Protein interactions regulating HIV replication in macrophages

摘要 巨噬细胞是HIV的储存库，这是治愈的障碍。然而，潜在的分子机制 病毒是如何在这种细胞类型中建立和维持感染的，人们对此知之甚少。澄清 巨噬细胞中的艾滋病毒复制机制对根除艾滋病毒的努力至关重要。 我们先前证明了周期蛋白L2是巨噬细胞感染HIV-1所必需的，与巨噬细胞相互作用并 靶向HIV限制因子SAM结构域和含HD结构域的蛋白1(SAMHD1)进行降解。 然而，细胞周期蛋白L2-SAMHD1相互作用的细节尚不清楚。此外，细胞周期蛋白L2是如何被调控的 在巨噬细胞中的作用尚不清楚。在我们最近的研究中，我们发现周期蛋白L2与细胞周期蛋白L2相互作用并被磷酸化 通过双特异性酪氨酸磷酸化调节的激酶1A(DYRK1A)。而Cyclin L2的基因敲除 将HIV感染减少10倍，DYRK1A的耗尽增加了原代巨噬细胞中的HIV感染 10倍。这项建议的首要目标是确定细胞周期蛋白L2、DYRK1A之间的相互作用 和SAMHD1调节巨噬细胞中的艾滋病毒复制。我们的具体目标是： 目的1：明确DYRK1A在巨噬细胞中调节细胞周期蛋白L2的机制。我们 假设DYRK1A通过磷酸化或降解抑制细胞周期蛋白L2。首先，我们将确定 DYRK1A介导的细胞周期蛋白L2的磷酸化是否能阻止HIV感染的促进 巨噬细胞。其次，我们将定义细胞周期蛋白L2和DYRK1A的分子结构域 互动。第三，我们将确定DYRK1A调节细胞周期蛋白L2水平的机制。 巨噬细胞。 目的：研究细胞周期蛋白L2和HIV-1在病毒感染过程中对巨噬细胞SAMHD1的调节作用。艾滋病毒-- 1不具有VPX，但能够在巨噬细胞中建立感染，尽管SAMHD1含量丰富。 因此，HIV可能通过细胞周期蛋白L2或其他途径来协调SAMHD1的降解。为了实现这一目标，我们 将(I)确定这种相互作用所需的细胞周期蛋白L2和SAMHD1的分子结构域(Ii)确定 病毒或细胞因子(S)，分别诱导细胞周期蛋白L2和SAMHD1水平在 早期HIV感染和(Iii)确定巨噬细胞中SAMHD1的降解是否受细胞周期蛋白L2的影响 DYRK1a的磷酸化。 这些研究将揭示Cyclin L2-DYRK1A-SAMHD1复合体如何调控巨噬细胞中的艾滋病毒感染。 我们的努力将对理解一种新的途径具有重要意义，这种途径决定了艾滋病毒复制 在巨噬细胞中被限制或启用。由于这些蛋白质的解剖，潜在的影响是显著的 相互作用可能揭示对抗艾滋病毒感染的潜在治疗目标。

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.