Date of Award

5-1-2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Biomedical Sciences

Department

Graduate School of Biomedical Sciences

First Advisor

Johnny J. He

Second Advisor

Anuja Ghorpade

Third Advisor

Robert J. Wordinger

Abstract

Acquired immune deficiency syndrome (AIDS) is a pandemic caused by human immunodeficiency virus type 1 (HIV-1). It is a major health issue in many parts of the world ever since its discovery in 1981. The most devastating effect of HIV-1 infection is the graduate loss of CD4+ T cells, which eventually leads to the dysfunction of the immune system, susceptibility to opportunistic infections and cancer. HIV-1 Nef protein is long known as an essential pathogenic factor for HIV-1/AIDS pathogenesis. A few recent studies including ours have demonstrated that Nef can be transferred to neighboring cells and alters the function of these cells. However, the underlying mechanism of intercellular Nef transfer is in dispute. In the first part of our study, we characterized two potential underlying mechanisms for intercellular Nef transfer: direct cell-cell contact and exosomes using several complementary strategies and a panel of exosomal markers. First, we showed that Nef was transferred from Nef-expressing or HIV-infected CD4+ T lymphocytes to CD4+ T lymphocytes and astrocytes, and that the transfer was mainly associated with tunneling nanotube formation. Then we determined that Nef enhanced virological synapse formation and induced cytoskeleton re-arrangement and cell surface protrusions, suggesting that Nef promotes the establishment of intercellular connection and communication between infected cells and uninfected cells. Thirdly, we examined the possibility of Nef transfer through exosomes. In the exosome uptake assay, Nef transfer was undetectable while exosome marker CD81 transferred rapidly. In contrast, Nef was detected in crude exosomes collected from Nef-transfected 293T. In addition, two different populations of exosomes were successfully separated by OptiPrep gradient fractionation and determined as AChE+/CD81low/TSG101low exosomes and AChE- /CD81high/TSG101high exosomes. We determined that Nef was selectively secreted into the AChE+/CD81low/TSG101low population. Lastly, microscopic imaging showed no significant Nef detection in exosomal vesicle-like structures in and out the cell. Taken together, this study shows that Nef transfer requires direct cell-cell contact such as tunneling nanotubes, not cell-free exosomes. In addition, this study reveals existence of two types of exosomes: AChE+/CD81low/TSG101low exosomes and AChE/CD81high/TSG101high exosomes. In the second part, we characterized HIV-1 infection of astrocytes. Astrocytes are the most abundant cells in the central nervous system (CNS) and play important roles in HIV-1/neuroAIDS. Detection of HIV-1 proviral DNA, RNA and early gene products but not late structural gene products in astrocytes in vivo and in vitro indicates that astrocytes are susceptible to HIV-1 infection albeit in a restricted manner. We, as well as others have shown that cell-free HIV-1 is capable of entering CD4- astrocytes through human mannose receptor-mediated endocytosis. In this study, we took advantage of several newly developed fluorescence protein-based HIV-1 reporter viruses and further characterized HIV-1 interaction with astrocytes. First, we found that HIV-1 was successfully transferred to astrocytes from HIV-infected CD4+ T cells in a cell-cell contact- and gp120-dependent manner. In addition, we demonstrated that compared to endocytosis-mediated cell-free HIV-1 entry and subsequent degradation of endocytosed virions, cell-cell contact between astrocytes and HIV-infected CD4+ T cells led to robust HIV-1 infection of astrocytes but retained the restricted nature of viral gene expression. Furthermore, we showed that HIV-1 latency was established in astrocytes. Lastly, we demonstrated that infectious progeny HIV-1 was readily recovered from latently infected astrocytes in a cell-cell contact-mediated manner. Taken together, our studies point to the importance of the cell-cell contact-mediated HIV-1 interaction with astrocytes and provide direct evidence to support the notion that astrocytes are HIV-1 latent reservoirs in the CNS.