The Experts below are selected from a list of 906 Experts worldwide ranked by ideXlab platform
Eric Hunter - One of the best experts on this subject based on the ideXlab platform.
-
mason pfizer Monkey Virus envelope glycoprotein cycling and its vesicular co transport with immature particles
Viruses, 2018Co-Authors: Petra Grznarova Proksova, Eric Hunter, Michaela Rumlová, Jan Lipov, Jaroslav Zelenka, Hana Langerova, Tomáš RumlAbstract: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.
-
rock1 and lim kinase modulate retroVirus particle release and cell cell transmission events
Journal of Virology, 2014Co-Authors: Xiaoyun Wen, Eric Hunter, Lingmei Ding, Jaang Jiun Wang, Jason Hammonds, Hin Chu, Xuemin Chen, Paul SpearmanAbstract:The assembly and release of retroViruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retroVirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retroVirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also Virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1- and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retroVirus release from host cells and in cell-cell transmission events. IMPORTANCE Viruses often interact with the cellular cytoskeletal machinery in order to deliver their components to the site of assembly and budding. This study indicates that a key regulator of actin dynamics at the plasma membrane, LIM kinase, is important for the release of viral particles for HIV as well as for particle release by a distantly related retroVirus, Mason-Pfizer Monkey Virus. Moreover, disruption of LIM kinase greatly diminished the spread of HIV from cell to cell. These findings suggest that LIM kinase and its dynamic modulation of the actin cytoskeleton in the cell may be an important host factor for the production, release, and transmission of retroViruses.
-
A Mason-Pfizer Monkey Virus Gag-GFP Fusion Vector Allows Visualization of Capsid Transport in Live Cells and Demonstrates a Role for Microtubules
PloS one, 2013Co-Authors: Jasmine Clark, Tomáš Ruml, Petra Grznarova, Paul Spearman, Jan Lipov, Elizabeth Stansell, William E. Diehl, Eric HunterAbstract: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.
-
Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules.
Virology, 2013Co-Authors: Lara E. Pereira, Tomáš Ruml, Jasmine Clark, Petra Grznarova, Xiaoyun Wen, Rachel Lacasse, Paul Spearman, Eric HunterAbstract: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 4 h 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.
-
The impact of altered polyprotein ratios on the assembly and infectivity of Mason-Pfizer Monkey Virus.
Virology, 2009Co-Authors: Zdena Kohoutová, Eric Hunter, Michaela Rumlová, Martin Andreansky, Michael Sakalian, Iva Pichová, Tomáš RumlAbstract: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.
Tomáš Ruml - One of the best experts on this subject based on the ideXlab platform.
-
mason pfizer Monkey Virus envelope glycoprotein cycling and its vesicular co transport with immature particles
Viruses, 2018Co-Authors: Petra Grznarova Proksova, Eric Hunter, Michaela Rumlová, Jan Lipov, Jaroslav Zelenka, Hana Langerova, Tomáš RumlAbstract: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.
-
structure of the immature hiv 1 capsid in intact Virus particles at 8 8 a resolution
Nature, 2015Co-Authors: Florian K. M. Schur, Hans-georg Kräusslich, Barbara Müller, Michaela Rumlová, Tomáš Ruml, Wim J. H. Hagen, John A G BriggsAbstract:Human immunodeficiency Virus type 1 (HIV-1) assembly proceeds in two stages. First, the 55 kilodalton viral Gag polyprotein assembles into a hexameric protein lattice at the plasma membrane of the infected cell, inducing budding and release of an immature particle. Second, Gag is cleaved by the viral protease, leading to internal rearrangement of the Virus into the mature, infectious form. Immature and mature HIV-1 particles are heterogeneous in size and morphology, preventing high-resolution analysis of their protein arrangement in situ by conventional structural biology methods. Here we apply cryo-electron tomography and sub-tomogram averaging methods to resolve the structure of the capsid lattice within intact immature HIV-1 particles at subnanometre resolution, allowing unambiguous positioning of all α-helices. The resulting model reveals tertiary and quaternary structural interactions that mediate HIV-1 assembly. Strikingly, these interactions differ from those predicted by the current model based on in vitro-assembled arrays of Gag-derived proteins from Mason-Pfizer Monkey Virus. To validate this difference, we solve the structure of the capsid lattice within intact immature Mason-Pfizer Monkey Virus particles. Comparison with the immature HIV-1 structure reveals that retroviral capsid proteins, while having conserved tertiary structures, adopt different quaternary arrangements during Virus assembly. The approach demonstrated here should be applicable to determine structures of other proteins at subnanometre resolution within heterogeneous environments.
