The role of Vpu and tetherin in HIV/AIDS pathogenesis

Human Immunodeficiency Virus type 1 (HIV-1) is the cause of the worldwide AIDS epidemic. In the absence of an effective vaccine, understanding the mechanisms by which the virus replicates has provided several targets for the design of potent antiviral drugs. HIV-1 encodes a number of accessory genes whose function is to subvert host defence systems that have evolved to combat viral infections. We have shown that one such accessory protein, Vpu, is required by the virus to overcome the action of a human protein known as tetherin or CD317. Tetherin levels in cells are activated by viral infection. HIV-1 viruses that lack a Vpu gene are blocked in their replication because tetherin prevents new virus particles leaving the cell, thereby inhibiting the spread of the virus. However, the importance of Vpu and tetherin in the pathogenesis of HIV-1 disease in infected individuals has not been studied.
This grant proposal aims to study the role of Vpu and tetherin in patients by using samples stored in the King?s College Infectious Disease BioBank. We will isolate Vpu genes from the blood of HIV positive individuals with different rates of disease progression and examine their ability to counteract tetherin, to see if variation in Vpu function is associated with clinical outcomes. We will also examine the regulation of tetherin expression in various primary cells that HIV-1 targets, and if genetic variations in and around the tetherin gene can affect these expression levels. Finally, we will use functional Vpu variants isolated from patients as tools to identify cellular proteins required for its ability to overcome tetherin. These studies will achieve the following objectives: (1) To determine whether Vpu and tetherin functions affect the susceptibility of patients to faster rates of disease progression; (2) Identification of mutant Vpu genes that encode non-functional proteins will allow us understand the basis for its anti-tetherin function; (3) Finally, determining an in vivo role of Vpu and tetherin in HIV/AIDS pathogenesis will provide compelling rationale to develop strategies to target Vpu function for new antiviral drug design.

Human Immunodeficiency Virus type 1 (HIV-1), the aetiological agent of pandemic AIDS, encodes a number of accessory genes whose protein products allow the virus to avoid effectors of the innate and adaptive immune response. Understanding the importance of these factors in vivo will highlight whether these accessory proteins will be viable targets for novel antiretroviral therapeutics. The Vpu protein of HIV-1 has recently been shown to overcome the actions of an interferon-induced membrane protein tetherin (CD317) that can be expressed on many HIV-1 primary target cells. In the absence of Vpu, HIV-1 replication is potently inhibited in tetherin expressing cells because new viral particles fail to be released from the plasma membrane. The purpose of this grant is to examine Vpu and tetherin function in HIV-1-infected patients. Using patient material stored in the KCL Infectious Disease BioBank, we will clone Vpu alleles from HIV-1 positive patients defined as rapid progressors and long-term non-progressors/controllers, as well as longitudinal samples from patients from acute to chronic HIV infection. We will assess the function of these Vpu alleles for their ability to antagonize tetherin in vitro, identify the molecular determinants that confer functional variation in Vpu, and assess whether variation in Vpu is associated with any HIV-1 pathogenic outcomes. We will also determine tetherin expression levels on primary HIV-1 target cells, the regulation of these expression levels by proinflammatory stimuli, and the effects of single-nucleotide polymorphisms in the tetherin promoter on its transcriptional regulation. In addition, we will develop methods to isolate tetherin and Vpu interacting factors from host cells in an effort to identify cellular proteins required for their respective functions. These studies will provide in vivo evidence of the importance of Vpu/tetherin interactions in HIV-1 pathogenesis, their suitability as markers for clinical HIV-1 patient management, and rationale for the design of drugs to disrupt Vpu function.