THE EFFECTS OF DOMAIN SWAPPING IN HIV-1 CAPSID PROTEIN

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 HIV-1的组装是一个多步骤的过程，其中几个单独的结构蛋白经历了实质性的形态学重排。衣壳（CA）蛋白在这一组装中起着至关重要但知之甚少的作用。 CA含有两个独立折叠的结构域，N-末端（NTD）和C-末端（CTD）结构域，通过柔性接头连接。 已显示HIV-1的CTD在成熟衣壳蛋白和分离的结构域中在溶液中二聚化（Gamble等，1997）。 成熟病毒粒子的当前结构模型表明，成熟核心的基本结构单元归因于通过CTD二聚化连接在一起的NTD的自缔合形成的CA六聚体（Gamble et al. 1997）。 此外，CTD二聚化已被证明是这种六聚体形成所必需的（Lanman et al. 2003）。 HIV的CTD包含主要同源区（MHR），这是一个在不同属的逆转录病毒中高度保守的20个氨基酸序列（Wills and Craven 1991）。 有充分证据表明，病毒组装对MHR突变高度敏感，但从结构角度来看，尚不清楚为什么MHR突变如此有害（凯恩斯和Craven 2001）。最近的研究表明，CA的CTD和二聚锌指相关结构域SCAN之间存在结构同源性（Ivanov等，2005）。 然而，与CA的CTD相比，SCAN结构域二聚体是结构域交换的。在结构域交换的二聚体中，来自一个亚基的结构元件与来自另一个亚基的相应结构元件交换（Liu和Eisenberg 2002）。 SCAN二聚体的结构域交换区域对应于CTD的MHR。结构域交换的二聚体将提供MHR的重要性的解释，因为在该区域中的氢键将发生在二聚体界面上。为了确定当C-末端结构域被结构域交换时CA是否可以组装，工程化融合构建体，其中HIV-1CA的C-末端结构域被SCAN结构域替换。 将使用H/D交换和快速光化学氧化来研究这些相互作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The assembly of HIV-1 is a multi-step process in which several individual structural proteins undergo a substantial morphological rearrangement. The capsid (CA) protein plays a crucial but poorly understood role in this assembly. CA contains two-independently folded domains, the N-terminal (NTD) and C-terminal (CTD) domains, connected by a flexible linker. The CTD of HIV-1 has been shown to dimerize in solution both in the mature capsid protein and in the isolated domain (Gamble et al. 1997). The current structural model of the mature virion indicates the fundamental building block of the mature core is attributed to a CA hexamer formed by the self-association of the NTD tied together by dimerization of the CTD (Gamble et al. 1997). Additionally, CTD dimerization has been shown to be required for this hexamer formation (Lanman et al. 2003). The CTD of HIV contains the major homology region (MHR), a sequence of 20 amino acids that is highly conserved across different genera of retroviruses (Wills and Craven 1991). It is well documented that viral assembly is highly sensitive to MHR mutations but from a structural perspective it is not clear why MHR mutations are so deleterious (Cairns and Craven 2001). Recent studies have shown that a structural homology exists between the CTD of CA and the dimeric zinc finger associated domain SCAN (Ivanov et al. 2005). However, the SCAN domain dimer is domain swapped in comparison to the CTD of CA. In a domain swapped dimer, a structural element from one subunit is exchanged with the corresponding structural element from another subunit (Liu and Eisenberg 2002). The domain swapped region of the SCAN dimer corresponds to the MHR of the CTD. A domain swapped dimer would provide an explanation for the importance of the MHR because hydrogen bonding in this region would occur across the dimer interface. To determine whether CA can assemble when the C-terminal domain is domain swapped a fusion construct was engineered where the C-terminal domain of HIV-1 CA was replaced with the SCAN domain. These interactions will be studied using H/D exchange and fast photochemical oxidation.