For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

• Keywords
• betaretrovirus, infectious disease, matrix protein, maturation, microbiology, myristoyl switch, protease, viruses,
• MeSH
• Cell Membrane MeSH
• Endopeptidases MeSH
• Gene Products, gag chemistry   MeSH
• Mason-Pfizer monkey virus * chemistry   physiology   MeSH
• Proteins MeSH
• Virus Assembly MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Endopeptidases MeSH
• Gene Products, gag MeSH
• Proteins MeSH

BACKGROUND: Myristoylation of the matrix (MA) domain mediates the transport and binding of Gag polyproteins to the plasma membrane (PM) and is required for the assembly of most retroviruses. In betaretroviruses, which assemble immature particles in the cytoplasm, myristoylation is dispensable for assembly but is crucial for particle transport to the PM. Oligomerization of HIV-1 MA stimulates the transition of the myristoyl group from a sequestered to an exposed conformation, which is more accessible for membrane binding. However, for other retroviruses, the effect of MA oligomerization on myristoyl group exposure has not been thoroughly investigated. RESULTS: Here, we demonstrate that MA from the betaretrovirus mouse mammary tumor virus (MMTV) forms dimers in solution and that this process is stimulated by its myristoylation. The crystal structure of N-myristoylated MMTV MA, determined at 1.57 Å resolution, revealed that the myristoyl groups are buried in a hydrophobic pocket at the dimer interface and contribute to dimer formation. Interestingly, the myristoyl groups in the dimer are mutually swapped to achieve energetically stable binding, as documented by molecular dynamics modeling. Mutations within the myristoyl binding site resulted in reduced MA dimerization and extracellular particle release. CONCLUSIONS: Based on our experimental, structural, and computational data, we propose a model for dimerization of MMTV MA in which myristoyl groups stimulate the interaction between MA molecules. Moreover, dimer-forming MA molecules adopt a sequestered conformation with their myristoyl groups entirely buried within the interaction interface. Although this differs from the current model proposed for lentiviruses, in which oligomerization of MA triggers exposure of myristoyl group, it appears convenient for intracellular assembly, which involves no apparent membrane interaction and allows the myristoyl group to be sequestered during oligomerization.

• MeSH
• Models, Biological MeSH
• Cell Line MeSH
• Rats MeSH
• Crystallography, X-Ray MeSH
• Humans MeSH
• Models, Molecular MeSH
• Protein Multimerization * MeSH
• Protein Processing, Post-Translational * MeSH
• Viral Matrix Proteins chemistry   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Mammary Tumor Virus, Mouse chemistry   physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Viral Matrix Proteins MeSH

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein.

• MeSH
• Cell Membrane metabolism   MeSH
• Phosphatidylinositol 4,5-Diphosphate metabolism   MeSH
• Mason-Pfizer monkey virus chemistry   MeSH
• Myristates chemistry   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Viral Matrix Proteins chemistry   metabolism   MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Phosphatidylinositol 4,5-Diphosphate MeSH
• Myristates MeSH
• Viral Matrix Proteins MeSH