The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform

Maxine L Linial - One of the best experts on this subject based on the ideXlab platform.

  • expression of prototype foamy virus Pol as a gag Pol fusion Protein does not change the timing of reverse transcription
    Journal of Virology, 2013
    Co-Authors: Dana L Jackson, Maxine L Linial
    Abstract:

    Foamy viruses are retroviruses whose Pol Protein is synthesized without Gag from a spliced mRNA. Unlike orthoretroviruses, reverse transcription occurs during viral assembly, leading to DNA-containing virions. When prototype foamy virus Pol is expressed as an orthoretroviral-like Gag-Pol fusion Protein, reverse transcription also occurs late in viral replication, as measured by the timing of reverse transcriptase sensitivity to the inhibitor 3′-azido-3′deoxythymidine (AZT). Thus, timing of reverse transcription is intrinsic to Pol itself.

  • foamy virus Pol Protein expressed as a gag Pol fusion retains enzymatic activities allowing for infectious virus production
    Journal of Virology, 2012
    Co-Authors: Amber Sinicrope, Dana L Jackson, Shuyuarn F Yu, Maxine L Linial
    Abstract:

    Foamy viruses (FV) synthesize Pol from a spliced Pol mRNA independently of Gag, unlike orthoretroviruses, which synthesize Pol as a Gag-Pol Protein that coassembles with Gag. We found that prototype FV (PFV) mutants expressing Gag and Pol only as a Gag-Pol Protein without the spliced Pol contain protease activity equivalent to that of wild-type (WT) Pol. Regardless of the presence or absence of the spliced Pol, the PFV Gag-Pol Proteins can assemble into virus-like particles (VLPs), in contrast to the orthoretroviral Gag-Pol Proteins, which cannot form VLPs. However, the PFV Gag-Pol VLPs have aberrant morphologies and are not infectious. In the absence of the spliced Pol, coexpression of a PFV Gag-Pol Protein with Gag can produce infectious virions. Our results suggest that enzymes encoded by PFV Pol (protease, reverse transcriptase, and integrase) are enzymatically active if they are synthesized as part of a Gag-Pol Protein.

  • the c terminus of foamy retrovirus gag contains determinants for encapsidation of Pol Protein into virions
    Journal of Virology, 2008
    Co-Authors: Maxine L Linial
    Abstract:

    Foamy viruses (FV) differ from orthoretroviruses in many aspects of their replication cycle. A major difference is in the mode of Pol expression, regulation, and encapsidation into virions. Orthoretroviruses synthesize Pol as a Gag-Pol fusion Protein so that Pol is encapsidated into virus particles through Gag assembly domains. However, as FV express Pol independently of Gag from a spliced mRNA, packaging occurs through a distinct mechanism. FV genomic RNA contains cis-acting sequences that are required for Pol packaging, suggesting that Pol binds to RNA for its encapsidation. However, it is not known whether Gag is directly involved in Pol packaging. Previously our laboratory showed that sequences flanking the three glycine-arginine-rich (GR) boxes at the C terminus of FV Gag contain domains important for RNA packaging and Pol expression, cleavage, and packaging. We have now shown that both deletion and substitution mutations in the first GR box (GR1) prevented neither the assembly of particles with wild-type density nor packaging of RNA genomes but led to a defect in Pol packaging. Site-directed mutagenesis of GR1 indicated that the clustered positively charged amino acids in GR1 play important roles in Pol packaging. Our results suggest that GR1 contains a Pol interaction domain and that a Gag-Pol complex is formed and binds to RNA for incorporation into virions.

  • Role of the C Terminus of Foamy Virus Gag in RNA Packaging and Pol Expression
    Journal of Virology, 2004
    Co-Authors: Carolyn R. Stenbak, Maxine L Linial
    Abstract:

    Foamy viruses (FV) are complex retroviruses that possess several unique features that distinguish them from all other retroviruses. FV Gag and Pol Proteins are expressed independently of one another, and both Proteins undergo single cleavage events. Thus, the mature FV Gag Protein does not consist of the matrix, capsid, and nucleocapsid (NC) Proteins found in orthoretroviruses, and the putative NC domain of FV Gag lacks the hallmark Cys-His motifs or I domains. As there is no Gag-Pol fusion Protein, the mechanism of Pol packaging is different but unknown. FV RNA packaging is not well understood either. The C terminus of FV Gag has three glycine-arginine motifs (GR boxes), the first of which has been shown to have nucleic acid binding properties in vitro. The role of these GR boxes in RNA packaging and Pol packaging was investigated with a series of Gag C-terminal truncation mutants. GR box 1 was found to be the major determinant of RNA packaging, but all three GR boxes were required to achieve wild-type levels of RNA packaging. In addition, Pol was packaged in the absence of GR box 3, but GR boxes 1 and 2 were required for efficient Pol packaging. Interestingly, the Gag truncation mutants demonstrated decreased Pol expression levels as well as defects in Pol cleavage. Thus, the C terminus of FV Gag was found to be responsible for RNA packaging, as well as being involved in the expression, cleavage, and incorporation of the Pol Protein.

