Designing a smart protein-protein interaction assay for HIV research
Expert opinion
Of all the scientific disciplines in modern medicine, virology has been a major center of attention over the past several decades, notably with significant focus on HIV. The term “virology” is a generic definition that encompasses the study of virus biology and viral diseases per se, including the distribution, biochemistry, physiology, molecular biology, ecology, evolution, and clinical aspects of viruses. Finding new ways to treat viral diseases represents a major challenge in medicine.
Viruses rely heavily on interactions with their host to sustain their life cycle. Several steps in the cycle have long been considered as desirable targets for assay development and drug discovery. Viral infection follows the sequence: attachment and entry, uncoating, integration of viral genome in host genome, replication, and assembly and release of newly assembled viral particles.
In this article, Fawzi Khoder-Agha thoroughly describes the strategy he used to characterize the molecular interactions involved in the reverse transcription of HIV-1 virus. Using recombinant tagged proteins, produced either in E.coli or in baculovirus-infected insect cells, he performed an in-vitro study on the mechanism of interaction of key proteins involved in the process: mitochondrial Lysyl-tRNA synthase (mLysRS), HIV-1 GagPol, and tRNA3Lys. Combining Western-blot, circular dichroism spectroscopy Fluorescent Polarization, and TR-FRET, he illustrated the different protein-protein interactions in detail. By using a C-terminal truncated form of mLysRS (mLysRSΔC), he identified how the subdomains of GagPol (namely IN and TF) are involved in binding to mLysRS. He also demonstrated how the binding of mLysRS to GagPol could be enhanced in the presence of tRNA3Lys.
These highly specific and high affinity protein-protein interactions (all with Kd in the nanomolar range) are mandatory for ensuring the cascade of events leading to HIV-1 replication. Now, full understanding of this stable and complex protein assembly enables a clear strategy for developing specific inhibitors.
Put all the pieces of this protein puzzle together in 96-well plates and run your biochemical assays!
Abstract
An important step in human immunodeficiency virus type 1 (HIV-1) replication is the packaging of tRNA3Lys from the host cell, which plays the role of primer RNA in the process of initiation of reverse transcription. The viral GagPol polyprotein precursor, and the human mitochondrial lysyl-tRNA synthetase (mLysRS) from the host cell, have been proposed to be involved in the packaging process. More specifically, the catalytic domain of mLysRS is supposed to interact with the transframe (TF or p6*) and integrase (IN) domains of the Pol region of the GagPol polyprotein.
In this work, we report a quantitative characterization of the protein:protein interactions between mLysRS and its viral partners, the Pol polyprotein, and the isolated integrase and transframe domains of Pol. A dissociation constant of 1.3 ± 0.2 nM was determined for the Pol:mLysRS interaction, which exemplifies the robustness of this association. The protease and reverse transcriptase domains of GagPol are dispensable in this association, but the TF and IN domains have to be connected by a linker polypeptide to recapitulate a high affinity partner for mLysRS. The binding of the viral proteins to mLysRS does not dramatically enhance the binding affinity of mLysRS for tRNA3Lys.
These data support the conclusion that the complex formed between GagPol, mLysRS and tRNA3Lys, which involves direct interactions between the IN and TF domains of Pol with mLysRS, is more robust than suggested by the previous models supposed to be involved in the packaging of tRNA3Lys into HIV-1 particles.