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.

• Klíčová slova
• betaretrovirus, infectious disease, matrix protein, maturation, microbiology, myristoyl switch, protease, viruses,
• MeSH
• buněčná membrána MeSH
• endopeptidasy MeSH
• genové produkty gag chemie   MeSH
• Masonův-Pfizerův opičí virus * chemie   fyziologie   MeSH
• proteiny MeSH
• sestavení viru MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endopeptidasy MeSH
• genové produkty gag MeSH
• proteiny MeSH

Retroviral envelope glycoprotein (Env) is essential for the specific recognition of the host cell and the initial phase of infection. As reported for human immunodeficiency virus (HIV), the recruitment of Env into a retroviral membrane envelope is mediated through its interaction with a Gag polyprotein precursor of structural proteins. This interaction, occurring between the matrix domain (MA) of Gag and the cytoplasmic tail (CT) of the transmembrane domain of Env, takes place at the host cell plasma membrane. To determine whether the MA of Mason-Pfizer monkey virus (M-PMV) also interacts directly with the CT of Env, we mimicked the in vivo conditions in an in vitro experiment by using a CT in its physiological trimeric conformation mediated by the trimerization motif of the GCN4 yeast transcription factor. The MA protein was used at the concentration shifting the equilibrium to its trimeric form. The direct interaction between MA and CT was confirmed by a pulldown assay. Through the combination of nuclear magnetic resonance (NMR) spectroscopy and protein cross-linking followed by mass spectrometry analysis, the residues involved in mutual interactions were determined. NMR has shown that the C terminus of the CT is bound to the C-terminal part of MA. In addition, protein cross-linking confirmed the close proximity of the N-terminal part of CT and the N terminus of MA, which is enabled in vivo by their location at the membrane. These results are in agreement with the previously determined orientation of MA on the membrane and support the already observed mechanisms of M-PMV virus-like particle transport and budding.IMPORTANCE By a combination of nuclear magnetic resonance (NMR) and mass spectroscopy of cross-linked peptides, we show that in contrast to human immunodeficiency virus type 1 (HIV-1), the C-terminal residues of the unstructured cytoplasmic tail of Mason-Pfizer monkey virus (M-PMV) Env interact with the matrix domain (MA). Based on biochemical data and molecular modeling, we propose that individual cytoplasmic tail (CT) monomers of a trimeric complex bind MA molecules belonging to different neighboring trimers, which may stabilize the MA orientation at the membrane by the formation of a membrane-bound net of interlinked Gag and CT trimers. This also corresponds with the concept that the membrane-bound MA of Gag recruits Env through interaction with the full-length CT, while CT truncation during maturation attenuates the interaction to facilitate uncoating. We propose a model suggesting different arrangements of MA-CT complexes between a D-type and C-type retroviruses with short and long CTs, respectively.

• Klíčová slova
• Env, M-PMV, Retrovirus, cytoplasmic tail, protein interactions,
• MeSH
• genové produkty env chemie   genetika   MeSH
• genové produkty gag chemie   genetika   MeSH
• Masonův-Pfizerův opičí virus chemie   genetika   MeSH
• proteinové domény MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty env MeSH
• genové produkty gag MeSH

Retroviral Gag polyproteins are targeted to the inner leaflet of the plasma membrane through their N-terminal matrix (MA) domain. Because retroviruses of different morphogenetic types assemble their immature particles in distinct regions of the host cell, the mechanism of MA-mediated plasma membrane targeting differs among distinct retroviral morphogenetic types. Here, we focused on possible mechanistic differences of the MA-mediated plasma membrane targeting of the B-type mouse mammary tumor virus (MMTV) and C-type HIV-1, which assemble in the cytoplasm and at the plasma membrane, respectively. Molecular dynamics simulations, together with surface mapping, indicated that, similarly to HIV-1, MMTV uses a myristic switch to anchor the MA to the membrane and electrostatically interacts with phosphatidylinositol 4,5-bisphosphate to stabilize MA orientation. We observed that the affinity of MMTV MA to the membrane is lower than that of HIV-1 MA, possibly related to their different topologies and the number of basic residues in the highly basic MA region. The latter probably reflects the requirement of C-type retroviruses for tighter membrane binding, essential for assembly, unlike for D/B-type retroviruses, which assemble in the cytoplasm. A comparison of the membrane topology of the HIV-1 MA, using the surface-mapping method and molecular dynamics simulations, revealed that the residues at the HIV-1 MA C terminus help stabilize protein-protein interactions within the HIV-1 MA lattice at the plasma membrane. In summary, HIV-1 and MMTV share common features such as membrane binding of the MA via hydrophobic interactions and exhibit several differences, including lower membrane affinity of MMTV MA.

