Human immunodeficiency virus-like, nonreplicating, gag-env particles assemble in a recombinant vaccinia virus expression system

Abstract

We report the assembly of human immunodeficiency virus (HIV)-like particles in African green monkey kidney cells coinfected with two recombinant vaccinia viruses, one carrying the HIV-1 gag and protease genes and the other the env gene. Biochemical analysis of particles sedimented from culture supernatants of doubly infected cells revealed that they were composed of gag proteins, primarily p24, as well as the env proteins gpl20 and gp4l. Thin-section immunoelectron microscopy showed that these particles were 100 to 120 nm in diameter, were characterized by the presence of cylindrical core structures, and displayed the mature gpl20-gp41 complexes on their surfaces. Furthermore, thin-section immunoelectron microscopy analysis of infected cells showed that particle assembly and budding occurred at the plasma membrane. Nucleic acid hybridization suggested that the particles packaged only the gag mRNA but not the env mRNA. Therefore, the system we present is well suited for studies of HIV virion maturation. In addition, the HIV-like particles provide a novel and attractive approach for vaccine development. * Corresponding author. ing of gpl60 in mammalian cells (16a). The HIV p55 protein is anchored in cellular membranes by a myristic acid moiety added to the N-terminal penultimate glycine residue (2). Similar to other retrovirus systems (33), anchoring of the HIV-1 core protein has been shown to be necessary for production of virions (14). Development of an in vitro recombinant expression system that mimics the HIV-1 virus synthesis and maturation pathways would be advantageous for studying the role of env and gag protein association in regulating virus formation and release. We (S.

A replication-incompetent Rift Valley fever vaccine: Chimeric virus-like particles protect mice and rats against lethal challenge

“…RVF VLPs were generated by transient transfection of HEK-293 or 293-gag cells (HEK-293 cells that constitutively express Moloney murine leukemia virus (MoMLV) gag protein) with expression plasmids encoding the RVFV glycoproteins and the nucleoprotein (N). We initially focused upon the generation of chimeric (MoMLV gag-containing VLPs, designated RVF chimVLPs) because it has been previously shown that the inclusion of retroviral gag can increase the uniformity and quantity (Gheysen et al, 1989;Haffar et al, 1990;Haynes et al, 1991;Rovinski et al, 1992;Szecsi et al, 2006) and stability (Hammonds et al, 2003) of VLPs. In addition, MoMLV gag protein could have an adjuvant-like effect.…”

Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria

“…Unlike most other enveloped RNA viruses, in which the viral glycoproteins appear to catalyze virus particle formation, assembly and release of retrovirus particles occur when capsid proteins are produced in the absence of the other gene products. Studies have shown that expression of the gag gene alone in a number of systems results in the efficient assembly and release of membrane-enveloped virions (2,3,10,12,18,28,29,35). Thus, the product of this gene has the structural information necessary to mediate intracellular transport, direct assembly of the capsid shell, and catalyze the process of membrane extrusion known as budding.…”

The matrix protein of human immunodeficiency virus type 1 is required for incorporation of viral envelope protein into mature virions

“…Human immunodeficiency virus type 1 (HIV-1) assembly at the plasma membrane of infected cells is similar to the morphogenesis of type C retroviruses. The Gag precursor polyprotein of HIV-1 appears to be sufficient for virus assembly, since viruslike particles are formed in the absence of all other viral proteins (5,9,12,29,30). The Gag precursor of HIV-1 is first synthesized as a 55-kDa polyprotein (p55) and is subsequently cleaved by the viral protease to yield four mature Gag proteins: the matrix (MA) protein (p17); the capsid (CA) protein (p24); the nucleocapsid (NC) protein (p9); a proline-rich protein (p6); and two small peptides, p2 and pl (11).…”