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Paul A. Luciw - One of the best experts on this subject based on the ideXlab platform.
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Transactivation of the two promoters of SFV-3 by different mechanisms.
Virology, 1996Co-Authors: Rolf Renne, Paul A. Luciw, Ute Fleps, Dieter Neumann-haefelinAbstract:Abstract Simian foamy virus type 3 (SFV-3), a member of the Spumavirus genus of retroviruses, has a complex genome organization and encodes two open reading frames (ORFs), in addition to the structural genesgag, pol,andenv.ORF-1 encodes a viral transcriptional transactivator designated Taf (transactivator of foamy viruses) which augments transcription from the viral long terminal repeat (LTR). It was recently shown that human foamy virus, as well as the simian viruses SFV-1 and SFV-3, contains a second internal transcriptional promoter in the transmembrane domain of theenvgene; this promoter also is transactivated by Taf. Here we report the characterization of the internal promoter of SFV-3. The transcriptional start site of this promoter has been mapped in two different SFV-3-infected cell lines to nt position 9761 in the proviral genome of SFV-3. Allcis-regulatory elements required for transactivation by Taf are located between −202 and −32 (+1 representing the transcription initiation site in the internal promoter). Analysis of hybrid promoter constructs and deletion mutants in transient expression assays revealed that this region contains two elements which are independently responsive to Taf. In addition, we employed anin vivoDNA competition assay to determine whether the transactivation mechanisms of both SFV-3 promoters are similar or different. The differences observed utilizing this competition assay suggest that Taf transactivates the internal promoter and the LTR through different cellular transcription factors.
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Characterization of the internal promoter of simian foamy viruses.
Journal of virology, 1994Co-Authors: Mel Campbell, Lisa W. Renshaw-gegg, Rolf Renne, Paul A. LuciwAbstract:Simian and human foamy viruses (HFV and SFV), genetically related members of the Spumavirus genus of retroviruses, have complex genome structures which encode the gag, pol, and env genes for virion proteins as well as additional open reading frames. One of these open reading frames is a viral transactivator, encoded by genes designated taf for SFV and bel-1 for HFV, which augments transcription directed by the long terminal repeat (LTR) through cis-acting targets in the U3 domain of the LTR. Recently, an internal transcriptional promoter has been identified in sequences within the 3' end of the HFV env gene (M. Lochelt, W. Muranyi, and R. M. Flugel, Proc. Natl. Acad. Sci. 90:7317-7321, 1993). We have demonstrated by using transient expression assays in several tissue culture cell lines and by analyzing viral transcripts in infected cells that SFV-1 from a rhesus macaque and SFV-3 from an African green monkey also encode an internal promoter in the env gene. Transcription directed by the internal promoters of SFV-1 and SFV-3 is activated by the taf-1 and taf-3 gene products, respectively, in several cell types. The importance of a TATA box for the SFV-1 internal promoter was established by site-specific mutagenesis, and the 5' ends of transcripts initiating in the internal promoter have been determined. cis-acting sequences in the SFV-1 env gene required for the response to taf-1 are contained within a 121-bp element located 5' to the TATA box in the internal promoter. This taf-1-responsive element in the internal promoter functions in a position- and orientation-independent fashion in a heterologous promoter and thus has the properties of an enhancer which depends on taf-1 activity. Alignments reveal that the SFV-1 internal promoter and the SFV-1 LTR have little sequence relatedness. Cross-transactivation studies show that the transactivators of SFV-1 and HFV function on the internal promoter and LTR of the homologous virus but not on the heterologous virus. In summary, the genomes of simian and human foamy viruses direct viral transcription through both the promoter in the LTR and an internal promoter within the env gene, and each promoter contains unique enhancer-like elements regulated by the viral transactivator.
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Regulatory elements in the long terminal repeat (LTR) of simian foamy virus type 3 (SFV-3).
