Purification and characterization of HIV-human protein complexes

Abstract

To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen-host protein-protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10-100 proteins), generation of comprehensive host-virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral-host protein-protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host-pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome.

Fig. 1

Literature derived HIV–human protein–protein interaction map. A network displaying HIV-human interactions derived from the National Institute of Allergy and Infectious Diseases Division of AIDS (NIAID) HIV-1 Human Protein Interaction Database. HIV proteins correspond to red nodes whereas yellow nodes represent host factors. In total, 1785 unique HIV-human interactions among 1175 human and 15 HIV proteins are presented. Further work will be required to determine which interactions are direct or functionally relevant.

Fig. 2

The double pull-down approach to characterize HIV–human protein complexes. In this strategy, cell lines are expressing two proteins, one viral and one host, each with a different affinity tag. The first purification step enriches for the viral protein, and presumably all the complexes it is associated with whereas the second purification step targets a host protein and enriches for a specific and stoichiometric viral-host protein complex.

Fig. 3

The purification-mass spectrometry strategy to characterize HIV–human protein complexes. Cloned viral genes are inserted into a construct that fuses a 2XStrep3XFlag dual affinity tag on the C-terminus of each factor. These constructs can be used for transient transfection in HEK293 cells or for generating stably expressing Jurkat cells. After lysis, the extract is subjected to either Anti-Flag or Strep-Tactin IP beads where an aliquot of the beads, as well as the elution, is subjected to trypsin digestion and the material is analyzed using the OrbiTrap mass spectrometer. In both cases, a portion of the eluate is also subjected to SDS–PAGE analysis, where the gel is stained, bands excised, proteins extracted and the protein is digested with trypsin and analyzed using a Q-Star Elite mass spectrometer. The data obtained from both sets of purifications and multiple points during the isolation are then integrated together and subjected to an algorithm to derive quantitative viral-host protein–protein interactions. See text for a more detailed description.