Packaging DNA into th Herpes Simplex Virus Capsid

将 DNA 包装到单纯疱疹病毒衣壳中

• 批准号: 9904879
• 负责人: Jay Brown
• 金额: $ 36.75万
• 依托单位: University of Virginia Main Campus
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904879&HistoricalAwards=false
• 关键词: Packaging; DNA; into; th; Herpes

BrownMCB 9904879Technical Like all herpesviruses, herpes simplex virus 1 (HSV-1) consists of an icosahedral capsid surrounded by a membrane envelope. The virus DNA is contained inside the capsid. Infection with HSV-1 begins when the virus membrane binds to the cytoplasmic membrane of a host cell and fuses with it. Fusion results in introduction of the DNA-containing capsid (the nucleocapsid) into the cytoplasm where it migrates to the nucleus and injects its DNA through a nuclear pore. Virus DNA is then replicated and capsids are assembled in the nucleus. Further virus maturation includes packaging of DNA into a capsid which is enveloped at the inner nuclear membrane before exiting the host cell, a process that may proceed by de-envelopment at the outer nuclear membrane followed by re-envelopment in the cytoplasm. Packaging of HSV-1 DNA into a capsid is required for virus growth. The process involves participation of capsids, concatemeric DNA and the products of eight virus genes, UL6, UL15, UL17, UL21, UL25, UL28, UL32 and UL33. In cells infected with mutants (e.g. ts or null mutants) in any of the eight genes, capsids and HSV-1 DNA accumulate, but no encapsidation is observed. Previous studies have suggested potential roles for some of the eight processing/ packaging proteins. For example, the UL15 and UL28 gene products have been proposed to bind to DNA pac sites and translocate DNA into the capsid in a fuction analogous to that of the large subunit of bacteriophage terminase. The UL6 protein, a minor component of the capsid, may function like the bacteriophage portal or connector protein to mediate attachment of capsids to the terminase homolog. pUL25 may serve as a stopper or cap to keep DNA inside the capsid once it has entered.The goal of this research is to use ultrastructural and biochemical methods to clarify how HSV-1 DNA is encapsidated and how the processing/packaging proteins participate in encapsidation events. Ultrastructural studies will involve electron microscopic examination of HSV-1-infected cells after specific immuno-gold staining for capsid and processing/packaging proteins. It is expected that high resolution (~10 nm) information about the location of a protein in the infected cell nucleus will provide clues about its function. For instance, if pUL15 is involved in DNA translocation into the capsid, then it ought to be found near the site where DNA enters the capsid shell. Similarly, the cap protein should be localized to one particular site on the capsid shell after DNA packaging is complete, but possibly not before. Biochemical studies will be carried out to develop an in vitro system for HSV-1 DNA packaging. Such a system would facilitate more detailed analysis of the encapsidation process by providing the opportunity to manipulate the conditions of packaging outside an infected cell. Efforts to develop an in vitro packaging system will involve lysing infected cells, incubating the lysates and testing for evidence of DNA encapsidation during the incubation period. Evidence for packaging in crude cell lysates will be established before more purified systems are examined. Non-technical In herpes simplex virus 1 (HSV-1), viral DNA is contained inside an icosahedral capsid which in turn is surrounded by a membrane envelope. Infection involves the fusion of viral and host cell cytoplasmic membranes and the introduction of DNA-containing capsid into the cytoplasm. The viral DNA migrates into the nucleus by injection through a nuclear pore. Viral DNA replication and assemblage of the virus take place within the nucleus. A further step for the maturation of the virus is packaging of the newly replicated DNA into viral capsid, which is then enveloped at the inner nuclear membrane before exiting the host cell. This step may proceed by de-envelopment at the outer nuclear membrane followed by re-envelopment in the cytoplasm. A total of eight proteins coded by the virus genome have been identified to involve in these various processes. The goal of this study is to clarify specially how HSV-1 DNA is encapsidated and how some, if not all, of these proteins participate in encapsidation events. High resolution (~10 nm) electron microscopy with specific immuno-gold staining will be used to track these proteins in cells upon infection. This information is expected to provide clues about their function. Biochemical studies will also be carried out to develop an HSV-1 DNA packaging system using reconstituted cell extracts. Such a system would facilitate more detailed analysis of the encapsidation process by providing the opportunity to manipulate the conditions of packaging outside an infected cell.