Virology, 1993Co-Authors: Rolf Renne, Ayalew Mergia, Dieter Neumann-haefelin, Lisa W. Renshaw-gegg, Paul A. LuciwAbstract:Abstract Simian foamy virus type 3 (SFV-3) is a retrovirus that has a complex genome organization and encodes two open reading frames (ORF-1 and ORF-2) in addition to the genes coding for gag, pol , and env . In this report, we demonstrate that ORF-1 of SFV-3 encodes a transcriptional transactivator designated taf (transactivator of foamy virus) which augments gene expression directed by the viral long terminal repeat (LTR). The taf responsive elements have been mapped to the U3 region of the LTR, between positions —637 and —180 (+1 represents the transcription initiation site). Two regions between —637 and —180 in the LTR are targets for taf transactivation. These target sequences for taf confer responsiveness to a heterologous promoter independent of orientation; thus, they function like conditional enhancers. The R-U5 region of the viral LTR is shown to have an inhibitory effect on gene expression. SFV-1 is a related Spumavirus and encodes a taf gene that augments expression directed by the SFV-3 LTR as well as the SFV-1 LTR; however, the taf gene of SFV-3 transactivates the SFV-3 LTR but not the SFV-1 LTR. These data on regulatory elements in the SFV-3 LTR show that the mechanism of foamy virus transactivation is significantly different from lentiviruses as well as from the HTLV group of viruses.
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Identification of the simian foamy virus transcriptional transactivator gene (taf).
Journal of Virology, 1991Co-Authors: Ayalew Mergia, Karen E. S. Shaw, Elissa Pratt-lowe, Peter A. Barry, Paul A. LuciwAbstract:Simian foamy virus type 1 (SFV-1), a member of Spumavirus subfamily of retroviruses, encodes a transcriptional transactivator that functions to strongly augment gene expression directed by the viral long terminal repeat (LTR). The objective of this study was to identify the viral gene responsible for transactivation. Nucleotide sequences between the env gene and the LTR of SFV-1 were determined. The predicted amino acid sequence revealed two large open reading frames (ORFs), designated ORF-1 (311 amino acids) and ORF-2 (422 amino acids). In the corresponding region of the human foamy virus, three ORFs (bel-1, bel-2, and bel-3) have been identified (R. M. Flugel, A. Rethwilm, B. Maurer, and G. Darai, EMBO J. 6:2077-2084, 1987). Pairwise comparisons of the ORF-1 and ORF-2 with bel-1 and bel-2 show small clusters of homology; less than 39% overall homology of conserved amino acids is observed. A counterpart for human foamy virus bel-3 is not present in the SFV-1 sequence. Three species of viral RNA have been identified in cells infected with SFV-1; an 11.5-kb RNA representing full-length transcripts, a 6.5-kb RNA representing the env message, and a 2.8-kb RNA from the ORF region. Analysis of a cDNA clone encoding the ORF region of SFV-1 reveals that the 2.8-kb message is generated by complex splicing events involving the 3' end of the env gene. In transient expression assays in cell lines representing several species. ORF-1 was shown to be necessary and sufficient for transactivating viral gene expression directed by the SFV-1 LTR. The target for transactivation is located in the U3 domain of the LTR, upstream from position - 125 (+ 1 represents the transcription initiation site). We propose that OFF-1 of SFV-1 be designated the transcriptional transactivator of foamy virus (taf).
Peter Cherepanov - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for Spumavirus GAG tethering to chromatin.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Paul Lesbats, Erik Serrao, Alan Engelman, Dirk Lindemann, D.p. Maskell, Valerie E. Pye, Nicola O’reilly, Peter CherepanovAbstract:The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A–H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.
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Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function
Nature, 2016Co-Authors: Allison Ballandras-colas, Peter Cherepanov, Monica Brown, Nicola J. Cook, Tamaria G. Dewdney, Borries Demeler, Dmitry Lyumkis, Alan N. EngelmanAbstract:Integration of reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroviruses, catalysed by the protein integrase. Two studies, from the laboratories of Hideki Aihara and Alan Engelman, have used crystallography and cryo-electron microscopy to determine the structure of Rous sarcoma virus and mouse mammary tumour virus intasomes, the complex containing integrase, viral DNA, and target DNA. They find that integrase is an octameric assembly — not a tetramer as previously reported. A pair of core integrase dimers engages the viral DNA ends for catalysis, while another pair of non-catalytic integrase dimers bridge the two viral DNA molecules and help capture target DNA. The flanking, unanticipated pair of integrase dimers are required for target capture and strand transfer. An unexpected octameric integrase architecture for the betaretrovirus mouse mammary tumour virus intasome. Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses^ 1 . Previous structural characterization of integrase–viral DNA complexes, or intasomes, from the Spumavirus prototype foamy virus revealed a functional integrase tetramer^ 2 , 3 , 4 , 5 , and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase^ 6 , 7 , 8 , 9 . However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain–carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.
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Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function
Nature, 2016Co-Authors: Allison Ballandras-colas, Peter Cherepanov, Monica Brown, Nicola J. Cook, Tamaria G. Dewdney, Borries Demeler, Dmitry Lyumkis, Alan EngelmanAbstract:Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the Spumavirus prototype foamy virus revealed a functional integrase tetramer, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.
