The goal of this study is to understand the role of the HIV-1 protein Vpu in the regulation of the cytokine receptor CD40. Our previous studies demonstrated that HIV-1-infected endothelial cells (EC) support the firm attachment of non-Hodgkin's lymphoma (NHL) B cells. This mechanism was traced to viral enhancement of CD40, allowing for CD40-dependent induction of VCAM-1. In vivo, HIV-infected EC may thus contribute to the characteristic extranodal presentation of the AIDS-NHL. The HIV-1 accessory protein Vpu was found to be responsible for CD40 induction via a post-transcriptional pathway. While Vpu alters the expression of CD4 and certain other cellular proteins, the full scope of its influence has yet to be determined. Vpu-induction of immune regulatory proteins could have severe implications for AIDS-related conditions and is thus deserving of further study. We hypothesize that Vpu intersects with normal CD40 regulation pathways to increase the levels of functional CD40 receptor at the cell surface. Surprisingly little is known about the normal turnover pathways of CD40 in cells, regardless of the influence of a viral modulator. By studying Vpu-modulation of CD40 in CD4-negative as well as CD4-positive cells, we aim to i) clearly define normal CD40 regulation pathways, ii) clarify the role of Vpu in leukocyte and non-leukocyte targets, and iii) appreciate the clinical significance of these findings.
In Aim 1 we will use biochemical and imaging techniques to compare the biosynthetic and turnover pathways of CD40 in EC in the presence and absence of Vpu.
In Aim 2 we will use a series of Vpu mutant proteins to determine whether known structure-function motifs play a role in CD40 regulation, or if unique motifs and mechanisms are involved.
In Aim 3 we will extend these studies to include macrophages, a major CD4+/CD40+ HIV-1 target. Successful completion of these Aims will lead to an increased understanding of both CD40 regulation and Vpu function, and may identify targets of immune modulatory and/or anti-viral significance. ? ?
