The envelope glycoprotein (Env) plays a crucial role in the retroviral life cycle by mediating primary interactions with the host cell. As described previously and expanded on in this paper, Env mediates the trafficking of immature Mason-Pfizer monkey virus (M-PMV) particles to the plasma membrane (PM). Using a panel of labeled RabGTPases as endosomal markers, we identified Env mostly in Rab7a- and Rab9a-positive endosomes. Based on an analysis of the transport of recombinant fluorescently labeled M-PMV Gag and Env proteins, we propose a putative mechanism of the intracellular trafficking of M-PMV Env and immature particles. According to this model, a portion of Env is targeted from the trans-Golgi network (TGN) to Rab7a-positive endosomes. It is then transported to Rab9a-positive endosomes and back to the TGN. It is at the Rab9a vesicles where the immature particles may anchor to the membranes of the Env-containing vesicles, preventing Env recycling to the TGN. These Gag-associated vesicles are then transported to the plasma membrane.
- MeSH
- AIDS opičí virologie MeSH
- buněčná membrána metabolismus virologie MeSH
- endozomy metabolismus virologie MeSH
- genové produkty env genetika metabolismus MeSH
- Masonův-Pfizerův opičí virus genetika fyziologie MeSH
- sestavení viru MeSH
- transport proteinů MeSH
- transportní vezikuly metabolismus virologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Matrix proteins (MAs) play a key role in the transport of retroviral proteins inside infected cells and in the interaction with cellular membranes. In most retroviruses, retroviral MAs are N-terminally myristoylated. This modification serves as a membrane targeting signal and also as an anchor for membrane interaction. The aim of this work was to characterize the interactions anchoring retroviral MA at the plasma membrane of infected cell. To address this issue, we compared the structures and membrane affinity of the Mason-Pfizer monkey virus (M-PMV) wild-type MA with its two budding deficient double mutants, that is, T41I/T78I and Y28F/Y67F. The structures of the mutants were determined using solution NMR spectroscopy, and their interactions with water-soluble phospholipids were studied. Water-soluble phospholipids are widely used models for studying membrane interactions by solution NMR spectroscopy. However, this approach might lead to artificial results due to unnatural hydrophobic interactions. Therefore, we used a new approach based on the measurement of the loss of the (1)H NMR signal intensity of the protein sample induced by the addition of the liposomes containing phospholipids with naturally long fatty acids. HIV-1 MA was used as a positive control because its ability to interact with liposomes has already been described. We found that in contrast to HIV-1, the M-PMV MA interacted with the liposomes differently and much weaker. In our invivo experiments, the M-PMV MA did not co-localize with lipid rafts. Therefore, we concluded that M-PMV might adopt a different membrane binding mechanism than HIV-1.
- MeSH
- buněčná membrána metabolismus MeSH
- fosfolipidy metabolismus MeSH
- liposomy metabolismus MeSH
- magnetická rezonanční spektroskopie MeSH
- Masonův-Pfizerův opičí virus fyziologie MeSH
- mastné kyseliny metabolismus MeSH
- missense mutace MeSH
- mutantní proteiny chemie metabolismus MeSH
- proteiny virové matrix chemie metabolismus MeSH
- uvolnění viru z buňky * MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články 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.
- 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
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
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
Most retroviruses employ a frameshift mechanism during polyprotein synthesis to balance appropriate ratios of structural proteins and enzymes. To investigate the requirements for individual precursors in retrovirus assembly, we modified the polyprotein repertoire of Mason-Pfizer monkey virus (M-PMV) by mutating the frameshift sites to imitate the polyprotein organization of Rous sarcoma virus (Gag-Pro and Gag-Pro-Pol) or Human immunodeficiency virus (Gag and Gag-Pro-Pol). For the "Rous-like" virus, assembly was impaired with no incorporation of Gag-Pro-Pol into particles and for the "HIV-like" virus an altered morphogenesis was observed. A mutant expressing Gag and Gag-Pro polyproteins and lacking Gag-Pro-Pol assembled intracellular particles at a level similar to the wild-type. Gag-Pro-Pol polyprotein alone neither formed immature particles nor processed the precursor. All the mutants were non-infectious except the "HIV-like", which retained fractional infectivity.
- MeSH
- AIDS opičí virologie MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- financování organizované MeSH
- genové produkty gag genetika MeSH
- genové produkty pol genetika MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika patogenita MeSH
- messenger RNA genetika MeSH
- posunová mutace MeSH
- proteosyntéza MeSH
- RNA virová genetika MeSH
- transfekce MeSH
- virion genetika patogenita MeSH
- virové proteiny genetika MeSH
- virus Rousova sarkomu genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata 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
- financování organizované MeSH
- genomová oblast t-komplexu MeSH
- jaderné proteiny fyziologie chemie metabolismus MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus metabolismus MeSH
- mutace MeSH
- proteiny asociované s mikrotubuly fyziologie chemie metabolismus MeSH
- Retroviridae metabolismus MeSH
- terciární struktura proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
In contrast to other retroviruses, Mason-Pfizer monkey virus (M-PMV) assembles immature capsids in the cytoplasm. We have compared the ability of minimal assembly-competent domains from M-PMV and human immunodeficiency virus type 1 (HIV-1) to assemble in vitro into virus-like particles in the presence and absence of nucleic acids. A fusion protein comprised of the capsid and nucleocapsid domains of Gag (CANC) and its N-terminally modified mutant (DeltaProCANC) were used to mimic the assembly of the viral core and immature particles, respectively. In contrast to HIV-1, where CANC assembled efficiently into cylindrical structures, the same domains of M-PMV were assembly incompetent. The addition of RNA or oligonucleotides did not complement this defect. In contrast, the M-PMV DeltaProCANC molecule was able to assemble into spherical particles, while that of HIV-1 formed both spheres and cylinders. For M-PMV, the addition of purified RNA increased the efficiency with which DeltaProCANC formed spherical particles both in terms of the overall amount and the numbers of completed spheres. The amount of RNA incorporated was determined, and for both rRNA and MS2-RNA, quantities similar to that of genomic RNA were encapsidated. Oligonucleotides also stimulated assembly; however, they were incorporated into DeltaProCANC spherical particles in trace amounts that could not serve as a stoichiometric structural component for assembly. Thus, oligonucleotides may, through a transient interaction, induce conformational changes that facilitate assembly, while longer RNAs appear to facilitate the complete assembly of spherical particles.
- MeSH
- bezbuněčný systém MeSH
- financování organizované MeSH
- genom virový fyziologie MeSH
- genové produkty gag genetika metabolismus MeSH
- HIV-1 fyziologie izolace a purifikace ultrastruktura MeSH
- kapsida metabolismus ultrastruktura MeSH
- konformace proteinů MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus fyziologie izolace a purifikace MeSH
- mutace MeSH
- oligonukleotidy genetika metabolismus MeSH
- RNA ribozomální genetika metabolismus MeSH
- RNA virová genetika metabolismus ultrastruktura MeSH
- sestavení viru fyziologie MeSH
- terciární struktura proteinů účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
