<br/> <br/>With its global impact and complexity, a molecular understanding of the Human Immunodeficiency Virus -1 (HIV) requires a multi-disciplinary approach. Mass spectrometry (MS) coupled to the advent of bioinformatic technologies has come to the forefront as a powerful tool to elucidate the dynamic interactions of HIV proteins. In particular, understanding the integration of these HIV proteins by phosphorylation may give insight into how HIV’s life cycle is regulated. A top-down strategy to unearth the entire complement of HIV phosphorylation sites will be used to discover the kinases associated with each phosphorylation site, and to generate and test hypotheses for functionality.<br/>Kinase-specific candidate HIV protein phosphorylation sites will be identified using an integrated proteomic sequence analysis methodology. Because HIV RT is highly error prone, extensive strain-to-strain variation in amino acid sequence exists in HIV proteins. Amino acid residues conserved across many strains are likely to be functionally important, since mutating them can lead to reduced viability. Therefore, the bioinformatics analysis of putative phosphorylation sites begins with Ser, Thr and Tyr (STY) residues conserved across many strains. Results will be tabulated in a scoring matrix that indicates position-specific, kinase-specific likelihood of phosphorylation. The scores for each site will be summed over all HIV isolates.<br/>Our data supports the hypothesis that HIV contains a large proportion of phosphorylation sites that are still uncharacterized. To demonstrate feasibility, multiple isolates of the HIV proteins Vpu, Vif and Vpr were aligned and scored using the prediction algorithm NetPhosK 2.0, which generates a prediction probability score between 0 and 1, with higher scores indicating a higher likelihood of phosphorylation. These results were MS analyzed manually (Figure 1), which will generate scripts for automated data handling.

Analysis of HIV protein phosphorylation by host cell kinases using matrix assisted laser desorption mass spectrometry

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ACS Fall 2022

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