Alpha-helicoidal HEAT-like Repeat Proteins (\u03b1Rep) Selected as Interactors of HIV-1 Nucleocapsid Negatively Interfere with Viral Genome Packaging and Virus Maturation

Sudarat Hadpech,Wilhelm Furnon,Sawitree Nangola,Pierre Boulanger,Kanda Fanhchaksai,Marie Valerio-Lepiniec,Saw-See Hong,Philippe Minard,Chatchai Tayapiwatana,Koollawat Chupradit,Agathe Urvoas

doi:10.1038/s41598-017-16451-w

Abstract

A new generation of artificial proteins, derived from alpha-helicoidal HEAT-like repeat protein scaffolds (αRep), was previously characterized as an effective source of intracellular interfering proteins. In this work, a phage-displayed library of αRep was screened on a region of HIV-1 Gag polyprotein encompassing the C-terminal domain of the capsid, the SP1 linker and the nucleocapsid. This region is known to be essential for the late steps of HIV-1 life cycle, Gag oligomerization, viral genome packaging and the last cleavage step of Gag, leading to mature, infectious virions. Two strong αRep binders were isolated from the screen, αRep4E3 (32 kDa; 7 internal repeats) and αRep9A8 (28 kDa; 6 internal repeats). Their antiviral activity against HIV-1 was evaluated in VLP-producer cells and in human SupT1 cells challenged with HIV-1. Both αRep4E3 and αRep9A8 showed a modest but significant antiviral effects in all bioassays and cell systems tested. They did not prevent the proviral integration reaction, but negatively interfered with late steps of the HIV-1 life cycle: αRep4E3 blocked the viral genome packaging, whereas αRep9A8 altered both virus maturation and genome packaging. Interestingly, SupT1 cells stably expressing αRep9A8 acquired long-term resistance to HIV-1, implying that αRep proteins can act as antiviral restriction-like factors.

Highlights

Transcription activator-like effector nucleases (TALENS), or the clustered regularly interspaced short palindromic repeat/Cas[9] (CRISPR) system[8]
The biophysical properties of αRep proteins are highly favourable to biological and medical applications: (i) αRep proteins are expressed in bacteria as soluble proteins, implying a properly folded protein; (ii) they are functional in reducing and oxidative environments due to their disulfide independent folding, and can be active inside living cells as well as in the extracellular milieu; (iii) they are thermostable, with Tm >70 °C; (iv) heat denaturation is reversible and αRep proteins refold properly when cooled to 25 °C; (v) aggregation of αRep proteins does not occur at high concentrations; (vi) their small size allows the recognition of epitopes of low accessibility, increasing the diversity of binders for one target[28,29]
This region contained several elements identified as privileged targets for antiviral drugs. (i) SP1, the spacer peptide separating the capsid from the nucleocapsid domain, is a 14-residue long, alpha-helical domain which is critical for the self-assembly of Pr55Gag, and is the target of betulinic acid-derived assembly inhibitors30–35. (ii) The NC occupies a strategic position in the HIV-1 infectious cycle, as it possesses structural and catalytic properties, and acts as a nucleic acid chaperone[36,37,38,39,40]

Summary

Introduction

Transcription activator-like effector nucleases (TALENS), or the clustered regularly interspaced short palindromic repeat/Cas[9] (CRISPR) system[8]. The combined expression of 2LTRZFP and AnkGAG1D4 molecules in HIV-1-infected cells resulted in a significant negative effect on the viral replication[25] Another type of molecular scaffold, named alpha-repeat proteins (αRep), were tested as potential antivirals against HIV-1 in the present work. The biophysical properties of αRep proteins are highly favourable to biological and medical applications: (i) αRep proteins are expressed in bacteria as soluble proteins, implying a properly folded protein; (ii) they are functional in reducing and oxidative environments due to their disulfide independent folding, and can be active inside living cells as well as in the extracellular milieu; (iii) they are thermostable, with Tm >70 °C; (iv) heat denaturation is reversible and αRep proteins refold properly when cooled to 25 °C; (v) aggregation of αRep proteins does not occur at high concentrations; (vi) their small size allows the recognition of epitopes of low accessibility, increasing the diversity of binders for one target[28,29]. The multiple functions in which NC is involved[41,42,43,44,45], as well as its ALIX-mediated role in the virus particle release[46,47], justified the inclusion of the NC domain in our viral bait

Methods

Results

Conclusion