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. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI063938-04
Application #
7318340
Study Section
AIDS Molecular and Cellular Biology Study Section (AMCB)
Program Officer
Sharma, Opendra K
Project Start
2004-11-15
Project End
2009-10-31
Budget Start
2007-11-01
Budget End
2008-10-31
Support Year
4
Fiscal Year
2008
Total Cost
$316,025
Indirect Cost
Name
Oregon Health and Science University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Douglas, Janet L; Bai, Ying; Gustin, Jean K et al. (2013) A comparative mutational analysis of HIV-1 Vpu subtypes B and C for the identification of determinants required to counteract BST-2/Tetherin and enhance viral egress. Virology 441:182-96
Gustin, Jean K; Douglas, Janet L; Bai, Ying et al. (2012) Ubiquitination of BST-2 protein by HIV-1 Vpu protein does not require lysine, serine, or threonine residues within the BST-2 cytoplasmic domain. J Biol Chem 287:14837-50
Douglas, Janet L; Gustin, Jean K; Viswanathan, Kasinath et al. (2010) The great escape: viral strategies to counter BST-2/tetherin. PLoS Pathog 6:e1000913
Douglas, Janet L; Viswanathan, Kasinath; McCarroll, Matthew N et al. (2009) Vpu directs the degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism. J Virol 83:7931-47
Mansouri, Mandana; Viswanathan, Kasinath; Douglas, Janet L et al. (2009) Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated herpesvirus. J Virol 83:9672-81
Smith, J R; Henderson, W W; Rosenbaum, J T et al. (2008) Cultured human endothelial cells expressing HIV-1 Vpu and Tat support the expansion of malignant B cells from primary central nervous system lymphoma. Br J Ophthalmol 92:297-9