ABSTRACT Macrophages serve as HIV reservoir, which is a barrier to cure. However, the molecular mechanism underlying how the virus establishes and maintains infection in this cell type is much less understood. Elucidating the mechanisms of HIV replication in macrophages is of vital importance to the HIV eradication efforts. We previously demonstrated that cyclin L2 is required for HIV-1 infection in macrophages, interacts with and targets the HIV restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1) for degradation. However, the details of the cyclin L2-SAMHD1 interactions are unknown. In addition, how cyclin L2 is regulated in macrophages is unknown. In our recent studies we showed that cyclin L2 interacts with and is phosphorylated by the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 1A (DYRK1A). While knockout of cyclin L2 decreased HIV infection 10-fold, depletion of DYRK1A increased HIV infection in primary macrophages up to 10-fold. The overarching objective of this proposal is to determine how the interplay between cyclin L2, DYRK1A and SAMHD1 regulate HIV replication in macrophages. Our specific aims are: Aim 1: Define the mechanism by which the kinase DYRK1A regulates cyclin L2 in macrophages. We hypothesize that DYRK1A inhibits cyclin L2 through phosphorylation or degradation. First we will determine whether DYRK1A-mediated phosphorylation of cyclin L2 prevents the promotion of HIV infection in macrophages. Second, we will define the molecular domains of cyclin L2 and DYRK1A required for their interactions. Third, we will determine the mechanism by which DYRK1A regulate cyclin L2 levels in macrophages. Aim 2: Determine how cyclin L2 and HIV-1 regulate SAMHD1 in macrophages during viral infection. HIV- 1 possesses no Vpx, but is able to establish infection in macrophages despite the abundance of SAMHD1. Therefore, HIV may orchestrate the degradation of SAMHD1 through cyclin L2 or other means. In this aim, we will (i) determine the molecular domains of cyclin L2 and SAMHD1 required for this interaction (ii) identify the viral or cellular factor(s) that induce elevation and reduction in cyclin L2 and SAMHD1 levels respectively during early HIV infection and (iii) determine if SAMHD1 degradation in macrophages is influenced by cyclin L2 phosphorylation by DYRK1A. These studies will reveal how the cyclin L2-DYRK1A-SAMHD1 complex regulate HIV infection in macrophages. Our efforts will be highly significant for understanding a novel pathway that determines whether HIV replication is restricted or enabled in macrophages. The potential impact is significant since the dissection of these protein interactions could reveal potential therapeutic targets against HIV infection.