HIV-1 Capsid Stabilization Assay

Abstract

The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro.

A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors

“…There are several available methods to monitor and quantify the stability and uncoating of HIV-1 core, including in situ uncoating assays to monitor uncoating within infected cells (for review, see 11,28,29 ). The available in vitro methods use either authentic cores laboriously isolated from virions released from infected cells 13,20,30–32 or those assembled in vitro from purified, recombinant CA or capsid-nucleocapsid (CANC) proteins 33,34 . In vitro assembled CANC tubular structures have a mature-like arrangement of CA subunits 35 and can thus serve as surrogates for the HIV-1 core 34 .…”

“…In vitro assembled CANC tubular structures have a mature-like arrangement of CA subunits 35 and can thus serve as surrogates for the HIV-1 core 34 . For example, in the capsid stabilization assay 33,34 , in vitro assembled CANC tubular structures are spontaneously disassembled upon incubation in a destabilization buffer and ultracentrifuged through a 70% sucrose cushion. The CA content in the pelleted fraction is compared to the input by Western blot.…”

The small‐molecule 3G11 inhibits HIV‐1 reverse transcription

“…To test whether the ability of 3G11 to inhibit HIV‐1 infection is early during infection, we challenged Cf2Th cells with single‐round replication HIV‐1‐GFP viruses. As shown in Figure , 3G11 blocked HIV‐1 infection as potent as the small‐molecule inhibitor PF74, and is much stronger when compared to the small‐molecule BI‐2 . The small‐molecule 3G11 used at 10 μ m completely inhibited infection of single‐round replication HIV‐1 viruses; these results suggested that 3G11 blocks HIV‐1 infection at an early step such as reverse transcription, nuclear import, or integration.…”

“…This particular pocket has been previously described for other drugs . Although 3G11 binds to capsid, we were unable to detect an effect of the drug on capsid stability by using the fate of the capsid and capsid stability assays . Furthermore, by using HIV‐1/SIV mac capsid chimeras, we attempted to functionally test the role of capsid in the sensitivity of HIV‐1 to 3G11; these experiments suggested that capsid is not the determinant for the sensitivity of HIV‐1 to 3G11.…”

“…CA–NC stability assay was performed as described previously . Briefly, CA–NC tubes [assembled in 50 m m Tris‐Hcl pH 8.0, 0.5 m NaCl, and 2 mg/ml DNA oligo(Tg)25 (50‐bp long)] were incubated with either stabilization buffer (10 m m Tris‐HCl pH 8.0, 10 n m KCl, 2 m m MgCl 2 , 0.5 m m DTT) or destabilization buffer (20 m m Tris‐HCl pH 8.0, 10 n m KCl, 2 m m MgCl 2 , 0.5 m m DTT, 1% glycerol, 0.1% NP40) for 1 hr at room temperature in the presence of increasing concentrations of 3G11.…”