Diaz-Griffero Lab

The study of HIV-1 uncoating in vitro has been hindered by the unstable nature of the HIV-1 core outside the cellular environment. This evidence together with work mentioned above suggests that the HIV-1 core is stabilized by cellular factors. Future work on this area will use biochemical and genetic approaches to identify factors that stabilize the HIV-1 core in the cellular environment. To this end, we will biochemically isolate HIV-1 cores from infected cells and identify the proteins associated to the core by mass spectrometry. We will compare the protein content of HIV-1 cores stabilized by different conditions: using reverse transcription inhibitors (Yang et al., 2012), viruses containing a defective reverse transcriptase enzyme (Yang et al., 2012), the microtubule disruptive drug nocodazol (Lukic et al., 2014; Malikov et al., 2015), cells expressing cytoplasmic CPSF6(Fricke et al., 2013b), and cells expressing MxB/Mx2(Fricke et al., 2014). As a negative control, we will mock isolate cores from cells expressing rhesus TRIM5a, which accelerates uncoating (Diaz-Griffero et al., 2007a; Perron et al., 2007; Stremlau et al., 2005). The assays we have developed to study capsid stability in vitro and in vivo will be used to confirm these interactions(Fricke et al., 2013a). Finally, the contribution to uncoating and infection will be evaluated in cells where the candidate proteins are knockout using the Cas9/CRISPR technology that is already working in our lab. Finding proteins that stabilize the HIV-1 core during infection will provide fundamental understanding on the uncoating process of HIV-1.