Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein

Séverine Freisz,Joelle Mezher,Lamine Hafirassou,Philippe Wolff,Yves Nominé,Christophe Romier,Philippe Dumas,Eric Ennifar

doi:10.4161/rna.20483

Abstract

The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

Highlights

The HIV-1 Virion Infectivity Factor (Vif) is a small basic protein of 192 amino acids (23 KDa, pI = 10.7)
Determinants for Vif binding to the dimerization initiation site (DIS) loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-Tat-responsive element (TAR) inhibitors or DIS small molecule binders might be effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions
APOBEC3G, formerly known as CEM15, was first identified as a potent endogenous inhibitor for replication of several retroviruses, including HIV1.8,12,13 Later on, other members of the APOBEC3 family have been identified as HIV-1 restriction factors: APOBEC3F,12,14,15 APOBEC3B,12,16 APOBEC3DE17 and APOBEC3H.18

Summary

Introduction

We used Isothermal Titration Calorimetry (ITC) to study the binding thermodynamics of Vif binding to the HIV-1 genomic RNA TAR and Dimerization Initiation Site (DIS) stem-loops (Fig. 1B). In our experimental conditions and in presence of 200 mM KCl, we observed a specific Vif binding to the TAR27 RNA

Results

Conclusion