Elucidating the Molecular Bases of Nef-Alix and Nef-PTPN23 Interactions

by Linh Dan Nguyen

 

Introduction

The Human Immunodeficiency Virus-1 (HIV-1) is a retrovirus that causes significant threats to the human population. The HIV-1 protein Nef is a key factor in viral replication. Among Nef’s many functions, the most conserved is the downregulation of the surface protein receptor, CD4. Current research has shown that Nef hijacks clathrin-AP2-dependent endocytosis to internalize CD41. Nef then mediates the downregulation of CD4 towards the multivesicular bodies (MVBs) and eventually towards lysosomal degradation2, a process facilitated by Nef’s hijacking of Alix, an ESCRT (endosomal sorting complex required for transport) adaptor protein3. Alix involvement promotes the binding of Vps-28 of ESCRT-1 to CHMP2A of ESCRT-III leading to the formation of intra-luminal vesicles (ILVs) where CD4 is retained during the MVB lysosome transitions. Disruption of the Nef-Alix interaction may therefore rescue CD4 in infected cells and thereby impede HIV-1 infection. Our project here aims to elucidate the mechanistic details of the Nef-Alix interaction. In addition, PTPN23, also known as HD-PTP, is a paralog protein of Alix and utilizes a similar ESCRT mechanism for the downregulation of MHC-1 in Kaposi’s sarcoma-associated herpes virus (KSHV)4. Preliminary data shows that Nef also binds to PTPN23 in vitro. However, the molecular details, and cellular effects, of this interaction is unknown. Our project also aims at elucidating the molecular basis of the Nef-PTPN23 interaction.

Methodology and Purpose

My summer research involved utilizing a series of approaches to closely examine the Nef-Alix and Nef-PTPN23 interactions. The first portion of my summer research focused on the protein expression and purification of Alix and PTPN23 that will later be used to conduct a gel filtration binding test against Nef to determine whether binding occurs. The second portion of my summer research focused on the cloning of three constructs, which required a significant amount of troubleshooting. Purified Alix is prone to a high degree of degradation due to an unstable PRD domain; our previous Alix construct has a histag at its N-terminus (furthest away from the PRD domain). We therefore tried to relocate the histag tail to the C-terminus (adjacent to the PRD domain), which should allow us to fish out intact, non-degraded Alix during purification. The next two cloning constructs involve creating individual domains (Bro1 and CC) of PTPN23 from a didomain construct (provided by our collaborator, John Guatelli). Preliminary data from Dr. Guatelli’s lab has indicated that there is a strong binding between Nef and didomain PTPN23. The cloning of individual PTPN23 domains would allow further assessment of how Nef interacts with each domain of PTPN23 to navigate through the ESCRT pathway. The remaining portion of my summer work shifted the focus back to protein expression of two successfully cloned PTPN23 constructs (Bro1, CC) and four Alix constructs (Bro1 domain, CC domain, didomain, and fulllength Alix that were provided by our collaborator, Dr. DaSilva). We currently have all Alix constructs and PTPN23-CC expressed and prepared for the next portion of the experiment.

Conclusion and Future Direction

Our current data on the binding between Nef and Alix is inconclusive: this binding was apparent in some gel filtration binding tests while not apparent in other types of binding assays. Future direction of the experiment is to closely examine the interaction of Nef to the new constructs of Alix and PTPN23 to examine the Nef interaction with the different domains and full-length molecules. We suspect that some conformational change occurs within full length Alix to allow Nef-binding. Our next set of binding tests using the purified individual domains of Alix will test this. If our hypothesis is verified, future steps of the experiment aim to use an activator to open Alix into a conformation capable of Nef-binding. We will then seek to use Cryo EM to elucidate the structural of Nef-Alix interaction. Work toward understanding the Nef-PTPN23 interaction will follow a similar path.

 

References

 

1. Kwon, Y. et al. Structural basis of CD4 downregulation by HIV-1 Nef. Nat Struct Mol Biol 27, 822-828 (2020).
2. daSilva, L.L.P. et al. Human Immunodeficiency Virus Type 1 Nef Protein Targets CD4 to the Multivesicular Body Pathway. Journal of Virology 83, 6578-6590 (2009).
3. Amorim, N.A. et al. Interaction of HIV-1 Nef protein with the host protein Alix promotes lysosomal targeting of CD4 receptor. J Biol Chem 289, 27744-56 (2014).
4. Parkinson, M. D., Piper, S. C., Bright, N. A., Evans, J. L., Boname, J. M., Bowers, K., … & Luzio, J. P. (2015). A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I. Biochemical Journal, 471(1), 79-88.