Packaging DNA into th Herpes Simplex Virus Capsid

像所有疱疹病毒一样，单纯疱疹病毒1 （HSV-1）由一个被膜包膜包围的二十面体衣壳组成。病毒DNA包含在衣壳内。1型单纯疱疹病毒的感染开始于病毒膜与宿主细胞的细胞质膜结合并融合。融合导致含有DNA的衣壳（核衣壳）进入细胞质，在那里它迁移到细胞核，并通过核孔注入DNA。病毒DNA随后被复制，衣壳在细胞核中组装。进一步的病毒成熟包括在离开宿主细胞之前将DNA包装到被包裹在内层核膜上的衣壳中，这一过程可能先在外核膜上脱包膜，然后在细胞质上再被包膜。将HSV-1 DNA包装到衣壳中是病毒生长所必需的。这一过程涉及衣壳、串联DNA和8个病毒基因UL6、UL15、UL17、UL21、UL25、UL28、UL32和UL33的产物。在被8个基因中的任何一个突变体（如ts或零突变体）感染的细胞中，衣壳和HSV-1 DNA积累，但未观察到衣壳化。先前的研究已经提出了这八种加工/包装蛋白中的一些的潜在作用。例如，UL15和UL28基因产物已被提出与DNA pac位点结合并将DNA转运到衣壳中，其功能类似于噬菌体末端酶的大亚基。UL6蛋白是衣壳的一个次要成分，其功能类似于噬菌体入口蛋白或连接蛋白，介导衣壳与末端酶同源物的附着。一旦DNA进入衣壳，pUL25可以作为一个塞子或盖子将其保持在衣壳内。本研究的目的是利用超微结构和生化方法阐明HSV-1 DNA是如何被封装的，以及加工/包装蛋白是如何参与封装事件的。超微结构研究将包括对hsv -1感染细胞进行衣壳和加工/包装蛋白特异性免疫金染色后的电子显微镜检查。预计高分辨率（~ 10nm）的蛋白质在感染细胞核中的位置信息将为其功能提供线索。例如，如果pUL15参与了DNA进入衣壳的易位，那么它应该在DNA进入衣壳壳的位置附近找到。类似地，在DNA包装完成后，帽蛋白应该定位在衣壳壳上的一个特定位置，但可能不是在此之前。将进行生化研究，以建立HSV-1 DNA包装的体外系统。这种系统将通过提供操纵感染细胞外包装条件的机会，促进对封装过程进行更详细的分析。开发体外包装系统的努力将包括裂解感染细胞，孵育裂解物和在孵育期间检测DNA封装的证据。在检查更多纯化系统之前，将建立在粗细胞裂解物中包装的证据。在单纯疱疹病毒1 （HSV-1）中，病毒DNA被包含在二十面体衣壳内，而衣壳又被膜包膜包围。感染涉及到病毒和宿主细胞质膜的融合，并将含有dna的衣壳引入细胞质。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。病毒成熟的进一步步骤是将新复制的DNA包装到病毒衣壳中，然后在离开宿主细胞之前将其包裹在内膜上。这一步骤可以先在外核膜上脱包膜，然后在细胞质上重新包膜。共有8种由病毒基因组编码的蛋白质被确定参与这些不同的过程。本研究的目的是特别澄清HSV-1 DNA是如何被封装的，以及这些蛋白质中的一些（如果不是全部的话）是如何参与封装事件的。高分辨率（~10 nm）电子显微镜与特异性免疫金染色将用于跟踪这些蛋白质在细胞感染。这些信息有望为它们的功能提供线索。还将进行生化研究，以开发使用重组细胞提取物的HSV-1 DNA包装系统。这种系统将通过提供操纵感染细胞外包装条件的机会，促进对封装过程进行更详细的分析。

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.