Human immunodeficiency virus type-1 (HIV) pathogenesis is associated with immune activation and chronic inflammation during active viral replication as well as during antiretroviral viral suppression. Even in cases where levels of replication competent virus are undetectable due to effective antiretroviral therapy, non-replicative virus is still produced with release of non-infectious viral RNA. This RNA is sensed by surveying innate immune cells, such as macrophages, which then induce pro-inflammatory cytokines such as interleukin-1 beta (IL-1β). Processing of IL-1β is associated with nod-like receptor protein-3 (NLRP3) inflammasome and caspase activation in many inflammatory diseases; however, the mechanism of IL-1β induction in human primary macrophages exposed to HIV RNA is unknown. To elucidate the mechanism of IL-1β activation by HIV RNA, we exposed human primary macrophages to RNA40 (a GU-rich ssRNA derived from the HIV long-terminal repeat region) and observed a significant induction of IL-1β. Processing of IL-1β in macrophages exposed to RNA40 required activation of the NLRP3 inflammasome independent of potassium efflux or reactive oxygen species production. RNA40 also triggered caspase-4 and -5 activation to mediate IL-1β release in a one-step event that occurs shortly after exposure to RNA40.

In total, these findings indicate that following exposure of human primary macrophages to GU-rich ssRNA from the HIV LTR region, the NLRP3 inflammasome is activated through a non-canonical pathway involving caspase-4 and caspase-5 to process IL-1β. This mechanism provides further insight into HIV pathogenesis, and suggests that targeting caspase-4 and caspase-5 could be useful in controlling HIV-associated chronic inflammation.

ABSTRACT OF THE THESIS

Trehalose, an mTOR-independent Inducer of Autophagy, Inhibits HIV Infection in Primary Human Macrophages

by

Simson Hon

Master of Science in Biology

University of California, San Diego, 2017

Professor Stephen Spector, Chair

Professor Li-Fan Lu, Co-Chair

While tremendous progress has been made in terms of prevention, detection, and treatment of human immunodeficiency virus type-1 (HIV), the agent that causes acquired immunodeficiency syndrome (AIDS), concerns remain as strains of HIV that are resistant to antiretroviral therapy (ART) have begun to emerge. In the face of this new threat, other methods must be considered in combination with medical treatment. Autophagy, a highly conserved pathway that enable cells to recycle cytoplasmic content to promote survival during periods of stress, has been receiving renewed attention for its role in neurodegenerative diseases and immune response to pathogen challenge. In fact, chemically induced autophagy has been shown to inhibit HIV replication in human primary macrophages. However, autophagy has been classically studied as a pathway regulated by the mammalian target of rapamycin complex 1 (mTORC1), which recently, has been shown to have other modulatory effects beyond autophagy.

As such, we determined if trehalose, small-molecule enhancer of rapamycin 28 (SMER28), and spermidine, which are compounds that have been shown to induce autophagy in an mTOR-independent fashion, are able to inhibit HIV replication in human primary macrophages. Here, I show that all three mTOR-independent inducers of autophagy have an inhibitory effect on HIV replication. Furthermore, I demonstrate that trehalose can induce autophagic flux in human primary macrophages. Yet, perhaps the most striking result was that trehalose downregulates CD4 and chemokine CC receptor 5 (CCR5) expression, both of which are key receptors for HIV entry. In support of this, I observed decreased HIV entry into human primary macrophages following trehalose treatment. Taken together, these results support further investigation into the beneficial effects that trehalose may have as part of standard HIV treatment.

Human immunodeficiency virus type-1 (HIV-1) is a global pandemic that has infected millions across the globe and is the precursor to acquired immunodeficiency syndrome (AIDS). Antiretroviral therapy (ART) allows HIV-1 infected individuals to live a full life; however, ART does not remove HIV-1 infected cells, allowing infected cells to become latent. These latent HIV-1 reservoirs cannot only reactivate once ART administration ends, but these latent HIV-1 reservoirs despite producing no replication competent virus can produce chronic inflammation in an individual leading to an assortment of other health conditions. A strategy to eradicate latent reservoirs is designed to reactivate HIV-1 from latently infected cells and kill the virus prior to it infecting uninfected bystander cells (often called “shock and kill”). Here, we identify Tat-Vpu peptide as a potential “shock” in the “shock and kill” cure strategy. We show that Tat-Vpu is able to increase HIV-1 viral release in infected macrophages through the LVEM66 amino acid sequence that is not observed in the tat-scramble. To analyze the inflammatory pathway induced by HIV-1, we used a GU-rich single stranded RNA (RNA40) derived from the long terminal repeat (LTR) region of HIV-1. We identified that TLR8 is critical in causing “bystander” CD4+ T cells to elicit IL-1β production. We also show a lack of cell death in RNA40 stimulated macrophages, and identify that caspase-8 and RIPK3 are involved in efficient IL-1β release suggesting the involvement of an alternative inflammasome. We also show that potassium efflux is essential in producing an inflammatory response in RNA40 stimulated macrophages. The involvement of TLR8, no pyroptosis, potassium efflux, and involvement of proteins in the alternative inflammasome indicate that a novel inflammatory pathway is involved with the induction of IL-1β by RNA40.

Antiretroviral therapy (ART) has ameliorated substantially human immunodeficiency virus type 1 (HIV-1) associated disease. Even so, persons living with HIV-1 (PLWH) on ART still exhibit chronic inflammation and various forms of HIV-1-associated disorders. The continued existence of HIV-1-associated disorders is thought to result from persistent inflammation present in PLWH despite sustained viral suppression and normalization of the CD4+ T cell count. Recent literature suggests that inflammasome activation is central to persistent inflammation with the NLRP3 inflammasome being the most studied. Upon formation of the NLRP3 inflammasome complex, the cytokines IL-1β and IL-18 are produced. PLWH have increased activation of the complex that results in excess cytokine production, causing excessive inflammation. This activation is thought to contribute to a broad range of associated diseases including the early onset of aging, cardiovascular disease, and cognitive disorders. In this review, the mechanisms associated with the activation of the NLRP3 inflammasome in HIV-1 infected cells as well as the role of NLRP3 in persistent inflammation and HIV-associated pathogenesis are discussed. A summary of the NLRP3 inflammasome in HIV pathogenesis and inflammation as well as its role in other diseases will be given. Consequently, possible therapeutics using NLRP3 inflammasome inhibitors will also be discussed. By understanding the NLRP3 inflammasome and its connection to HIV-1, effective methods to treat HIV-1-associated diseases caused by excessive inflammation can be identified.

As an obligatory intracellular pathogen, human immunodeficiency virus type-1 (HIV) is dependent upon its ability to exploit host cell machinery for replication and dissemination, and to circumvent cellular processes that prevent its growth. One such intracellular process is autophagy, a component of the host defense against HIV with roles in innate immune signaling, adaptive immunity and intracellular degradation of HIV. During permissive infection, HIV down-modulates autophagy, promoting its own replication. Inducers of autophagy can overcome this suppression and inhibit HIV. This review summarizes recent advances in understanding the antiviral and replicative roles of autophagy during HIV infection. Dissecting the molecular mechanisms by which HIV utilizes autophagy may lead to the identification of novel drug candidates to treat and potentially eradicate HIV infection.