Delineating HIV-1 nuclear import mechanisms through capsid interaction with MxB and the nuclear pore complex

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ABSTRACT Human Immunodeficiency Virus 1 (HIV-1) causes life-long infection, which requires taking antiviral drugs to control the infection. No vaccines are available against this virus. The HIV-1 cone-shaped capsid core, made up of capsid protein (CA), protects the HIV-1 genome, plays a major role in HIV-1 infection and interacts with several host factors involved in nuclear import of viral complexes and integration into the host genome. The myxovirus resistance protein B (MxB) is thought to restrict HIV-1 infection by inhibiting uncoating and blocking nuclear entry, the latter measure by 2-Long Terminal Repeats (2-LTR) circles inside the nucleus. However, MxB does not inhibit the appearance of CA inside the nucleus of infected cells, even when infection is inhibited. Additionally, measuring 2-LTR circles in the nucleus, which is a marker for HIV-1 nuclear import, does not correlate with productive infection. Currently, little is known about the kinetics and sites of HIV-1 uncoating and the role of MxB in regulating this process. HIV-1 nuclear import is also not completely understood. Several nucleoporins (NUPs), which form the nuclear pore complex (NPC), interact with CA to promote HIV-1 nuclear import. The NPC was previously thought to be a static structure with an inner diameter of ~40nm, too small to allow nuclear import of the 60nm-wide HIV-1 capsid core, leading to an assumption that HIV-1 has to uncoat before entering the nucleus. However, recent evidence suggests that NPC is a dynamic structure that may expand and that HIV-1 cone- shaped cores can enter the nucleus. Little is known about how the NPC and MxB interactions with HIV-1 CA respectively promote or inhibit HIV-1 infection. Therefore, I hypothesize that the NPC-associated MxB blocks nuclear import by inhibiting uncoating and that the nuclear import of HIV-1 core requires expansion of the nuclear pore complex. In the two Specific Aims of this proposal, I will investigate the HIV-1 nuclear import and CA core disassembly in the presence of MxB (Aim 1) and delineate the changes in nuclear pore architecture caused by HIV-1 (Aim 2) using advance microscopy techniques. In Aim 1, I will determine HIV-1 nuclear entry inhibition by MxB through visualizing the presence of fluorescently labeled integrase-superfolderGFP (INsfGFP) inside the nucleus. I will also examine HIV-1 uncoating by visualizing loss of a fluorescently labeled cyclophilin A derivative, which binds to CA with high avidity. In Aim 2, I will examine changes in the nuclear pore size upon HIV-1 infection using super-resolution microscopy. I will infect NUP-labeled cells with HIV-1 CA mutant, which fails to transport to the nucleoplasm and remains associated with NPC. I will then visualize the NPC with Stimulated Emission Depletion (STED) microscopy and Stochastic Optical Reconstruction Microscopy (STORM). This project will provide me with diverse training in virology, cell biology and advanced microscopy techniques and will advance our understanding of HIV-1 nuclear import mechanisms. In the long-term, my work may support development of novel antiviral drugs that can mitigate HIV-1 infection.