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

Abstract

Intracellular capsid transport and release of Mason-Pfizer monkey virus are dependent on myristylation of the Gag matrix domain (MA). A myristylated MA mutant, in which Thr41 and Thr78 are replaced with isoleucines, assembles capsids that are transported to the plasma membrane but are blocked in an early budding step. Since the nuclear magnetic resonance structure of MA showed that these Thr residues point into the hydrophobic core of the protein, it was hypothesized that the T41I/T78I mutant was defective in release of myristic acid from the more hydrophobic core. In order to further investigate whether an increase in the hydrophobicity of the MA core modulates capsid-membrane interactions and viral budding, three tyrosine residues (11, 28, and 67), oriented toward the MA core, were replaced individually or in a pair-wise combination with the more hydrophobic phenylalanine residue(s). As a control, Tyr82, oriented toward the outer surface of MA, was also replaced with phenylalanine. These Tyr-to-Phe substitutions did not alter capsid assembly compared to wild type in a capsid assembly assay. Pulse-chase, immunofluorescence, and electron microscopy studies demonstrated that single substitutions of Tyr11, Tyr28, and Tyr67 recapitulated the T41I/T78I mutant phenotype of decreased budding kinetics and accumulation of capsids at the plasma membrane. MA double mutants with a combination of these Tyr substitutions exhibited a phenotype that was even more defective in budding. In contrast, MA mutants with Tyr82 replaced by Phe resulted in a transportdefective phenotype. These results strongly support the hypothesis that myristic acid is sequestered inside MA prior to capsid-membrane interactions.

Basic Residues in the Mason-Pfizer Monkey Virus Gag Matrix Domain Regulate Intracellular Trafficking and Capsid-Membrane Interactions

“…MA mutants that have a more hydrophobic core are myristylated and transported to the plasma membrane but are defective at an early stage of budding. The phenotype of these mutants is consistent with the hypothesis that myristic acid is sequestered within the MA domain upon capsid assembly to yield a protein conformation that is conducive for both capsid transport and initiation of envelopment at the plasma membrane (40,56).…”

“…This is in contrast to wild-type kinetics, in which 50% of Gag was cleaved at 1.5 h and 60% was released as p27 in the 4-h chase. Within the cell, K25A immature capsids were primarily dispersed throughout the cytoplasm; this phenotype is similar to the previously characterized Y82F transport-defective MA mutant (56). In contrast to Y82F, however, the K25A mutant capsids did not accumulate in the pericentriolar region of the cell, suggesting that capsids can initiate intracellular transport but are inefficiently transferred to the plasma membrane.…”

“…Mutant derivatives of the M-PMV proviral vector were constructed using pNCS (56), a plasmid containing a gag fragment corresponding to nucleotides 351 to 1167 of pSARM4 (51). The desired codon(s) was generated in pNCS by PCR-directed mutagenesis.…”

“…Unlike the latter retroviruses, which simultaneously assemble their protein shell and extrude the plasma membrane, M-PMV must wrap the lipid bilayer around a 90-nm preformed protein shell. We have hypothesized previously (40,56) that a driving force for this wrapping process could be the exposure and insertion of the 14-carbon saturated fatty acid (myristate) moiety, which is covalently attached to a glycine residue at position 2 of the Gag polyprotein, into the plasma membrane. Myristylation of MA is not required for immature capsid assembly but is needed for transport and release of capsids; a glycine-to-valine mutant that is defective for myristic acid attachment assembles capsids, but these remain at a perinuclear region of the cell (41).…”

Defect of Human Immunodeficiency Virus Type 2 Gag Assembly in Saccharomyces cerevisiae

“…Mutations of hydrophobic residues near the N terminus of MA to less hydrophobic residues severely impaired membrane binding of Gag without inhibiting N-terminal myristoylation, suggesting a failure of membrane insertion of the myristoyl moiety (36). Interestingly, in Mason-Pfizer monkey virus, a prototype for capsid formation prior to membrane relocation, an increase in the hydrophobicity of MA led to arrest at an early stage of particle budding, possibly by inhibiting exposure of the N-terminal myristoyl moiety (45). Thus, it cannot be ruled out that in our study, the absence of salt may have caused a disruption of the gross conformation of Gag (e.g., unfolding of the hydrophobic core of MA) and/or led to sequestration of the N-terminal myristoyl moiety.…”