We recently used a systematic affinity tag/purification-mass spectrometry (AP-MS) approach to purify all 18 HIV-1 polyproteins and processed proteins from two different cell types (HEK293 and Jurkat) and characterized the HIV–human complexes by mass spectrometry. Using a novel scoring algorithm we developed that quantitatively reports on mass spectrometry-derived protein-protein interactions, termed MiST (mass spectrometry interaction statistics), we derived a set of 497 HIV-human protein-protein interactions involving 435 distinct human proteins.
We are using other systems approaches to gain a deeper understanding of the HIV infection process. These include analyzing global changes in post-translational modifications in the HIV-infected host proteome, performing genetic interaction analyses with RNAi and mutant viruses. Finally, we are using this platform to interrogate other pathogenic organisms and how they infect their host, including hepatitis C, influenza and West Nile virus.
More than 95% of the protein interactions we derived via MiST had not been reported, and several deepened our understanding of how HIV proteins function during the course of infection. For example, HIV protease associates with the translational initiation factor, eIF3, and cleaves one of its subunits, eIF3d, in its RNA-binding domain. Knockdown of eIF3d leads to an increase of HIV infection, and the inhibitory activity of eIF3 was further mapped to the reverse-transcription stage of infection. This finding has led us to a model in which the eIF3d subunit of eIF3 binds to viral RNA in the early stages of infection and inhibits it from being reverse transcribed into DNA, an effect overcome by cleavage of eIF3d by the HIV protease.
Additionally we uncovered a new subunit of the Vif/Cul5 protein complex that targets the APOBEC3 family of enzymes, restriction factors that inhibit HIV infection, for ubiquitination and degradation. This protein subunit, CBFb, is a transcription factor and heterodimerizes with the RUNX family of transcription factors. CBFb allows for the reconstitution of an active, hexameric, Vif-containing ubiquitin ligase complex, and its knockdown by RNAi revealed that HIV absolutely requires it for infection.
Some questions addressed in ongoing studies
- What global changes can be seen in post-translational modification in the host proteome infected by HIV?
- What genetic interactions exist between RNAi and mutant viruses?
- Will hepatitis C, influenza, and West Nile virus protein interactions with human proteins reflect similar or differing methods of host infection to HIV?