-
role of mason pfizer Monkey Virus ca nc spacer peptide like domain in assembly of immature particles
Journal of Virology, 2014Co-Authors: Karolina Strohalmovabohmova, Iva Pichová, Tomáš Ruml, Pavel Ulbrich, Vojtěch Spiwok, Martin Lepsik, Romana Hadravova, Ivana Křižova, Lucie Bednarova, Michaela RumlováAbstract:ABSTRACT The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroViruses. Published models of supramolecular structures of immature particles suggest that in lentiViruses and alpharetroViruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer Monkey Virus (M-PMV), a betaretroVirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroViruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-A cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE RetroViruses such as HIV-1 are of great medical importance. Using Mason-Pfizer Monkey Virus (M-PMV) as a model retroVirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retroVirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiViruses, alpharetroViruses, and betaretroViruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroViruses, such as HIV, Rous sarcoma Virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.
-
Interaction of Mason-Pfizer Monkey Virus matrix protein with plasma membrane
Frontiers in microbiology, 2014Co-Authors: Jan Prchal, Tomáš Ruml, Tomáš Kroupa, Richard HrabalAbstract:Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane through its N-terminal domain, the matrix protein. However, single generas of Retroviridae family differ in the way how they interact with plasma membrane. While in case of lentiViruses (e.g. human immunodeficiency Virus (HIV)) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, betaretroViruses (Mason-Pfizer Monkey Virus (M-PMV)) first assemble their Virus-like particles in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroViruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV matrix protein with plasma membrane with emphasis on the structural aspects of the interaction with single phospholipids.
-
Reviewed by:
2014Co-Authors: Jan Prchal, Tomáš Ruml, Tomáš Kroupa, Richard Hrabal, Atsushi KoitoAbstract:Budding is the final step of the late phase of retroviral life cycle. It begins with the interaction of Gag precursor with plasma membrane (PM) through its N-terminal domain, the matrix protein (MA). However, single genera of Retroviridae family differ in the way how they interact with PM. While in case of LentiViruses (e.g., human immunodeficiency Virus) the structural polyprotein precursor Gag interacts with cellular membrane prior to the assembly, BetaretroViruses [Mason-Pfizer Monkey Virus (M-PMV)] first assemble their Virus-like particles (VLPs) in the pericentriolar region of the infected cell and therefore, already assembled particles interact with the membrane. Although both these types of retroViruses use similar mechanism of the interaction of Gag with the membrane, the difference in the site of assembly leads to some differences in the mechanism of the interaction. Here we describe the interaction of M-PMV MA with PM with emphasis on the structural aspects of the interaction with single phospholipids
Hunter Eric - One of the best experts on this subject based on the ideXlab platform.
