Confirmation of the Cell-Free System in Cells:

Other studies from the Lingappa lab, mainly done by Lingappa lab Ph.D. students, have shown that HIV capsid assembly intermediates contain other viral proteins that would be expected in nascent virions (Zimmerman et al. 2002). Furthermore, these intermediates can be trapped and identified in primate cell lines expressing the HIV virus (Dooher and Lingappa, 2004), as was predicted from the initial cell-free studies. Similar ABCE1-containing intermediates are found when related viruses such as HIV-2 and SIV are expressed in primate cells, suggesting that this pathway is universal, at least among primate lentiviruses (Dooher and Lingappa, 2004). In addition, mapping studies have revealed that the HIV nucleocapsid domain (NC) is critical for binding of ABCE1 to Gag (Lingappa et al., 2006). Recent studies using pulse-chase labeling in cells and immunogold double labeling electron microscopy further confirm the existence of ABCE1-containing assembly intermediates in cells expressing the HIV genome (Dooher et al., 2007). Together these studies indicate that HIV Gag progresses through an energy-dependent pathway of assembly intermediates containing cellular factors that are critical for capsid assembly in cells (Klein et al., 2007).

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