A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids

Abstract

Retrovirus assembly involves a complex series of events in which a large number of proteins must be targeted to a point on the plasma membrane where immature viruses bud from the cell. Gag polyproteins of most retroviruses assemble an immature capsid on the cytoplasmic side of the plasma membrane during the budding process (C-type assembly), but a few assemble immature capsids deep in the cytoplasm and are then transported to the plasma membrane (B-or D-type assembly), where they are enveloped. With both assembly phenotypes, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form ( The assembly of an infectious retrovirus requires the coordinated expression of a minimum of three open reading frames found within all retroviral genomes: gag, pol, and env (55). Retrovirus capsid assembly is directed by the gag gene product, and expression of the gag gene product alone is sufficient for retroviral immature capsid assembly in the cytoplasm, followed by the budding and release of enveloped virus-like particles (VLPs) from the plasma membrane (10,17,22,23,28,39,44,48,59). Retroviral Gag polyproteins, the product of gag gene expression, are synthesized in the cytoplasm and self-associate with one another by undefined molecular mechanisms to form a spherical immature capsid containing genomic RNA. The Gag polyproteins contain a series of basic amino acids near the amino terminus, and most are modified by cotranslational amino-terminal myristoylation, promoting an association with cell membranes (21). In most retroviruses, Gag polyproteins interact specifically with the plasma membrane to initiate the viral budding process, culminating in the release of an extracellular immature VLP (reviewed in reference 25). During or immediately following viral budding, the Gag polyproteins are cleaved by a viral proteinase into the structural proteins that constitute a mature infectious virion, and the spherical immature capsid condenses to form an asymmetric mature viral core (reviewed in reference 56). In all retroviruses, these mature structural proteins include the matrix protein associated with the viral membrane; the major capsid protein forming the asymmetric viral core; and the nucleocapsid protein associated with the viral genomic RNA within the core (26).

Identification of a Conserved Residue of Foamy Virus Gag Required for Intracellular Capsid Assembly

“…The CTRS is proposed to target and retain Gag molecules at a specific site within the cell, allowing for localized increases in protein concentrations such that GagGag interactions and assembly may proceed (7). It is also likely that a cellular factor(s) is involved in the targeting process, a hypothesis supported by the recent report of the discovery of an insect cell line defective in the transport of assembled capsids to the PM (32). We propose that FV assembly occurs free of membranes in a fashion similar to the B-and D-type retroviruses and that capsid formation is mediated by a signal akin to the CTRS domain.…”

“…While the mechanism of intracellular capsid assembly is not fully understood for the B-and D-type retroviruses, recent experiments with the D-type retrovirus Mason-Pfizer monkey virus (MPMV) have show that an 18-amino-acid domain near the N terminus of Gag, termed the cytoplasmic targeting and retention signal (CTRS) and centered on a highly conserved arginine (Arg) residue at position 55, is required to direct the cytoplasmic assembly of capsids (7,32,38,40). Mutation of Arg55 of Gag to tryptophan (R55W) results in a switch of morphology to the default type C capsid assembly at the plasma membrane (PM), a process dependent on the N-terminal myristylation signal (37,38).…”

Analysis of Mason-Pfizer Monkey Virus Gag Domains Required for Capsid Assembly in Bacteria: Role of the N-Terminal Proline Residue of CA in Directing Particle Shape

“…We have previously shown that the Gag polyprotein of M-PMV can assemble into procapsids in eukaryotic cells (mammalian, insect, and yeast), in prokaryotic cells, and in an in vitro cell-free system (22,25,28,29,31,33; T. Ruml, unpublished data). The gag gene encodes the three domains, MA (p10), CA (p27), and NC (p14), found in all replication-competent retroviruses, as well as additional proteins, pp24/16 (hereafter designated PP), p12, and p4; these are arranged in the following order: NH 2 -MA (p10)-PP-p12-CA (p27)-NC (p14)-p4-COOH.…”

Analysis of Mason-Pfizer Monkey Virus Gag Particles by Scanning Transmission Electron Microscopy

“…A recombinant baculovirus expressing the M-PMV gag, pro, and pol reading frames, with an A18V point mutation in the matrix domain of the corresponding Gag polyprotein, has been described previously (22). Spodoptera frugiperda (Sf9) cells were grown at 27°C in suspension in Grace's supplemented insect medium in a 1-liter spinner flask to a density of 0.5 ϫ 10 6 to 1.0 ϫ 10 6 cells per ml.…”