-
ROCK1 and LIM kinase modulate retroVirus particle release and cell-cell transmission events
'American Society for Microbiology', 2014Co-Authors: Wen Xiaoyun, Hunter Eric, Wang, Jaang Jiun, Ding Lingmei, Spearman Paul, Chen Xuemin, Chu Hin, Hammonds Jason, Qi MingliAbstract:The assembly and release of retroViruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retroVirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retroVirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer Monkey Virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also Virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1-and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retroVirus release from host cells and in cell-cell transmission events. © 2014, American Society for Microbiology.Link_to_subscribed_fulltex
-
The effect of point mutations within the N-terminal domain of Mason-Pfizer Monkey Virus capsid protein on Virus core assembly and infectivity
Elsevier Inc. Published by Elsevier Inc., 2008Co-Authors: Wildová Marcela, Hunter Eric, Ruml Tomáš, Pichová Iva, Hadravová Romana, Štokrová Jitka, Křížová Ivana, Rumlova MichaelaAbstract:AbstractRetroviral capsid protein (CA) mediates protein interactions driving the assembly of both immature viral particles and the core of the mature virions. Structurally conserved N-terminal domains of several retroViruses refold after proteolytic cleavage into a β-hairpin, stabilized by a salt bridge between conserved N-terminal Pro and Asp residues. Based on comparison with other retroviral CA, we identified Asp50 and Asp57 as putative interacting partners for Pro1 in Mason-Pfizer Monkey Virus (M-PMV) CA. To investigate the importance of CA Pro1 and its interacting Asp in M-PMV core assembly and infectivity, P1A, P1Y, D50A, T54A and D57A mutations were introduced into M-PMV. The P1A and D57A mutations partially blocked Gag processing and the released viral particles exhibited aberrant cores and were non-infectious. These data indicate that the region spanning residues Asp50–Asp57 plays an important role in stabilization of the β-hairpin and that Asp57 likely forms a salt-bridge with P1 in M-PMV CA
-
Distinct Roles for Nucleic Acid in In Vitro Assembly of Purified Mason-Pfizer Monkey Virus CANC Proteins
American Society for Microbiology, 2006Co-Authors: Ulbrich Pavel, Hunter Eric, Nermut Milan, Rumlova Michaela, Haubova Sarka, Ruml TomášAbstract: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 (ΔProCANC) 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 ΔProCANC 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 ΔProCANC 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 ΔProCANC 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
-
Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions
American Society for Microbiology, 2005Co-Authors: Song Chisu, Pichová Iva, Micoli Keith, Bauerova Helena, Hunter EricAbstract:Assembly of an infectious retroVirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer Monkey Virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on Virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions
-
An Early Stage of Mason-Pfizer Monkey Virus Budding Is Regulated by the Hydrophobicity of the Gag Matrix Domain Core
American Society for Microbiology, 2004Co-Authors: Stansell Elizabeth, Tytler Ewan, Walter, Mark R., Hunter EricAbstract:Intracellular capsid transport and release of Mason-Pfizer Monkey Virus are dependent on myristylation of the Gag matrix domain (MA). A myristylated MA mutant, in which Thr41 and Thr78 are replaced with isoleucines, assembles capsids that are transported to the plasma membrane but are blocked in an early budding step. Since the nuclear magnetic resonance structure of MA showed that these Thr residues point into the hydrophobic core of the protein, it was hypothesized that the T41I/T78I mutant was defective in release of myristic acid from the more hydrophobic core. In order to further investigate whether an increase in the hydrophobicity of the MA core modulates capsid-membrane interactions and viral budding, three tyrosine residues (11, 28, and 67), oriented toward the MA core, were replaced individually or in a pair-wise combination with the more hydrophobic phenylalanine residue(s). As a control, Tyr82, oriented toward the outer surface of MA, was also replaced with phenylalanine. These Tyr-to-Phe substitutions did not alter capsid assembly compared to wild type in a capsid assembly assay. Pulse-chase, immunofluorescence, and electron microscopy studies demonstrated that single substitutions of Tyr11, Tyr28, and Tyr67 recapitulated the T41I/T78I mutant phenotype of decreased budding kinetics and accumulation of capsids at the plasma membrane. MA double mutants with a combination of these Tyr substitutions exhibited a phenotype that was even more defective in budding. In contrast, MA mutants with Tyr82 replaced by Phe resulted in a transport-defective phenotype. These results strongly support the hypothesis that myristic acid is sequestered inside MA prior to capsid-membrane interactions
Iva Pichová - One of the best experts on this subject based on the ideXlab platform.
