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

In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription.IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse transcription. Mutations that change the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, an enhancement of the basic character of this region increased both the efficiency of gRNA packaging and the infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. In summary, the basic region in M-PMV Gag plays a key role in the packaging of genomic RNA and, consequently, in assembly and reverse transcription.

• Klíčová slova
• M-PMV, RNA packaging, assembly, basic residues, human immunodeficiency virus, infectivity, nucleocapsid, retroviruses, reverse transcription,
• MeSH
• buněčné linie MeSH
• genové produkty gag genetika   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   fyziologie   MeSH
• mutace genetika   MeSH
• nukleokapsida - proteiny genetika   MeSH
• reverzní transkripce genetika   MeSH
• RNA virová genetika   MeSH
• sekvence aminokyselin genetika   MeSH
• sestavení viru genetika   MeSH
• zinkové prsty genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• genové produkty gag MeSH
• nukleokapsida - proteiny MeSH
• RNA virová MeSH

The intracellular transport of Mason-Pfizer monkey virus (M-PMV) assembled capsids from the pericentriolar region to the plasma membrane (PM) requires trafficking of envelope glycoprotein (Env) to the assembly site via the recycling endosome. However, it is unclear if Env-containing vesicles play a direct role in trafficking capsids to the PM. Using live cell microscopy, we demonstrate, for the first time, anterograde co-transport of Gag and Env. Nocodazole disruption of microtubules had differential effects on Gag and Env trafficking, with pulse-chase assays showing a delayed release of Env-deficient virions. Particle tracking demonstrated an initial loss of linear movement of GFP-tagged capsids and mCherry-tagged Env, followed by renewed movement of Gag but not Env at 4h post-treatment. Thus, while delayed capsid trafficking can occur in the absence of microtubules, efficient anterograde transport of capsids appears to be mediated by microtubule-associated Env-containing vesicles.

• Klíčová slova
• Anterograde transport, Cytoskeleton, Envelope, Gag, Live cell-imaging, M-PMV,
• MeSH
• AIDS opičí metabolismus   virologie   MeSH
• buněčná membrána virologie   MeSH
• Cercopithecus aethiops MeSH
• genové produkty env genetika   metabolismus   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• Macaca mulatta MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• transport proteinů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• genové produkty env MeSH
• genové produkty gag MeSH

Immature capsids of the Betaretrovirus, Mason-Pfizer Monkey virus (M-PMV), are assembled in the pericentriolar region of the cell, and are then transported to the plasma membrane for budding. Although several studies, utilizing mutagenesis, biochemistry, and immunofluorescence, have defined the role of some viral and host cells factors involved in these processes, they have the disadvantage of population analysis, rather than analyzing individual capsid movement in real time. In this study, we created an M-PMV vector in which the enhanced green fluorescent protein, eGFP, was fused to the carboxyl-terminus of the M-PMV Gag polyprotein, to create a Gag-GFP fusion that could be visualized in live cells. In order to express this fusion protein in the context of an M-PMV proviral backbone, it was necessary to codon-optimize gag, optimize the Kozak sequence preceding the initiating methionine, and mutate an internal methionine codon to one for alanine (M100A) to prevent internal initiation of translation. Co-expression of this pSARM-Gag-GFP-M100A vector with a WT M-PMV provirus resulted in efficient assembly and release of capsids. Results from fixed-cell immunofluorescence and pulse-chase analyses of wild type and mutant Gag-GFP constructs demonstrated comparable intracellular localization and release of capsids to untagged counterparts. Real-time, live-cell visualization and analysis of the GFP-tagged capsids provided strong evidence for a role for microtubules in the intracellular transport of M-PMV capsids. Thus, this M-PMV Gag-GFP vector is a useful tool for identifying novel virus-cell interactions involved in intracellular M-PMV capsid transport in a dynamic, real-time system.

• MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• genetické vektory genetika   MeSH
• genové produkty gag genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• kapsida metabolismus   MeSH
• kinetika MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus genetika   metabolismus   fyziologie   MeSH
• mikrotubuly metabolismus   virologie   MeSH
• molekulární zobrazování MeSH
• pohyb MeSH
• proviry genetika   metabolismus   fyziologie   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• sestavení viru MeSH
• transport proteinů MeSH
• viabilita buněk MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• fluorescenční barviva MeSH
• genové produkty gag MeSH
• rekombinantní fúzní proteiny MeSH
• zelené fluorescenční proteiny MeSH

Despite extensive data demonstrating that immature retroviral particle assembly can take place either at the plasma membrane or at a distinct location within the cytoplasm, targeting of viral precursor proteins to either assembly site still remains poorly understood. Biochemical data presented here suggest that Tctex-1, a light chain of the molecular motor dynein, is involved in the intracellular targeting of Mason-Pfizer monkey virus (M-PMV) polyproteins to the cytoplasmic assembly site. Comparison of the three-dimensional structures of M-PMV wild-type matrix protein (wt MA) with a single amino acid mutant (R55F), which redirects assembly from a cytoplasmic site to the plasma membrane, revealed different mutual orientations of their C- and N-terminal domains. This conformational change buries a putative intracellular targeting motif located between both domains in the hydrophobic pocket of the MA molecule, thereby preventing the interaction with cellular transport mechanisms.

• MeSH
• biologické modely MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   virologie   MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• cytoplazma metabolismus   MeSH
• dyneiny metabolismus   MeSH
• fenotyp MeSH
• genomová oblast t-komplexu MeSH
• jaderné proteiny chemie   metabolismus   fyziologie   MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus metabolismus   MeSH
• mutace MeSH
• proteiny asociované s mikrotubuly chemie   metabolismus   fyziologie   MeSH
• Retroviridae metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dyneiny MeSH
• jaderné proteiny MeSH
• proteiny asociované s mikrotubuly MeSH

Mason-Pfizer monkey virus (M-PMV) Gag protein contains a domain p12 that is unique to this virus (simian retrovirus-3) and its close relatives. The alpha-helical N-terminal half of p12, which contains a leucine zipper-like region, forms ordered structures in E. coli and the C-terminal half can form SDS-resistant oligomers in vitro. Together these properties suggest that p12 is a strong protein-protein interaction domain that facilitates Gag-Gag oligomerization. We have analyzed the oligomerization potential of a panel of p12 mutants, including versions containing substituted dimer, trimer, and tetramer leucine zippers, expressed in bacteria and in the context of the Gag precursor expressed in vitro and in cells. Purified recombinant p12 and its mutants could form various oligomers as shown by chemical cross-linking experiments. Within Gag these same mutants could assemble when overexpressed in cells. In contrast, all the mutants, including the leucine zipper mutants, were assembly defective in a cell-free system. These data highlight the importance of a region containing alternating leucines and isoleucines within p12, but also indicate that this domain's scaffold-like function is more complex than small number oligomerization.

• MeSH
• Cercopithecus aethiops MeSH
• COS buňky MeSH
• genové produkty gag genetika   metabolismus   MeSH
• leucinové zipy MeSH
• Masonův-Pfizerův opičí virus genetika   fyziologie   ultrastruktura   MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• rekombinantní proteiny genetika   izolace a purifikace   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transmisní elektronová mikroskopie MeSH
• vazba proteinů MeSH
• virion ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• genové produkty gag MeSH
• p12 protein, Mason-Pfizer monkey virus MeSH Prohlížeč
• rekombinantní proteiny MeSH

The capsid precursor protein (Gag) of Mason-Pfizer monkey virus, the prototype type D retrovirus, has been expressed to high levels in bacteria under the control of the phage T7 promoter. Electron microscopic studies of induced cells revealed the assembly of capsid-like structures within inclusion bodies that formed at the poles of the cells 6 h after induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). The inclusion bodies and enclosed capsid-like structures were solubilized completely in 8 M urea, but following renaturation, we observed assembly in vitro of capsid-like structures that demonstrated apparent icosahedral symmetry. These results demonstrate for the first time that retroviral capsid precursors have the propensity to self-assemble in vitro and point to new approaches for the analysis of retroviral assembly and structure.

• MeSH
• buněčné inkluze virové MeSH
• Escherichia coli genetika   ultrastruktura   MeSH
• genové produkty gag metabolismus   MeSH
• HeLa buňky MeSH
• kapsida metabolismus   MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus metabolismus   ultrastruktura   MeSH
• proteinové prekurzory metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• genové produkty gag MeSH
• proteinové prekurzory MeSH