Retroviruses are associated with a wide range of clinical diseases, including leukemia, tumors, and acquired immunodeficiency syndrome. Human immunodeficiency virus (HIV) is an exceptionally deadly retrovirus that has caused more than 20 million deaths over the past two decades. Due to the emergence of resistant strains, it is unlikely that current drug strategies, which target individual proteins, will lead to a cure. There is a serious need for superior approaches: for example, targeting anti-viral drugs towards essential RNA-protein or protein-protein interactions. To this end, I am interested in studying the structural determinants of reverse transcription and gene translation in retroviruses like HIV, Human T-cell leukemia virus and Moloney murine leukemia virus (MLV). Although aspects of reverse transcription and gene translation have received considerable attention, understanding of RNA structures and events that promote or regulate these steps remain primitive.
Nuclear magnetic resonance (NMR) and other biophysical and biochemical methods are the principal tools used in my laboratory to study these relatively large RNA and protein molecules. The structure determination of large protein- RNA complexes by NMR is extremely challenging, but the potential payoff is high and should ultimately lead to detailed models of steps involved in retroviral replication. The following are some of the projects that are currently being studied in my lab:
Primer placement and initiation of reverse transcription
For successful retroviral infection to occur, the RNA genome must be reverse-transcribed into DNA by the enzyme reverse transcriptase. The primer needed for the first synthetic step of reverse transcription is a specific tRNA molecule from the host that is packaged into the virion during assembly. This tRNA is annealed to the 5’ end of the viral genome called the Primer Binding Site (PBS), and requires the presence of a viral nucleocapsid protein (NC) to facilitate the necessary nucleic acid rearrangements. Studies in my laboratory are intended to provide the structural details of these tRNA-PBS-NC complexes and elucidate mechanisms that promote the initiation of reverse transcription.
In retroviruses, the structural Gag protein and the enzymatic Pol proteins are generated from the same precursor mRNA either by a frameshift or a readthrough mechanism. For example, MLV uses the readthorugh strategy and has the two genes separated by a single UAG stop codon. About nintey-five percent of the translating ribosomes recognize this codon and end the Gag protein. The other five percent, however, insert a glutamine residue at this position before resuming downstream translation into the pol gene. This 20:1 ratio of Gag to Gag-Pol is known to be vital for viral infectivity. Interestingly, the Gag stop codon is immediately followed by a sequence of RNA predicted to form a pseudoknot. Studies have shown this sequence to be critical for the readthrough function.
In collaboration with Dr. Stephen Goff’s lab at Columbia University, my goal is to elucidate the solution structure and the mechanisms by which this pseudoknot functions to maintain the Gag/Gag-Pol ratio.