CELLULAR INTERACTIONS OF HIV-2 VPX

描述：(申请人提供)复杂逆转录病毒的辅助蛋白通常具有多种功能，有助于体内致病。人类免疫缺陷病毒2型(HIV-2)的VPX蛋白是病毒前整合复合体核输入的必要条件和充分条件，最近被证明结合并促进MHC II类相关蛋白不变链(II)的降解。本提案的目的是描述VPX的这两个不同的功能。本研究的第一个目的是确定VPX的核定位信号(NLS)，并确定VPX进入细胞核的途径。NLS将通过核定位研究进行鉴定，然后在酵母双杂交筛选中用作“诱饵”，以鉴定对VPX核进口重要的蛋白质。随后将通过一系列检测确认阳性克隆是否与VPX相互作用。第二个目的是确定VPX如何促进II类的降解，并描述VPX对II类途径的影响。将在感染VPX(+)和VPX(-)病毒的细胞中分析包括II在内的MHC II类途径成分。将建立VPX诱导的细胞系，并用于剖析VPX促进II的降解和影响MHC I类途径的机制。这些研究将进一步了解VPX在HIV-2复制和发病机制中的作用，并有助于开发HIV抑制疗法。

DESCRIPTION: (provided by applicant) The accessory proteins of complex retroviruses typically have multiple functions that contribute to pathogenesis in vivo. The Vpx protein of human immunodeficiency virus type 2 (HIV-2) is known to be necessary and sufficient for nuclear import of the viral pre-integration complex and has been recently shown to bind and enhance degradation of the MHC class II associated protein, invariant chain (Ii). The goal of this proposal is to characterize these two distinct functions of Vpx. The first aim of this study is to identify the nuclear localization signal (NLS) of Vpx and define the pathway through which Vpx accesses the nucleus. The NLS will be identified by nuclear localization studies and then used as "bait" in a yeast two-hybrid screen to identify proteins important for Vpx nuclear import. Positive clones will be subsequently confirmed for Vpx-interaction by a battery of assays. The second aim is to determine how Vpx enhances Ii degradation and delineate the effects of Vpx on the class II pathway. MHC class II pathway components, including Ii, will be analyzed in cells infected with Vpx(+) and Vpx(-) viruses. A Vpx-inducible cell line will be developed and used to dissect the mechanisms by which Vpx enhances degradation of Ii and affects the MHC class I pathway. These studies will further the understanding of the role of Vpx in HIV-2 replication and pathogenesis and contribute to the development of HIV inhibitory therapies.