-
role of mason pfizer Monkey Virus ca nc spacer peptide like domain in assembly of immature particles
Journal of Virology, 2014Co-Authors: Karolina Strohalmovabohmova, Iva Pichová, Tomáš Ruml, Pavel Ulbrich, Vojtěch Spiwok, Martin Lepsik, Romana Hadravova, Ivana Křižova, Lucie Bednarova, Michaela RumlováAbstract:ABSTRACT The hexameric lattice of an immature retroviral particle consists of Gag polyprotein, which is the precursor of all viral structural proteins. Lentiviral and alpharetroviral Gag proteins contain a peptide sequence called the spacer peptide (SP), which is localized between the capsid (CA) and nucleocapsid (NC) domains. SP plays a critical role in intermolecular interactions during the assembly of immature particles of several retroViruses. Published models of supramolecular structures of immature particles suggest that in lentiViruses and alpharetroViruses, SP adopts a rod-like six-helix bundle organization. In contrast, Mason-Pfizer Monkey Virus (M-PMV), a betaretroVirus that assembles in the cytoplasm, does not contain a distinct SP sequence, and the CA-NC connecting region is not organized into a clear rod-like structure. Nevertheless, the CA-NC junction comprises a sequence critical for assembly of immature M-PMV particles. In the present work, we characterized this region, called the SP-like domain, in detail. We provide biochemical data confirming the critical role of the M-PMV SP-like domain in immature particle assembly, release, processing, and infectivity. Circular dichroism spectroscopy revealed that, in contrast to the SP regions of other retroViruses, a short SP-like domain-derived peptide (SPLP) does not form a purely helical structure in aqueous or helix-promoting solution. Using 8-A cryo-electron microscopy density maps of immature M-PMV particles, we prepared computational models of the SP-like domain and indicate the structural features required for M-PMV immature particle assembly. IMPORTANCE RetroViruses such as HIV-1 are of great medical importance. Using Mason-Pfizer Monkey Virus (M-PMV) as a model retroVirus, we provide biochemical and structural data confirming the general relevance of a short segment of the structural polyprotein Gag for retroVirus assembly and infectivity. Although this segment is critical for assembly of immature particles of lentiViruses, alpharetroViruses, and betaretroViruses, the organization of this domain is strikingly different. A previously published electron microscopic structure of an immature M-PMV particle allowed us to model this important region into the electron density map. The data presented here help explain the different packing of the Gag segments of various retroViruses, such as HIV, Rous sarcoma Virus (RSV), and M-PMV. Such knowledge contributes to understanding the importance of this region and its structural flexibility among retroviral species. The region might play a key role in Gag-Gag interactions, leading to different morphological pathways of immature particle assembly.
-
The impact of altered polyprotein ratios on the assembly and infectivity of Mason-Pfizer Monkey Virus.
Virology, 2009Co-Authors: Zdena Kohoutová, Eric Hunter, Michaela Rumlová, Martin Andreansky, Michael Sakalian, Iva Pichová, Tomáš RumlAbstract: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.
-
the rna binding g patch domain in retroviral protease is important for infectivity and d type morphogenesis of mason pfizer Monkey Virus
Journal of Biological Chemistry, 2005Co-Authors: Helena Bauerovazabranska, Eric Hunter, Tomáš Ruml, Jitka Stokrova, Kvido Strisovsky, Iva PichováAbstract:Retroviral proteases (PRs) cleave the viral polyprotein precursors into functional mature proteins late during particle release and are essential for viral replication. Unlike most retroViruses, beta-retroViruses, including Mason-Pfizer Monkey Virus (M-PMV), assemble immature capsids within the cytoplasm of the cell. The activation of beta-retroviral proteases must be highly regulated, because processing of the Gag-related polyprotein precursors occurs only after transport of immature capsids to the plasma membrane and budding. Several beta-retroviral proteases have unique C-terminal extension sequences, containing a glycine-rich motif (G-patch), which specifically binds in vitro to single-stranded nucleic acids. In M-PMV PR the G-patch is removed in vitro as well as in vivo by autoproteolytic processing to yield truncated active forms of PR. To investigate the role of the G-patch domain on the Virus life cycle, we introduced mutations within the C-terminal domain of protease. We found that the G-patch domain of M-PMV PR is not required for the processing of viral polyproteins, but it significantly influences the infectivity of M-PMV, the activity of reverse transcriptase, and assembly of immature capsid within the cells. These results demonstrate for the first time that the G-patch domain of M-PMV PR is critical for the life cycle of beta-retroViruses, and its evolutionary conservation within members of this genus suggests its importance for retroViruses that display D-type morphology.
