HIV Vpr Targets PU.1 in Macrophages to Evade the Innate Immune Response and Promote Viral Spread

Abstract

Human immunodeficiency virus (HIV-1) establishes a persistent infection despite the host immune response and treatment with antiretroviral therapy. HIV evades the immune response primarily due to the activity of several accessory proteins. One of these accessory proteins is Viral protein R (Vpr). The importance of Vpr for successful infection is reflected in its universal conservation amongst all primate lentiviruses, including HIV-1. Several studies have demonstrated Vpr is paradoxically unnecessary for viral spread between CD4+ T lymphocytes, the main cellular target of HIV. However, it remains necessary for maximal spread between macrophages and from macrophages to CD4+ T lymphocytes in both in vitro and in vivo systems. Vpr suppresses intrinsic and innate immune responses through mechanisms not well understood. Efficient viral spread from infected macrophages to uninfected cells is achieved by virological synapse formation when HIV Env on the surface of the infected cell binds HIV receptors on the surface of the uninfected cell, forming a channel through which HIV can be directly passed. Therefore, efficient Env production is vital for maximal viral spread. Previous studies determined that Vpr counteracts a macrophage-specific restriction factor, which we demonstrate in Chapter 2 to be the macrophage mannose receptor (MR). MR augments virion entry by binding mannose residues on HIV Env, likely stabilizing the virion at the cell membrane, but restricts HIV infection upon egress. After infection, MR restricts viral spread by meditating the transfer of Env and Env containing virions to the lysosome for degradation. Two HIV accessory proteins target MR. Nef interacts with the cytoplasmic tail of MR at the cell surface, facilitating lysosomal degradation of MR. And Vpr counteracts the detrimental effects of MR on Env by reducing transcription of the MR gene, MRC1. In Chapter 3, we demonstrate that transcriptional suppression of MRC1 is caused by Vpr-mediated degradation of the master myeloid transcription factor, PU.1. PU.1 acts alone and in coordination with other transcription factors to regulate the expression of MR and many additional antiviral factors. PU.1 regulated factors are intimately involved in pathogen sensing and immune signaling through Toll-like receptors, Type I interferons, interferon-stimulated genes, and other host restriction factors capable of targeting HIV Env for degradation such as IFITM3. The Vpr-mediated degradation of PU.1 requires Vpr’s cellular co-factor, DCAF1 in the Cul4A-E3 ubiquitin ligase complex and results in degradation of PU.1 which can be prevented with pharmacological inhibition of the proteasome. Like Vpr itself, Vpr’s ability to counteract PU.1 is highly conserved across several HIV-1, HIV-2, and SIV vpr alleles. The innate immune response is suppressed both in infected macrophages and uninfected bystander cells from vpr-expressing HIV cultures. Virion-associated Vpr can rapidly degrade PU.1 after virion fusion, which partially explains the bystander effect. Knockdown of PU.1 in infected macrophages with HIV lacking Vpr restored Env expression and viral spread. Altogether, Vpr counteracts PU.1 in macrophages, suppressing the immune response during HIV infection and promoting viral spread.