• Klíčová slova
• Gag polyprotein, HIV-1, coarse-grained molecular dynamics, covalent labeling–mass spectrometry, human immunodeficiency virus (HIV), lipid–protein interaction, mass spectrometry (MS), matrix protein, membrane binding, molecular dynamics, mouse mammary tumor virus (MMTV), particle assembly, retrovirus, viral replication,
• MeSH
• buněčná membrána metabolismus   patologie   MeSH
• HIV infekce metabolismus   patologie   MeSH
• HIV-1 fyziologie   MeSH
• infekce onkogenními viry metabolismus   patologie   MeSH
• interakce hostitele a patogenu MeSH
• lidé MeSH
• molekulární modely MeSH
• myši MeSH
• retrovirové infekce metabolismus   patologie   MeSH
• sestavení viru MeSH
• virus myšího tumoru prsní žlázy fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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
• biologické modely MeSH
• buněčné linie MeSH
• krysa rodu Rattus MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární modely MeSH
• multimerizace proteinu * MeSH
• posttranslační úpravy proteinů * MeSH
• proteiny virové matrix chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• virus myšího tumoru prsní žlázy chemie   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny virové matrix 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
• buněčná membrána metabolismus   MeSH
• fosfatidylinositol-4,5-difosfát metabolismus   MeSH
• Masonův-Pfizerův opičí virus chemie   MeSH
• myristáty chemie   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny virové matrix chemie   metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fosfatidylinositol-4,5-difosfát MeSH
• myristáty MeSH
• proteiny virové matrix MeSH

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

• MeSH
• acyltransferasy chemie   genetika   izolace a purifikace   MeSH
• Escherichia coli enzymologie   genetika   MeSH
• exprese genu MeSH
• kyselina myristová chemie   MeSH
• Masonův-Pfizerův opičí virus chemie   genetika   MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny virové matrix chemie   genetika   izolace a purifikace   MeSH
• rekombinantní fúzní proteiny chemie   genetika   izolace a purifikace   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acyltransferasy MeSH
• glycylpeptide N-tetradecanoyltransferase MeSH Prohlížeč
• kyselina myristová MeSH
• proteiny virové matrix MeSH
• rekombinantní fúzní proteiny MeSH

The oligomerization capacity of the retroviral matrix protein is an important feature that affects assembly of immature virions and their interaction with cellular membrane. A combination of NMR relaxation measurements and advanced analysis of molecular dynamics simulation trajectory provided an unprecedentedly detailed insight into internal mobility of matrix proteins of the Mason-Pfizer monkey virus. Strong evidence have been obtained that the oligomerization capacity of the wild-type matrix protein is closely related to the enhanced dynamics of several parts of its backbone on a nanosecond time scale. Increased flexibility has been observed for two regions: the loop between α-helices α2 and α3 and the C-terminal half of α-helix α3 which accommodate amino acid residues that form the oligomerization interface. On the other hand, matrix mutant R55F that has changed structure and does not exhibit any specific oligomerization in solution was found considerably more rigid. Our results document that conformational selection mechanism together with induced fit and favorable structural preorganization play an important role in the control of the oligomerization process.

• MeSH
• aminokyseliny chemie   genetika   MeSH
• kvarterní struktura proteinů MeSH
• Masonův-Pfizerův opičí virus chemie   MeSH
• multimerizace proteinu * MeSH
• nukleární magnetická rezonance biomolekulární MeSH
• proteiny virové matrix chemie   MeSH
• simulace molekulární dynamiky MeSH
• substituce aminokyselin MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• aminokyseliny MeSH
• proteiny virové matrix MeSH