-
Localization of self-interacting domains within betaretroVirus Gag polyproteins.
Virology, 2005Co-Authors: Aleš Zábranský, Michael Sakalian, Iva PichováAbstract:The BetaretroVirus genus is characterized by the ability to preassemble immature capsids within the cytoplasm. For Mason-Pfizer Monkey Virus (M-PMV) this ability depends in part upon the unique Internal Scaffold Domain (ISD) within the p12 region of Gag. In this study, we have further characterized the ability of M-PMV p12 to promote Gag-Gag interaction and have examined the Gag polyprotein of the related mouse mammary tumor Virus (MMTV) to potentially identify a region with equivalent function. Using the yeast two-hybrid system, we confirmed that both Gag polyproteins strongly interact, primarily through the CA-NC regions, but also through additional domains N-terminal to CA. For M-PMV, this auxiliary interaction domain was p12. For MMTV, no single strongly self-interacting protein was identified. Instead, MMTV Gag appears to utilize the weak contributions of several protein domains to support the main interaction of its CA-NC. Our findings suggest that, in addition to the canonical NC "I-domain" interaction, MMTV Gag self-association results from the concerted action of multiple regions of the polyprotein while M-PMV Gag relies mainly on its p12 domain.
-
and Biochemistry, Academy of Sciences of
2004Co-Authors: Zdeněk Krejčík, Tomáš Ruml, Věra Jenčová, Ivan Rosenberg, Jerry Alex, Alla Gustchina, Iva PichováAbstract:doi:10.1111/j.1432-1033.2004.04386.x The gene encoding an integrase of Mason–Pfizer Monkey Virus (M-PMV) is located at the 3¢-end of the pol open reading frame. The M-PMV integrase has not been previously isolated and characterized. We have now cloned, expressed, isolated, and characterized M-PMV integrase and compared its activities and primary structure with those of HIV-1 and other retroviral integrases. M-PMV integrase prefers untranslated 3¢-regionderived long-terminal repeat sequences in both the 3¢-processing and the strand transfer activity assays. While the 3¢-processing reaction catalyzed by M-PMV integrase was significantly increased in the presence of Mn 2+ and Co 2+ and was readily detectable in the presence of Mg 2+ and Ni 2+ cations, the strand transfer activity was strictly dependent only on Mn 2+. M-PMV integrase displays more relaxed substrate specificity than HIV-1 integrase, catalyzing the cleavage and the strand transfer of M-PMV and HIV-1 long-terminal repeat-derived substrates with simila
Bryan R. Cullen - One of the best experts on this subject based on the ideXlab platform.
-
The BetaretroVirus Mason-Pfizer Monkey Virus Selectively Excludes Simian APOBEC3G from Virion Particles
Journal of virology, 2006Co-Authors: Brian P Doehle, Heather L Wiegand, Nolwenn Jouvenet, Eric Hunter, Hal P Bogerd, Paul D Bieniasz, Bryan R. CullenAbstract:The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroViruses. Therefore, any retroVirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer Monkey Virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia Virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Schafer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroViruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroViruses.
-
inhibition of human immunodeficiency Virus rev and human t cell leukemia Virus rex function but not mason pfizer Monkey Virus constitutive transport element activity by a mutant human nucleoporin targeted to crm1
Journal of Virology, 1998Co-Authors: Hal P Bogerd, Bryan R. Cullen, Asier Echarri, Ted M RossAbstract:The hypothesis that the cellular protein Crm1 mediates human immunodeficiency Virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran · GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed ΔCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia Virus Rex-dependent gene expression. In contrast, ΔCAN had no significant affect on Mason-Pfizer Monkey Virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, ΔCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.