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