Amino acid substitutions within the matrix protein of type D retroviruses affect assembly, transport and membrane association of a capsid.

Abstract

The functional roles of the matrix (MA) protein in the assembly and maturation of retroviruses was investigated with a series of MA mutants of Mason‐Pfizer monkey virus (M‐PMV), an immunosuppressive type D retrovirus. The mutants we describe here were generated by the introduction of random point mutations within the MA coding domain by use of sodium bisulphite mutagenesis. Studies of these mutants show that the MA protein plays a critical role in three different, sequential events in the final stages of type D retrovirus replication: (i) folding of the gag gene‐encoded precursor poly‐proteins into a stable conformation for capsid assembly in the cytoplasm of infected cells; (ii) capsid transport from the site of assembly to the plasma membrane; and (iii) capsid association with, and extrusion of the membrane during virus budding. The mutants described here interfere with or block M‐PMV replication at each of these stages. Large numbers of preassembled capsids accumulate within the cytoplasm of transport‐defective mutant‐infected cells, suggesting that transport of M‐PMV capsids to the plasma membrane is an active and specific intracellular targeting process. The initial association of the capsid with the membrane may depend upon this intracytoplasmic transport process but additional protein‐lipid interactions that involve the MA protein are required for membrane extrusion around the preformed capsids; in cells infected with the budding‐defective mutant, assembled capsids accumulate under the inner surface of the cell plasma membrane, and are retarded in their release from the infected cell.

An Early Stage of Mason-Pfizer Monkey Virus Budding Is Regulated by the Hydrophobicity of the Gag Matrix Domain Core

“…Following separation of proteins by SDS-PAGE, the band intensity of Pr78 was measured for wild type and each mutant using phosphorimaging. Eighty percent of pulse-labeled Gag polyprotein was incorporated into immature capsids for wild type and A18V, a mutant that accumulates capsids in the cytoplasm (25). R55W, a mutant that assembles capsids at the plasma membrane (27), demonstrated only 50% Gag polyprotein was incorporated into capsids after the 30-min chase.…”

“…To better define the kinetics with which mutant Gag proteins were released and processed, a pulse-chase experiment with multiple chase times (1, 2, and 4 h) was carried out. This laboratory has previously shown that Gag precursor processing and virus release are temporally linked and that in the absence of capsid release Gag processing is not observed (25). Following analysis of samples with SDS-PAGE, OptiQuant software was used to obtain the band intensities for Gag (Pr78), Gag-Pro (p95), Gag-Pro-Pol (p180), and capsid (p27).…”

“…This means that instead of simultaneously extruding the membrane as they assemble, assembled betaretrovirus capsids must interact with the plasma membrane in a way that stimulates wrapping of the spherical structure with the lipid bilayer. The matrix (MA) domain of the M-PMV Gag polyprotein plays a critical role in this process (25), as well as in directing translating polysomes to the intracellular assembly site (32) and subsequent transport to the plasma membrane (8,25,27,32). MA is cotranslationally modified with myristic acid, a 14-carbon saturated fatty acid, and while myristylation of this domain is dispensable for capsid assembly, it is required for transport and release of capsids (26,29).…”

“…Based on a nuclear magnetic resonance (NMR) analysis of the M-PMV matrix protein, which is comprised of four helical domains arranged in two perpendicularly aligned pairs (9), and on an MA mutant (T41I/T78I) that accumulated immature capsids at the plasma membrane (25), it was previously hypothesized that the myristic acid moiety is sequestered within the MA domain prior to plasma membrane interactions (9). T41I/ T78I immature capsids are assembled and transported to the plasma membrane with wild-type kinetics but are defective at an early stage of budding (25).…”

“…T41I/ T78I immature capsids are assembled and transported to the plasma membrane with wild-type kinetics but are defective at an early stage of budding (25). Because the structural analyses showed that both of the threonine residues replaced in this mutant are oriented toward the core of the matrix protein, it was reasoned that substitution with the hydrophobic isoleucine residues might prevent release of myristic acid from the more hydrophobic MA core, even after the T41I/T78I capsid interacted with the lipid bilayer (9).…”