Little is known about the role human protein Tat-specific factor 1 (Tat-SF1) has in the production and regulation of Human Immunodeficiency Virus (HIV-1). What is known is that Tat-SF1 is a host factor for the virus. Upon Tat-SF1 knockdown, the relative levels of HIV RNAs change and HIV-1 infectivity is upregulated. These results are consistent with Tat-SF1 being involved in HIV-1 RNA regulation, although the exact mechanism is unknown. The hypothesis for this study was that if Tat-SF1 helps stabilize, splice, and/or export HIV-1 RNA, the protein must physically interact with the viral genome. This work attempts to demonstrate this possible interaction between Tat-SF1 and the HIV-1 RNA via in vitro RNA binding assays. Fragments of the HIV-1 genome were made through PCR and were then DNA sequenced before being used in in vitro transcription reactions. The HIV-1 RNA fragment was fluorescein labeled for easier visualization in the binding assay. Cell lysates made using E. coli plasmids contained the sequence for Tat-SF1 and also had a glutathione S-transferase (GST) tag inserted. The production of these proteins was induced using isopropyl β-D-1-thiogalactopyranoside (IPTG). For confirmation of protein expression, Western blotting was performed. These cell lysates and the HIV-1 RNA fragment were used to perform electrophoretic mobility shift assays (EMSA), a commonly used binding technique. Preliminary results showed that HIV-1 RNA was bound by Tat-SF1, supporting our hypothesis.

Investigating Tat-SF1 interactions with HIV-1 RNA

2019 Spring National Meeting

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