Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain

Pascal Jalaguier,Karine Turcotte,Alexis Danylo,Réjean Cantin,Michel J Tremblay,Jean-Luc E P H Darlix

doi:10.1371/journal.pone.0028314

Pascal Jalaguier, Karine Turcotte + Show 4 more

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https://doi.org/10.1371/journal.pone.0028314

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Abstract

It is now well accepted that the structural protein Pr55Gag is sufficient by itself to produce HIV-1 virus-like particles (VLPs). This polyprotein precursor contains different domains including matrix, capsid, SP1, nucleocapsid, SP2 and p6. In the present study, we wanted to determine by mutagenesis which region(s) is essential to the production of VLPs when Pr55Gag is inserted in a mammalian expression vector, which allows studying the protein of interest in the absence of other viral proteins. To do so, we first studied a minimal Pr55Gag sequence called Gag min that was used previously. We found that Gag min fails to produce VLPs when expressed in an expression vector instead of within a molecular clone. This failure occurs early in the cell at the assembly of viral proteins. We then generated a series of deletion and substitution mutants, and examined their ability to produce VLPs by combining biochemical and microscopic approaches. We demonstrate that the matrix region is not necessary, but that the efficiency of VLP production depends strongly on the presence of its basic region. Moreover, the presence of the N-terminal domain of capsid is required for VLP production when Gag is expressed alone. These findings, combined with previous observations indicating that HIV-1 Pr55Gag-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development.

Highlights

Virus-like particles (VLPs) have sparked an increasing interest over the last decade [1,2]
We show that a similar minimal sequence can result in different experimental observations when inserted in a mammalian expression vector instead of being part of a molecular clone
We tested in this study the VLP-producing ability of a minimal Pr55Gag sequence inserted in a strong mammalian expression vector, namely pcDNA3.1 (+)

Summary

Introduction

Virus-like particles (VLPs) have sparked an increasing interest over the last decade [1,2] These viral entities cannot perform a full replication cycle and cannot produce new progeny virus. This property has allowed them to be used and studied in different contexts. They serve notably in gene therapy investigations, as they can enter target cells and deliver a specific gene [3]. They are employed for the study of chemical compounds that can block particle assembly and could prove to be useful in antiviral therapies. These recent successes underline the value of deepening our understanding of the mechanisms involved in VLP formation for many viruses, including human immunodeficiency virus type-1 (HIV-1)

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