)生一種類型的RNA——它被包裹進蛋白質(zhì)外殼中。在這種蛋白質(zhì)殼中,，逆轉(zhuǎn)錄過程制作出乙肝單鏈DNA拷貝,，它充當(dāng)?shù)诙lDNA鏈合成所需的模板。最終產(chǎn)生的顆粒通過細胞的內(nèi)質(zhì)網(wǎng)膜萌芽出來,，并被運送到病毒的外膜封套,。
在轉(zhuǎn)基因小鼠中，研究人員發(fā)現(xiàn)了兩種不同密度的病毒蛋白質(zhì)殼,。雖然兩種類型的蛋白質(zhì)殼都是二十面體結(jié)構(gòu),，但較低密度的蛋白質(zhì)殼含有病毒DNA的中間體。這些低密度的蛋白質(zhì)殼可能由細胞核釋放,。這些發(fā)現(xiàn)為人們了解活體狀態(tài)下的乙肝病毒復(fù)制提供新的線索,。
原始出處：
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
[Summary] [Full Text] [PDF] [Supplemental Data]
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.
關(guān)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.