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Functional and structural characterization of the integrase from the prototype foamy virus.
Nucleic Acids Research, 2008Co-Authors: Eugene Valkov, Anna Helander, Pietro Roversi, Stephen Hare, S.s. Gupta, Myra O Mcclure, Peter CherepanovAbstract:Establishment of the stable provirus is an essential step in retroviral replication, orchestrated by integrase (IN), a virus-derived enzyme. Until now, available structural information was limited to the INs of human immunodeficiency virus type 1 (HIV-1), avian sarcoma virus (ASV) and their close orthologs from the Lentivirus and Alpharetrovirus genera. Here, we characterized the in vitro activity of the prototype foamy virus (PFV) IN from the Spumavirus genus and determined the three-dimensional structure of its catalytic core domain (CCD). Recombinant PFV IN displayed robust and almost exclusively concerted integration activity in vitro utilizing donor DNA substrates as short as 16 bp, underscoring its significance as a model for detailed structural studies. Comparison of the HIV-1, ASV and PFV CCD structures highlighted both conserved as well as unique structural features such as organization of the active site and the putative host factor binding face. Despite possessing very limited sequence identity to its HIV counterpart, PFV IN was sensitive to HIV IN strand transfer inhibitors, suggesting that this class of inhibitors target the most conserved features of retroviral IN-DNA complexes.
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LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro
Nucleic acids research, 2006Co-Authors: Peter CherepanovAbstract:Transcriptional co-activator LEDGF/p75 is the major cellular interactor of HIV-1 integrase (IN), critical to efficient viral replication. In this work, a series of INs from the Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Spumavirus and Lentivirus retroviral genera were tested for interaction with the host factor. None of the non-lentiviral INs possessed detectable affinity for LEDGF in either pull-down or yeast two-hybrid assays. In contrast, all lentiviral INs examined, including those from bovine immunodeficiency virus (BIV), maedi-visna virus (MVV) and equine infectious anemia virus (EIAV) readily interacted with LEDGF. Mutation of Asp-366 to Asn in LEDGF ablated the interaction, suggesting a common mechanism of the host factor recognition by the INs. LEDGF potently stimulated strand transfer activity of divergent lentiviral INs in vitro. Unprecedentedly, in the presence of the host factor, EIAV IN almost exclusively catalyzed concerted integration, whereas HIV-1 IN promoted predominantly half-site integration, and BIV IN was equally active in both types of strand transfer. Concerted BIV and EIAV integration resulted in 5 bp duplications of the target DNA sequences. These results confirm that the interaction with LEDGF is conserved within and limited to Lentivirus and strongly argue that the host factor is intimately involved in the catalysis of lentiviral DNA integration.
Dirk Lindemann - One of the best experts on this subject based on the ideXlab platform.
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Structural basis for Spumavirus GAG tethering to chromatin.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Paul Lesbats, Erik Serrao, Alan Engelman, Dirk Lindemann, D.p. Maskell, Valerie E. Pye, Nicola O’reilly, Peter CherepanovAbstract:The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A–H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards.
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A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and
2016Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Melvyn W. Yap, Roksana W. Ogrodowicz, Juliane Reh, Jonathan P. StoyeAbstract:The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5a restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergen
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Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture
PLoS Pathogens, 2016Co-Authors: Grégory Effantin, Leandro F Estrozi, Nick Aschman, Patricia Renesto, Nicole Stanke, Dirk Lindemann, Guy Schoehn, Winfried WeissenhornAbstract:Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.
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Characterisation of a Spumavirus Gag protein
Retrovirology, 2013Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Roksana W. Ogrodowicz, Juliane Reh, Melvin W Yap, Jonathan P. StoyeAbstract:Background Human prototypic foamy virus (HPFV) belongs to the spumaretrovrinae subfamily and is an attractive vector candidate for gene therapy [1] as it is apathogenic. The Gag protein is not cleaved into matrix (MA), capsid (CA) and nucleocapsid (NC) as occurs in orthoretroviruses; rather, it is able to perform the roles of these proteins as a single polypeptide [2]. Foamy virus Gag proteins are targets for restriction factors such as Trim5a [3] and also interact with the aminoterminal leader peptide of the envelope protein (Env). This Gag-Env interaction is essential for budding of viral particles from the host cell [4,5].
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A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and Capsid
PLoS pathogens, 2013Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Melvyn W. Yap, Roksana W. Ogrodowicz, Juliane Reh, Jonathan P. StoyeAbstract:The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.
