Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain Pascal Jalaguier1, Karine Turcotte1, Alexis Danylo1, Re´ jean Cantin1, Michel J. Tremblay1,2* 1 Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Que´bec – CHUL, Que´bec, Canada, 2 De´partement de Microbiologie-Infectiologie et Immunologie, Faculte´ de me´decine, Universite´ Laval, Que´bec, Canada 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. Citation: Jalaguier P, Turcotte K, Danylo A, Cantin R, Tremblay MJ (2011) Efficient Production of HIV-1 Virus-Like Particles from a Mammalian Expression Vector Requires the N-Terminal Capsid Domain. PLoS ONE 6(11): e28314. doi:10.1371/journal.pone.0028314 Editor: Jean-Luc EPH Darlix, Institut National de la Sante´ et de la Recherche Me´dicale, France Received August 23, 2011; Accepted November 6, 2011; Published November 30, 2011 Copyright: 2011 Jalaguier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by an operating grant to M.J.T. from the Canadian Institutes of Health Research (CIHR) (MOP-14438). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michel.j.tremblay@crchul.ulaval.ca 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 therefore cannot produce new progeny virus. This property has allowed them to be used and studied in different contexts. They serve notably in gene therapy investiga- tions, as they can enter target cells and deliver a specific gene [3]. They are also employed for the study of chemical compounds that can block particle assembly and could prove to be useful in antiviral therapies. Finally, VLPs have recently been used with success as a tool for vaccination against human papillomavirus [4,5,6]. 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). HIV-1 can form VLPs by sole virtue of its Gag polyprotein precursor, also known as Pr55Gag. This structural viral constituent is indeed the main actor in HIV-1 particle formation as it drives assembly through protein-protein, protein-RNA, and protein-lipid interactions, orchestrating the incorporation of each of the major virus-encoded components into assembled particles [7,8,9,10]. Pr55Gag is translated from unspliced viral mRNA on free ribosomes in the cytoplasm and encodes the internal structural components of the virion, i.e. matrix (MA), capsid (CA) and nucleocapsid (NC) along with the C-terminal p6 domain and two spacer peptides SP1 and SP2 [11]. In the absence of any other viral proteins, Pr55Gag can self-assemble into VLPs [12] by ordered multimerization of monomers and/or oligomers to produce a spherical shell, which forms the structural framework of the immature virus particle [10,13]. It is noteworthy that HIV-1 Pr55Gag-based VLPs elicit strong cellular and humoral responses in non-human primate research models for AIDS [1,14,15]. Several regions of the Pr55Gag polyprotein have been reported to be crucial for particle formation. Notably, previous structural and genetic analyses have demonstrated that the regions responsible for membrane binding are located at the N-terminal region of MA, which encompasses a cluster of basic residues and a myristyl group interacting with acidic phospholipids of the membrane [16,17]. However, several studies have shown that MA can be largely deleted or even entirely replaced by a heterologous myristyl anchor without compromising the formation of extracellular viruses [18,19,20,21]. This suggests that the role of MA in assembly is not as important as its role in Pr55Gag trafficking [22,23] and in the incorporation of virus-encoded envelope glycoproteins (ENV) [24,25]. CA provides structural stability to HIV-1 particles and also plays a key role in the formation of protein-protein contacts required for a productive particle assembly. CA comprises two globular domains: an N-terminal domain (NTD) and a C-terminal PLoS ONE | www.plosone.org 1 Month 2011 | Volume 6 | Issue 11 | e28314