Background:  Given the limited genetic capacity of HIV-1 and complex molecular events occurring during its infection, it is likely that the virus must interact with an extensive variety of cellular factors to complete its passage through the cell.

Methods:  To identify the subset of host factors required for HIV-1 infection we have taken a proteomic approach using affinity purification coupled with mass spectrometry. In our study, stringent genetic selection was imposed to recover viral clones that could incorporate a potent antigenic (3xFLAG) or biochemical tag (biotin) into selected translated regions of the viral genome yet remain fully viable through multiple rounds of infection. This modification allows for the facile recovery of cellular proteins encountered in complex formation with the tagged viral protein during ongoing infection.

Results:  To date, we have recovered viruses that can accommodate a tag within the integrase, viral infectivity factor (Vif), and viral envelope proteins (Env). These modified viruses are replication-competent, indicating the tagged viral proteins are fully functional during viral passage. As expected, the tagged viral proteins are stably expressed and retained within the viral population over an extended period. Moreover, the tagged integrase and Env proteins could easily be captured from infected culture by magnetic resins conjugated with the appropriate ligand (i.e., anti-FLAG antibody or streptavidin).

Conclusions:  Using our panel of independently tagged, replication-competent derivatives, we seek to recover host proteins that interact specifically with the virus as it progresses through its natural life cycle. Mass spectrometry techniques are in progress to determine the identity of host proteins and their complexes captured via their interaction with the targeted viral protein.

Keywords: Viral-Host Interactions; Replication-Competent Tagged Viruses; Affinity Purification