PROJECT SUMMARY Autophagy is an important cellular antiviral defense mechanism that potentially affects HIV infection and transmission. The antiviral properties of autophagy are two-fold: (i) autophagy targets virions for lysosomal degradation and (ii) this pathway aids in antigen presentation of virus pathogens. Despite its antiviral potential, efforts to capitalize on autophagy against HIV have been limited due to a gap in our understanding of the role of autophagy in HIV infection. Our long-term goal is to manipulate the antiviral properties of autophagy to facilitate HIV clearance and improve treatments. Our overall objective is to identify the molecular interactions between HIV and the autophagy machinery. Our central hypothesis is that autophagy restricts HIV, but the virus overcomes this block using Nef, a notorious virulence factor that mediates immune evasion. This hypothesis has been formulated based on our published and novel work demonstrating that autophagy depletes the HIV proteins Gag, Vif and Vpr, but only when nef is defective 17. Gag is the driver of virion assembly; Vif counteracts the damaging effects of the restriction factor APOBEC3G; and Vpr enhances infectivity by facilitating viral transcription and arresting cell cycle at G2. Thus, a decrease in Gag, Vif and Vpr will impact virion production and infectivity. However, we found that Nef blocks autophagy, restoring in turn HIV proteins and particle release. Our studies showed that Nef sequesters the autophagosome initiator BECN1 at the endoplasmic reticulum (ER) by facilitating its association with the autophagy inhibitor BCL2. Mechanistically, we uncovered that Nef induces BCL2 mono-ubiquitination via the E3 ligase Parkin. This post-translational modification increases BCL2's inhibitory effect over BECN1. Hence, this activity of Nef impairs autophagosome formation. The significance for this research is that: (i) it will address an important knowledge gap concerning the interplay between HIV and autophagy, and will reveal how this pathway influences HIV infection and transmission; and (ii) it will provide the basis for the design of approaches aimed at enhancing autophagy to treat HIV infection and/or improve cure strategies. We will attain the overall objective by pursuing the following three specific aims: (1) Identify HIV molecules vulnerable to autophagy; (2) Dissect the mechanism by which Nef blocks autophagy; and (3) Elucidate the impact of autophagy antagonism on HIV infectivity. The proposed research is innovative because it represents a substantive departure from the status quo by revealing that HIV critically needs Nef to counteract autophagy restriction.