Uracil DNA glycosylase interacts with the p32 subunit of the replication protein A complex to modulate HIV-1 reverse transcription for optimal virus dissemination.

Cecile Herate,Marie Lambele,Serge Benichou,Carolin A Guenzel,Clarisse Vigne,Marie-Christine Rouyez

doi:10.1186/s12977-016-0257-x

Abstract

BackgroundThrough incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. We previously showed that this positive impact on reverse transcription was related to Vpr binding to the uracil DNA glycosylase 2 enzyme (UNG2), leading to enhancement of virus infectivity in established CD4-positive cell lines via a nonenzymatic mechanism.ResultsWe report here that Vpr can form a trimolecular complex with UNG2 and the p32 subunit (RPA32) of the replication protein A (RPA) complex and we explore how these cellular proteins can influence virus replication and dissemination in the primary target cells of HIV-1, which express low levels of both proteins. Virus infectivity and replication in peripheral blood mononuclear cells and monocyte-derived macrophages (MDMs), as well as the efficiency of the viral DNA synthesis, were significantly reduced when viruses were produced from cells depleted of endogenous UNG2 or RPA32. Moreover, viruses produced in macrophages failed to replicate efficiently in UNG2- and RPA32-depleted T lymphocytes. Reciprocally, viruses produced in UNG2-depleted T cells did not replicate efficiently in MDMs confirming the positive role of UNG2 for virus dissemination.ConclusionsOur data show the positive effect of UNG2 and RPA32 on the reverse transcription process leading to optimal virus replication and dissemination between the primary target cells of HIV-1.

Highlights

Through incorporation into virus particles, the Human immunodeficiency virus type 1 (HIV-1) Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process
uracil DNA glycosylase 2 enzyme (UNG2) and repair machinery such as the p32 subunit (RPA32) can be associated together in a trimolecular complex Previously, we reported that the ability of UNG2 to modulate the HIV-1 mutation rate was dependent on a 60-amino-acid domain located in the N-terminal regulatory region of the cellular protein containing the determinants for direct interaction with the RPA32 (i.e. p32) subunit of the replication protein A (RPA) complex [7]
We used in vitro binding assays performed with recombinant UNG2 and RPA32 expressed in E. coli in fusion with the glutathione S-transferase (GSTUNG2 and GST-RPA32, Fig. 1a, b, respectively)

Summary

Introduction

Through incorporation into virus particles, the HIV-1 Vpr protein participates in the early steps of the virus life cycle by influencing the reverse transcription process. HIV-1 utilizes or perturbs cellular pathways in order to optimize essential steps of the virus life cycle in T lymphocytes, macrophages or dendritic cells. The HIV-1 genome contains additional genes encoding regulatory proteins Nef, Vif, Vpu and Vpr, which are viral factors specialized in hijacking and perturbing essential cellular pathways during virus replication through interactions with host cell proteins. Vpr is the only HIV-1 auxiliary protein incorporated into virus particles through direct interaction with the Pr55Gag precursor, since its presence in the virion core will be subsequently required during the early steps of the virus life cycle in the newly infected cell (for review, [5])

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