The role of redox-dependent CD4 isomerization and membrane re-distribution in HIV-1 infection

Abstract

CD4, a key molecule of the immune system, is expressed on the surface of certain T lymphocytes (T cells) and participates in MHC class II driven lymphocyte activation. It is also the essential primary receptor for Human Immunodeficiency Virus (HIV) cell entry. Reactive oxygen species (ROS) and other redox-active molecules are important components of the immunological response. They initiate cytocidal responses of the pathogen defence scheme, and redox-activated signalling events ensure appropriate induction of adaptive immunological responses. A redox imbalance can result in failure of essential regulatory mechanisms and the development of pathological immune conditions. An increasing amount of evidence suggests that redox active enzymes such as thioredoxin (Trx) are implicated in CD4 immunological function and in HIV entry at the cell surface, and the dynamic localization of CD4 in specific plasma membrane microdomains, like detergent resistant membrane microdomains (DRM) or lipid rafts, has been shown to play a key role in these regards. However, the biological utility of both the microdomain distribution and the disulphide reduction of CD4, together with the interplay between these processes and the role of cellular oxidoreductases therein remain poorly understood. In this study, we investigated a cell surface-based Trx redox system, and asked whether a relationship exists between these two fundamental aspects of CD4 function by analysing how manipulating cell surface redox conditions affects CD4 membrane domain localization and HIV entry into host CD4-positive (CD4 +) cells. Our investigation of the role of a cell surface redox system in regulating CD4 function was prefaced by research into the membrane association of a variant of Thioredoxin reductase 1 (TrxR1), the enzyme responsible for reducing (and thereby recharging) the active site cysteines of Trx. These studies, carried out in the laboratory of Prof. E Arner (Medical Biochemistry and Biophysics, Karolinska Institutet), were the first to show that a TrxR1 variant called TXNRD1_v3 (henceforth v3) is targeted to DRM domains via N-terminal acylation. Although the role(s) of v3 in this context remains poorly understood, the evidence suggesting that TrxR can associate with the plasma membrane under certain circumstances alludes to the importance of redox capacity at the cell surface, which increasingly suggests it is essential for the function of CD4. To this end, using a transgenic cell line that has been extensively used to model HIV entry and various HIV pseudoviruses, we then analysed the effects of manipulating cell surface redox conditions on CD4 membrane domain distribution and HIV entry. Our results showed that under normal cell growth conditions, the majority of CD4 is associated with detergent soluble regions of the plasma membrane (non-raft regions). Intriguingly, we found that the inhibition of cellular oxidoreductases, and specifically Trx1, results in a redistribution of CD4 into DRMs. CD4 DRM redistribution appears to be targeted, as other cell surface molecules (such as the HIV co-receptor, CCR5) remain unaffected. Furthermore, the redistribution of CD4 to the DRM’s correlates with reduced CD4-dependent HIV infection. Overall, these findings provide evidence for the presence of cell surface-acting redox systems and demonstrate how redox exchanges influence CD4 localization and function. In the context of HIV, our data support previous findings that the thioredoxin system plays an important role in regulating viral entry, which may be related to uncoupled trafficking of CD4 and the HIV co-receptor. Trx-mediated regulation of CD4 membrane domain trafficking may represent a redox switch for functional CD4 clustering during T cell activation.