Jonathan P. Stoye - One of the best experts on this subject based on the ideXlab platform.
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A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and
2016Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Melvyn W. Yap, Roksana W. Ogrodowicz, Juliane Reh, Jonathan P. StoyeAbstract:The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5a restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergen
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Characterisation of a Spumavirus Gag protein
Retrovirology, 2013Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Roksana W. Ogrodowicz, Juliane Reh, Melvin W Yap, Jonathan P. StoyeAbstract:Background Human prototypic foamy virus (HPFV) belongs to the spumaretrovrinae subfamily and is an attractive vector candidate for gene therapy [1] as it is apathogenic. The Gag protein is not cleaved into matrix (MA), capsid (CA) and nucleocapsid (NC) as occurs in orthoretroviruses; rather, it is able to perform the roles of these proteins as a single polypeptide [2]. Foamy virus Gag proteins are targets for restriction factors such as Trim5a [3] and also interact with the aminoterminal leader peptide of the envelope protein (Env). This Gag-Env interaction is essential for budding of viral particles from the host cell [4,5].
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A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and Capsid
PLoS pathogens, 2013Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Melvyn W. Yap, Roksana W. Ogrodowicz, Juliane Reh, Jonathan P. StoyeAbstract:The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.
Nicole Stanke - One of the best experts on this subject based on the ideXlab platform.
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A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and
2016Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Melvyn W. Yap, Roksana W. Ogrodowicz, Juliane Reh, Jonathan P. StoyeAbstract:The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5a restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergen
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Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture
PLoS Pathogens, 2016Co-Authors: Grégory Effantin, Leandro F Estrozi, Nick Aschman, Patricia Renesto, Nicole Stanke, Dirk Lindemann, Guy Schoehn, Winfried WeissenhornAbstract:Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae.
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Characterisation of a Spumavirus Gag protein
Retrovirology, 2013Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Roksana W. Ogrodowicz, Juliane Reh, Melvin W Yap, Jonathan P. StoyeAbstract:Background Human prototypic foamy virus (HPFV) belongs to the spumaretrovrinae subfamily and is an attractive vector candidate for gene therapy [1] as it is apathogenic. The Gag protein is not cleaved into matrix (MA), capsid (CA) and nucleocapsid (NC) as occurs in orthoretroviruses; rather, it is able to perform the roles of these proteins as a single polypeptide [2]. Foamy virus Gag proteins are targets for restriction factors such as Trim5a [3] and also interact with the aminoterminal leader peptide of the envelope protein (Env). This Gag-Env interaction is essential for budding of viral particles from the host cell [4,5].
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A Unique Spumavirus Gag N-terminal Domain with Functional Properties of Orthoretroviral Matrix and Capsid
PLoS pathogens, 2013Co-Authors: David C. Goldstone, Nicole Stanke, Dirk Lindemann, Neil J. Ball, Marta Sanz-ramos, Thomas G. Flower, Melvyn W. Yap, Roksana W. Ogrodowicz, Juliane Reh, Jonathan P. StoyeAbstract:The Spumaretrovirinae, or foamyviruses (FVs) are complex retroviruses that infect many species of monkey and ape. Although FV infection is apparently benign, trans-species zoonosis is commonplace and has resulted in the isolation of the Prototypic Foamy Virus (PFV) from human sources and the potential for germ-line transmission. Despite little sequence homology, FV and orthoretroviral Gag proteins perform equivalent functions, including genome packaging, virion assembly, trafficking and membrane targeting. In addition, PFV Gag interacts with the FV Envelope (Env) protein to facilitate budding of infectious particles. Presently, there is a paucity of structural information with regards FVs and it is unclear how disparate FV and orthoretroviral Gag molecules share the same function. Therefore, in order to probe the functional overlap of FV and orthoretroviral Gag and learn more about FV egress and replication we have undertaken a structural, biophysical and virological study of PFV-Gag. We present the crystal structure of a dimeric amino terminal domain from PFV, Gag-NtD, both free and in complex with the leader peptide of PFV Env. The structure comprises a head domain together with a coiled coil that forms the dimer interface and despite the shared function it is entirely unrelated to either the capsid or matrix of Gag from other retroviruses. Furthermore, we present structural, biochemical and virological data that reveal the molecular details of the essential Gag-Env interaction and in addition we also examine the specificity of Trim5α restriction of PFV. These data provide the first information with regards to FV structural proteins and suggest a model for convergent evolution of gag genes where structurally unrelated molecules have become functionally equivalent.