The role of mammalian STE20-like (MST) kinases in HIV-1 Vpr-mediated manipulation of NF-κB signalling

• Applicant: Dr. Kristina Hopfensperger
• Subject Area: Clinical Infectiology and Tropical Medicine
• Term: since 2026
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 574125491

Ubiquitination is a post-translational modification mediated by E3 ubiquitin ligases. It plays a dual role during human immunodeficiency virus (HIV) infection. On the one hand, it is required for efficient virus sensing and the induction of antiviral gene expression. On the other hand, HIV uses accessory proteins such as its viral protein U (Vpu) and viral protein R (Vpr) to manipulate the ubiquitination pattern of cellular proteins and thus enable efficient virus replication. Hence, analyses of ubiquitinome changes within HIV-infected cells have great potential to identify cellular proteins whose ubiquitination is altered by the virus to promote viral replication. Using mass spectrometry-based global ubiquitin pattern analyses of primary CD4+ T-cells infected with a clinically relevant HIV-1 strain, I have identified cellular proteins whose ubiquitination is likely manipulated by HIV-1 Vpr and/or Vpu. Among the identified potential targets of Vpr is the STE20-like (MST) kinase MST3. Interestingly, MST3 has been described to promote Toll-like receptor-mediated NF-κB activation by stabilizing the interaction between the IKK-complex and TAK1. I therefore hypothesize that HIV-1 Vpr induces MST3 ubiquitination and proteasomal degradation to inhibit MST3-mediated NF-κB activation, thereby dampening antiviral immune responses and promoting virus replication. My preliminary data confirms that MST3 promotes NF-κB activation. This induction is inhibited by HIV 1 Vpr. Furthermore, western blot analyses show that Vpr decreases MST3 protein levels and that this function is conserved among Vpr proteins from diverse HIV-1 strains. The fact that the protein levels of the MST3 paralog MST4 are not affected suggests high specificity. Finally, a Vpr mutant unable to bind DCAF-1, the E3 ligase substrate receptor, may still reduce MST3 levels. This finding could suggest that Vpr interacts with another, yet unknown substrate receptor. Within the proposed project, I aim to confirm that Vpr induces MST3 ubiquitination. I will characterize which type of ubiquitin chain is attached and analyse if Vpr directly interacts with MST3. In addition, I will characterize if Vpr-induced MST3 ubiquitination alters its stability, intracellular localization and/or interaction with TAK1. Finally, I will analyse if Vpr-mediated manipulation of MST3 boosts HIV/SIV replication in primary target cells of the virus. The results will not only shed light on the accessory protein Vpr, whose exact function has remained elusive and controversial. They will further elucidate a previously unknown Vpr-dependent viral immune evasion mechanism. In the future, specific inhibitors blocking Vpr-mediated manipulation of MST3 could be developed. Identification of new drug targets to inhibit HIV replication is highly warranted to fight HIV strains resistant to standard antiretroviral therapy.

• DFG Programme: Research Grants