Dynamic manipulation of NF-kappaB by HIV-1 in immune evasion and viral latency

Human Immunodeficiency Virus type 1 (HIV-1), the causative agent of the AIDS pandemic, encodes two accessory proteins (Vpr and Vpu) that potently suppress proinflammatory signalling via the NFkB family of transcription factors during the early and late phases of viral replication in CD4+ T cells and macrophages. This appears, at least in part, to be a mechanism to inhibit the induction of innate immune to viral replication at vulnerable stages of the lifecycle. Despite this, the integrated HIV-1 provirus is an NFkB-regulated gene which requires NFkB to be activated to initiate viral gene expression. Our recent publications on Vpu and unpublished work on Vpr from clinical isolates shows that the potency of NFkB suppression by these proteins has been underestimated. Both exert their functions through markedly different mechanisms that are incompletely understood at the molecular and cellular level. We have shown that Vpu promotes the degradation of the bTrCP1 adaptor of the SCF ubiquitin ligase, leading to a potent block of both classical and alternative NFkB pathways. But how this specificity is achieved is unclear. By contrast Vpr appears to block nuclear transit of inflammatory transcription factors by association with the nuclear pore and karyopherins. However, the requirement for its cognate ubiquitin ligase Cullin4DCAF1 implies Vpr targets an unknown factor to exert this activity. Since Vpr is a constituent of the incoming viral particle and Vpu is expressed 'late', these data imply that HIV-1 dynamically regulates NFkB across its replication cycle to balance innate immune evasion and viral production.
A key barrier to curing individuals of HIV-1 is the presence of a pool of infected CD4+T cells harbouring transcriptionally silent integrated proviruses - the so-called latent reservoir. While individuals living with HIV-1 are take combined antiretroviral therapy (cART) this latent reservoir is of little consequence. However, upon therapy withdrawal re-activation of some of these reservoir cells leads to rapid re-emergence of the virus. Therefore, aside from a handful of cases where HIV+ blood cancer patients were transplanted with bone marrow stem cells genetically resistant to infection, people living with HIV cannot be cured and must take cART for the rest of their lives. Thus, purging or silencing of this reservoir is viewed as the key goal of HIV-1 cure research, and ultimately this is dependent on the level of NFkB activation in individual latently infected cells. However, a key gap in knowledge is what viral and cellular factors promote the establishment of latent proviruses in the first place and whether these activities limit the success and sustainability of latency-reversing agents (LRAs) currently being developed to facilitate reservoir reduction - so called 'shock and kill' strategies.
It is our hypothesis that dynamic manipulation of NFkB in HIV-1-infected primary cells by Vpr and Vpu tips the balance to promote the establishment of a latent provirus. Furthermore, their potency at inhibiting NFkB may limit the sustained gene expression required for sufficient reservoir purging by LRAs/cure strategies activating these pathways.
We therefore seek MRC programmatic support for interlinked basic and translational studies to understand:
1. The consequences of dynamic regulation of NFkB for both viral and host gene expression across the viral replication cycle
2. The molecular mechanisms underlying the suppression of NFkB by HIV-1 accessory proteins
3. How accessory protein function affects the establishment, maintenance, and sustainable reversion of HIV-1 latency.

A key barrier to curing individuals of HIV-1 infection is the presence of infected CD4+T cells harbouring transcriptionally silent/quiescent integrated proviruses - the so-called latent reservoir. While PLWH are taking cART this is of little consequence. However, upon therapy withdrawal inflammatory responses lead to the re-activation of some of these cells and the re-emergence of the virus within weeks. A major cure strategy is to use latency reversing agents (LRAs) to reactivate these viruses in the presence of cART to purge the reservoir - so called Shock-and-Kill. However, to date these approaches have not shown sufficient efficacy in clinical trials.

HIV-1 encodes two accessory proteins (Vpr and Vpu) that suppress proinflammatory signalling via the NFkB family of transcription factors during the early and late phases of viral replication in CD4+ T cells and macrophages. This appears to be a mechanism to inhibit the induction of immune blocks to viral replication at vulnerable stages of the lifecycle. However, the integrated provirus is essentially an NFkB-dependent gen 8whose promoter requires either the classical or alternative NFkB-pathways to be activated to initiate viral gene expression. Our recent published data on Vpu and unpublished work on Vpr from clinical isolates shows that the potency of NFkB suppression by these proteins has been underestimated. Thus HIV-1 must dynamically regulate NFkB to balance immune evasion with viral production.

We hypothesize that Vpu and Vpr dynamically manipulate NFkB during replication, and in a minority of cells, promote the establishment of a latent/quiescent provirus. Furthermore, their potency at inhibiting NFkB may limit sustained gene expression required for reservoir purging by LRAs/cure strategies. If so, pharmacological targeting of accessory gene function may become important for curing HIV.

In this application we will test these hypotheses and the molecular bases of Vpu and Vpr function.