title: Efficient production of HIV-1 virus-like particles from a mammalian expression vector requires the N-terminal capsid domain.
abstract: It is now well accepted that the structural protein Pr55(Gag) 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 Pr55(Gag) 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 Pr55(Gag) 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 Pr55(Gag)-derived VLPs act as potent stimulators of innate and acquired immunity, make the use of this strategy worth considering for vaccine development.
authors.affiliation: Centre de Recherche en Infectiologie, Centre Hospitalier Universitaire de Québec-CHUL, Québec, Canada.
authors.forename: Pascal
authors.initials: P
authors.lastname: Jalaguier
authors.forename: Karine
authors.initials: K
authors.lastname: Turcotte
authors.forename: Alexis
authors.initials: A
authors.lastname: Danylo
authors.forename: Réjean
authors.initials: R
authors.lastname: Cantin
authors.forename: Michel J
authors.initials: MJ
authors.lastname: Tremblay
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002213:Capsid / Q000737:chemistry* / Q000648:ultrastructure; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry; D005822:Genetic Vectors / Q000235:genetics*; D057809:HEK293 Cells; D015497:HIV-1 / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008322:Mammals / Q000235:genetics*; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014763:Viral Matrix Proteins / Q000737:chemistry; D014771:Virion / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D011498:Protein Precursors; D014763:Viral Matrix Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1