Specific Interaction of DARPin with HIV-1 CANTD Disturbs the Distribution of Gag, RNA Packaging, and Tetraspanin Remodelling in the Membrane

Abstract

A designed repeat scaffold protein (AnkGAG1D4) recognizing the human immunodeficiency virus-1 (HIV-1) capsid (CA) was formerly established with antiviral assembly. Here, we investigated the molecular mechanism of AnkGAG1D4 function during the late stages of the HIV-1 replication cycle. By applying stimulated emission-depletion (STED) microscopy, Gag polymerisation was interrupted at the plasma membrane. Disturbance of Gag polymerisation triggered Gag accumulation inside producer cells and trapping of the CD81 tetraspanin on the plasma membrane. Moreover, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) experiments were performed to validate the packaging efficiency of RNAs. Our results advocated that AnkGAG1D4 interfered with the Gag precursor protein from selecting HIV-1 and cellular RNAs for encapsidation into viral particles. These findings convey additional information on the antiviral activity of AnkGAG1D4 at late stages of the HIV-1 life cycle, which is potential for an alternative anti-HIV molecule.

Dimeric Ankyrin with Inverted Module Promotes Bifunctional Property in Capturing Capsid to Impede HIV-1 Replication

“…An HIV RNA copy number in Ank GAG 1D4-S45Y, Ank GAG 1D4 NC-NC , and Ank GAG 1D4 NC-CN expressing cells was 1.76 × 10 7 , 6.14 × 10 5 and 3.69 × 10 4 copies/mL, respectively ( Figure 9 C). Corresponding to previous findings, the specific interaction of ankyrin to HIV-1 CAp24 interferes with the assembly process leading to a decrease in virion release and disturbing the viral RNA packaging [ 5 ]. This finding confers the improvement of anti-HIV-1 activity against HIV-1 NL4-3 WT replication in dimeric ankyrins.…”

“…This Gag-specific artificial ankyrin inhibits HIV-1 virion production in the late-stage HIV-1 life cycle. Additionally, the ability of Ank GAG 1D4 to disturb gag distribution in infected cells and interfere with RNA packaging of HIV-1 virion was demonstrated [ 5 ]. However, the HIV-1 protection by Ank GAG 1D4 in long-term culture is insufficient.…”

Designed Ankyrin Repeat Proteins: A New Class of Viral Entry Inhibitors

“…SupT1 cells that stably express an artificial ankyrin repeat molecule (AnkGAG1D4) binding to the human immunodeficiency virus (HIV) Gag polyprotein showed a reduced permissiveness to HIV-1 infection [ 34 ]. Subsequent studies investigated the molecular mechanisms whereby AnkGAG1D4 inhibited HIV replication, showing that the intracellular expression of AnkGAG1D4 affected the late stages of virus production [ 66 , 67 , 68 , 69 ]. Despite this as a promising strategy, intracellular DARPin delivery may limit in vivo administration of the molecule.…”