BrownMCB 9904879技术与所有疱疹病毒一样，单纯疱疹病毒1型（HSV-1）由二十面体衣壳和包膜组成。病毒DNA包含在衣壳内。当病毒膜与宿主细胞的细胞质膜结合并融合时，HSV-1感染开始。融合导致将含有DNA的衣壳（核衣壳）引入细胞质，在那里它迁移到细胞核并通过核孔注入其DNA。然后病毒DNA被复制，衣壳在细胞核中组装。进一步的病毒成熟包括将DNA包装到衣壳中，该衣壳在离开宿主细胞之前在内核膜处被包膜，该过程可以通过在外核膜处脱膜然后在细胞质中再结晶来进行。病毒生长需要将HSV-1 DNA包装到衣壳中。该过程涉及衣壳、多联体DNA和八种病毒基因UL 6、UL 15、UL 17、UL 21、UL 25、UL 28、UL 32和UL 33的产物的参与。在感染了8个基因中任何一个的突变体（例如ts或无效突变体）的细胞中，衣壳和HSV-1 DNA积累，但未观察到衣壳化。先前的研究已经表明了八种加工/包装蛋白中的一些的潜在作用。例如，已经提出UL 15和UL 28基因产物结合到DNA pac位点，并以类似于噬菌体末端酶大亚基的功能将DNA移位到衣壳中。UL 6蛋白是衣壳的次要组分，其功能可能与噬菌体门户或连接蛋白类似，以介导衣壳与末端酶同源物的附着。本研究的目的是利用超微结构和生物化学方法阐明HSV-1DNA是如何被衣壳化的，以及加工/包装蛋白是如何参与衣壳化的。超微结构研究将包括对衣壳和加工/包装蛋白进行特异性免疫金染色后对HSV-1感染细胞进行电子显微镜检查。预计有关蛋白质在感染细胞核中位置的高分辨率（~10 nm）信息将提供有关其功能的线索。例如，如果pUL 15参与DNA易位到衣壳中，那么它应该在DNA进入衣壳壳的位点附近被发现。类似地，在DNA包装完成后，帽蛋白应该定位于衣壳壳上的一个特定位点，但可能不在此之前。将进行生物化学研究以开发用于HSV-1 DNA包装的体外系统。这样的系统将通过提供操纵感染细胞外包装条件的机会来促进更详细地分析腺苷酸化过程。开发体外包装系统的努力将涉及裂解感染的细胞，孵育裂解物并测试孵育期间DNA降解的证据。在检测更纯化的系统之前，将建立在粗细胞裂解物中包装的证据。单纯疱疹病毒1型（HSV-1）的病毒DNA被包含在二十面体衣壳内，二十面体衣壳又被膜包膜包围。感染涉及病毒和宿主细胞质膜的融合以及将含DNA的衣壳引入细胞质中。 病毒DNA通过核孔注射进入细胞核。病毒DNA的复制和病毒的组装发生在细胞核内。病毒成熟的另一个步骤是将新复制的DNA包装到病毒衣壳中，然后在离开宿主细胞之前将病毒衣壳包裹在内核膜处。这一步骤可能是通过外核膜的去核，然后在细胞质中重新核化来进行的。已经鉴定出由病毒基因组编码的总共八种蛋白质参与这些不同的过程。本研究的目的是特别澄清HSV-1 DNA是如何被糖苷化的，以及这些蛋白质中的一些（如果不是全部）是如何参与糖苷化事件的。将使用具有特异性免疫金染色的高分辨率（~10 nm）电子显微镜来追踪感染后细胞中的这些蛋白质。这些信息有望提供有关其功能的线索。 还将进行生物化学研究，以开发使用重组细胞提取物的HSV-1 DNA包装系统。这样的系统将通过提供操纵感染细胞外包装条件的机会来促进更详细地分析腺苷酸化过程。