HIV-1 Vif Can Directly Inhibit Apolipoprotein B mRNA-editing Enzyme Catalytic Polypeptide-like 3G-mediated Cytidine Deamination by Using a Single Amino Acid Interaction and Without Protein Degradation

Joao Goncalves,Ana Margarida Fonseca,Frederico Aires da Silva,Mariana Santa-Marta

doi:10.1074/jbc.m409309200

Abstract

The human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G), also known as CEM-15, is a host-cell factor involved in innate resistance to retroviral infection. HIV-1 viral infectivity factor (Vif) protein was shown to protect the virus from APOBEC3G-mediated viral cDNA hypermutation. The mechanism proposed for protection of the virus by HIV-1 Vif is mediated by APOBEC3G degradation through ubiquitination and the proteasomal pathway. Here we show that in Escherichia coli the APOBEC3G-induced cytidine deamination is inhibited by expression of Vif without depletion of deaminase. Moreover, inhibition of deaminase-mediated bacterial hypermutation is dependent on a single amino acid substitution D128K that renders APOBEC3G resistant to Vif inhibition. This single amino acid was elegantly proven by other authors to determine species-specific sensitivity. Our results show that in bacteria this single amino acid substitution controls Vif-dependent blocking of APOBEC3G that is dependent on a strong protein interaction. The C-terminal region of Vif is responsible for this strong protein-protein interaction. In conclusion, our experiments suggest a complement to the model of Vif-induced degradation of APOBEC3G by bringing to relevance that deaminase inhibition can also result from a direct interaction with Vif protein.

Highlights

The viral infectivity factor (Vif)1 protein of human immunodeficiency virus type 1 (HIV-1) plays a dramatic importance in viral infectivity [1, 2]
When high ionic strength wash buffer was used, a decrease was observed in the amount of APOBEC3G or APOBEC3G-D128K retrieved by Vif-C114F and Vif, respectively. These results show a strong physical interaction that occurs between Vif and APOBEC3G, which is partially dependent on a single amino acid substitution D128K
The system described in the results aimed to co-express Vif protein with APOBEC3G in conditions where degradation by the ubiquitin-proteasome pathway or kinase regulation could not occur

Summary

Introduction

The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) plays a dramatic importance in viral infectivity [1, 2]. Previous studies found that non-permissive cells contain an anti-viral cellular factor capable of suppress the HIV infectivity [8, 9], recently identified as CEM15/ APOBEC3G, for which the antiviral action is overcome by Vif (10 –12). APOBEC3G is a virion-encapsidated cellular protein that deaminates dC to dU in minus-strand viral cDNA during reverse transcription [13,14,15,16], preferentially at CCCA sequences [17]. Our studies focused on this key question and investigated if Vif binding to APOBEC3G may affect deaminase function independently of the ubiquitin/proteasome pathway To examine this hypothesis, we have expressed both APOBEC3G and Vif in E. coli and regulated the expression of its target gene to quantify cytidine deaminase activity. We aimed to evaluate in bacteria if the mechanism involved in a putative APOBEC3G-Vif interaction could be mediated by the single-amino acid substitution in APOBEC3G involved in species-specific restriction of Vif function

Objectives

Results

Conclusion