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原帖由bigben发表于 2008-9-10 02:45
转载自肝胆相照，有修改，原文附在3楼，谢谢Rojjer兄
http://bbs.hbvhbv.com/forum/viewthread.php?tid=733854
针对鸭乙肝病毒cccDNA设计的锌指结构蛋白抑制组织培养中的DHBV病毒复制
简要翻译：
鸭乙肝病毒DHBV是研究感染愈3.6亿人的HBV的模式病毒。核苷类似物能通过抑制病毒聚合酶的方式减少病毒的复制，因为HBV cccDNA的存在，这些药物基本不能完全清除HBV。HBV cccDNA是HBV基因组的共价闭环形式，能够作为复制和转录的模板。cccDNA不是核苷类似物的靶标，因而在停止抗病毒治疗后，HBV能借助cccDNA重新感染（非cccDNA的HBV形式可被完全清除）。为了清除cccDNA，我们设计了6种不同的能够结合DHBV cccDNA增强子区域的锌指结构蛋白。利用凝胶迁移滞后实验和表面等离子体共振技术对结合动力学进行了评估，选定了2种解离常数为12.3nM和40.2nM的候选蛋白作进一步的研究。锌指结构蛋白克隆进真核表达质粒和能够复制完整DHBV病毒的pDHBV1.3质粒共转染LMH细胞系（鸡肝癌细胞系）。在锌指蛋白存在的情况下，病毒的RNA，蛋白显著减少。进而，胞内的病毒颗粒也显著下降。综述，锌指结构蛋白能够和DHBV cccDNA的增强子区域结合，并干扰病毒的转录，导致病毒产物和子代病毒的下降。
Zinc finger proteins designed to specifically target duck hepatitis B virus cccDNA inhibit viral transcription in tissue culture
http://jvi.asm.org/cgi/content/abstract/JVI.00366-08v1
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton Alberta, Canada
Duck hepatitis B virus (DHBV) is a model virus for human hepatitis B virus (HBV), which infects approximately 360 million individuals worldwide. Nucleoside analogs can decrease virus production by inhibiting the viral polymerase; however, complete clearance by these drugs is not common because of the persistence of the HBV episome. HBV DNA is present in the nucleus as a covalently closed circular (cccDNA) form, where it drives viral transcription and progeny virus production. cccDNA is not the direct target of antiviral nucleoside analogs and is the source of HBV re-emergence when antiviral therapy is stopped. To target cccDNA, six different zinc finger proteins (ZFP) were designed to bind DNA sequences in the DHBV enhancer region. After assessing binding kinetics using electrophoretic mobility shift assays and surface plasmon resonance, two candidates with dissociation constants of 12.3nM and 40.2nM were focused on for further study. The ZFPs were cloned into a eukaryotic expression vector and co-transfected into LMH (longhorn male hepatoma) cells with the plasmid pDHBV1.3, which replicates the DHBV life cycle. In the presence of each ZFP, viral RNA was significantly reduced and protein levels were dramatically decreased. As a result, intracellular viral particle production was also significantly decreased. In summary, designed ZFPs are able to bind to the DHBV enhancer and interfere with viral transcription, resulting in decreased production of viral products and progeny virus genomes.
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关于这篇文章的一个新闻报道
http://www.ahfmr.ab.ca/researchn ... archersinthemaking/
Researchers in the making:
Clamping down on hepatitis B
An AHFMR Student looks for ways to disable a stubborn form of the dangerous liver virus.
Hepatitis B is the world's most prevalent serious liver infection. About 2 billion people around the world have been infected, generally through the transmission of blood or other body fluids. With the help of current treatments, most of those 2 billion will manage to clear the virus and recover. But around 10% will not; they will develop chronic infections that can lead to even more serious long-term illnesses, such as liver cancer. For infants and children, the percentage is much higher: 90% of infants and 50% of young children infected with hepatitis B will develop chronic infections.
These chronic infections are caused by a special form of the hepatitis B virus that develops in the liver: a virus consisting of a very stable type of DNA (called cccDNA) that is extremely difficult to attack directly with drugs or treatments. But AHFMR Student Kimberley Zimmerman may have found a way to do just that.
Zimmerman studies zinc finger proteins-so called because each protein is composed of a number of finger-like structures, with zinc ions in the middle to hold them together. Each zinc finger can recognize and attach to a specific type of DNA; the more zinc fingers, the more DNA that can be recognized. Zimmerman designs these proteins to attach to specific DNA combinations-namely, the cccDNA that is the culprit behind chronic hepatitis B infections.
"Because we know the DNA sequence of hep B virus, we can decide what sequence of zinc finger protein is needed to bind it," says Zimmerman. She explains that the concept is similar in principle to the Denver boot, a type of wheel clamp used by some police departments to immobilize illegally parked vehicles. When a Denver boot is placed on a wheel, the car can't go anywhere. "The idea is that these zinc finger proteins are the clamp, and that particular form of hep B virus in the liver is the tire."
Once she was convinced that the proteins were attaching well to their targets, Zimmerman tried the process in a model of a hepatitis B infection to see how it affected production of the virus. And sure enough, she found that the proteins travelled to the hepatitis B DNA and bonded to it strongly, preventing the DNA from reproducing the hepatitis B virus. The next step is to determine whether the zinc finger proteins can make the DNA break down and disappear.
In September, Zimmerman filed a patent for the application of zinc finger proteins as future treatments. "The proteins are the first therapeutic to target cccDNA," she says. "We've seen very good results so far with inhibition of the virus, and it's exciting to take the next step."
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发表于 2015-9-1 17:39:46
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