In this project, we will dissect the activity of key host cofactor Tat-SF1 in human versus HIV-1 RNA splicing. Complex retroviruses such as HIV-1 co-opt the human spliceosome machinery for tightly coordinated production of their spliced mRNAs and genomic RNAs during the early and late stages of the viral life cycle. At present, the molecular mechanisms responsible for the dominant progression of HIV-1 through the host splicing cycle remain outstanding questions in the field. Tat-SF1 is a host protein that is critica in the production of fully spliced HIV-1 mRNAs in the early stage of the retroviral life cycle. Tat SF1 is known to associate with the U2 small nuclear ribonucleoprotein subunit (snRNP) of the spliceosome and regulate the splicing of specific human transcripts. As such, studies of Tat-SF1 will be important to understand host-virus interactions, thereby laying the groundwork for the development of new biochemical tools or therapeutic intervention approaches at the level of splicing. At present, the underlying structural and functional roles of Tat-SF1 in HIV-1 and human splicing remain unknown. We will address this knowledge gap by a strong collaboration involving multidisciplinary approaches that include structural biochemistry of pre-mRNA splicing factors, molecular biology, and virology. Specifically, we will determine Tat- SF1 interactions among the human U2 snRNA and U2 snRNP subunits, and test these interfaces for functions in human splicing versus HIV-1 splicing and infectivity. We also will investigate a new mechanism of action for HIV-1 to pre-empt the human splicing machinery, in which the early stage HIV-1 Rev protein promotes late stage unspliced HIV-1 RNAs by disrupting the host Tat-SF1-U2 snRNP complex. Already we have: (i) produced diffracting crystals of the Tat-SF1 RNA recognition motif, (ii) obtained an initial structure of a complex between the Tat-SF1 and the SF3b155 subunit of the U2 snRNP, and (iii) generated a stable knockdown of Tat-SF1 in a human cell line. Moreover, our preliminary results demonstrate that the HIV- 1 Rev protein associates with human Tat-SF1. In addition to elucidating new mechanisms of action for HIV-1 to manipulate host pathways of gene expression, successful completion of the proposed experiments will set the stage for new therapeutic strategies that target a host cofactor, thereby circumventing drug resistance challenges that plague existing anti-HIV-1 treatments.
Targeting host cofactors offers an alternative strategy to develop new anti-HIV therapeutics. Viruses such as HIV-1 use the human machinery to produce its RNA transcripts for protein expression and ultimately genomic replication. Here we will investigate the molecular mechanisms for a human protein to coordinate production of HIV-1 RNAs, by leveraging insights gained from our prior work. Results of these experiments will lay groundwork for understanding the interplay of a key host and viral process, and potential new avenues for therapeutic development.
|Loerch, Sarah; Leach, Justin R; Horner, Steven W et al. (2018) The pre-mRNA splicing and transcription factor Tat-SF1 is a functional partner of the spliceosome SF3b1 subunit via a U2AF homology motif interface. J Biol Chem :|
|Kielkopf, Clara L (2018) Insights from structures of cancer-relevant pre-mRNA splicing factors. Curr Opin Genet Dev 48:57-66|
|Loerch, Sarah; Kielkopf, Clara L (2016) Unmasking the U2AF homology motif family: a bona fide protein-protein interaction motif in disguise. RNA 22:1795-1807|