  • The roles of Pol and Env in the assembly pathway of human foamy virus.
    Journal of Virology, 1998
    Co-Authors: David N. Baldwin, Maxine L Linial
    Abstract:

    Human foamy virus (HFV) is the prototype of the Spumavirus genus of retroviruses. These viruses have a genomic organization close to that of other complex retroviruses but have similarities to hepadnaviruses such as human hepatitis B virus (HBV). Both HFV and HBV express their Pol Protein independently of their structural Proteins. Retroviruses and hepadnaviruses differ in their requirements for particle assembly and genome packaging. Assembly of retroviral particles containing RNA genomes requires only the Gag structural Protein. The Pol Protein is not required for capsid assembly, and the Env surface glycoProtein is not required for release of virions from the cell. In contrast, assembly of extracellular HBV particles containing DNA requires core structural Protein and Polymerase (P Protein) for assembly of nucleocapsids and requires surface glycoProteins for release from the cell. We investigated the requirements for synthesis of extracellular HFV particles by constructing mutants with either the Pol or env gene deleted. We found that the Pol Protein is dispensable for production of extracellular particles containing viral nucleic acid. In the absence of Env, intracellular particles are synthesized but few or no extracellular particles could be detected. Thus, foamy virus assembly is distinct from that of other reverse transcriptase-encoding mammalian viruses.

Axel Rethwilm - One of the best experts on this subject based on the ideXlab platform.

  • Biophysical and enzymatic properties of the simian and prototype foamy virus reverse transcriptases
    Retrovirology, 2010
    Co-Authors: Maximilian J Hartl, Axel Rethwilm, Florian Mayr, Birgitta M Wöhrl
    Abstract:

    Background The foamy virus Pol Protein is translated independently from Gag using a separate mRNA. Thus, in contrast to orthoretroviruses no Gag-Pol precursor Protein is synthesized. Only the integrase domain is cleaved off from Pol resulting in a mature reverse transcriptase harboring the protease domain at the N-terminus (PR-RT). Although the homology between the PR-RTs from simian foamy virus from macaques (SFVmac) and the prototype foamy virus (PFV), probably originating from chimpanzee, exceeds 90%, several differences in the biophysical and biochemical properties of the two enzymes have been reported (i.e. SFVmac develops resistance to the nucleoside inhibitor azidothymidine (AZT) whereas PFV remains AZT sensitive even if the resistance mutations from SFVmac PR-RT are introduced into the PFV PR-RT gene). Moreover, contradictory data on the monomer/dimer status of the foamy virus protease have been published. Results We set out to purify and directly compare the monomer/dimer status and the enzymatic behavior of the two wild type PR-RT enzymes from SFVmac and PFV in order to get a better understanding of the Protein and enzyme functions. We determined kinetic parameters for the two enzymes, and we show that PFV PR-RT is also a monomeric Protein. Conclusions Our data show that the PR-RTs from SFV and PFV are monomeric Proteins with similar biochemical and biophysical properties that are in some aspects comparable with MLV RT, but differ from those of HIV-1 RT. These differences might be due to the different conditions the viruses are confronted with in dividing and non-dividing cells.

  • Generation of an improved foamy virus vector by dissection of cis-acting sequences.
    Journal of General Virology, 2009
    Co-Authors: Tatiana Wiktorowicz, Katrin Peters, Nicole Armbruster, Andre F. Steinert, Axel Rethwilm
    Abstract:

    In contrast to other retroviruses, foamy viruses (FVs) generate their Pol Protein precursor independently of the Gag Protein from a spliced mRNA. The exact mechanism of Pol Protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central Poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells.

  • Determination of the relative amounts of Gag and Pol Proteins in foamy virus particles.
    Retrovirology, 2005
    Co-Authors: Marc Cartellieri, Dirk Lindemann, Wolfram W. Rudolph, Ottmar Herchenröder, Axel Rethwilm
    Abstract:

    We determined the relative ratios of Gag and Pol molecules in highly purified virions of spumaretroviruses or foamy viruses (FVs) using monoclonal antibodies and bacterially expressed reference Proteins. We found that the cleaved p68Gag moiety dominates in infectious FVs. Furthermore, approximate mean ratios in FV are 16:1 (pr71Gag plus p68Gag:p85RT),12:1 (p68Gag:p85RT), and 10:1 (pr71Gag plus p68Gag:p40IN). Thus, the results indicate that FVs have found a way to incorporate approximately as much Pol Protein into their capsids as orthoretroviruses, despite a completely different Pol expression strategy.

  • rna and Protein requirements for incorporation of the Pol Protein into foamy virus particles
    Journal of Virology, 2005
    Co-Authors: Katrin Peters, Tatiana Wiktorowicz, Martin Heinkelein, Axel Rethwilm
    Abstract:

    Foamy viruses (FVs) generate their Pol Protein precursor molecule independently of the Gag Protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol Protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76:10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol Protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5′ long terminal repeat and one at the 3′ end of the Pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the Protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor Proteins.

  • Complex Effects of Deletions in the 5′ Untranslated Region of Primate Foamy Virus on Viral Gene Expression and RNA Packaging
    Journal of Virology, 2000
    Co-Authors: Martin Heinkelein, Myra O Mcclure, Jana Thurow, Marco Dressler, Horst Imrich, Dieter Neumann-haefelin, Axel Rethwilm
    Abstract:

    Foamy viruses constitute the most divergent genus in the family Retroviridae. The primate foamy virus (PFV) replication strategy differs fundamentally from all other retroviruses and appears to bridge the retroviral and hepadnaviral replication pathway (for a recent review, see reference 31). PFVs have a genomic structure similar to other retroviruses and require provirus integration for replication (14, 42). However, the way in which Polymerase Protein is expressed (13, 57), the mode of viral capsid assembly and interaction with the cognate envelope (Env) glycoProtein (4, 18, 19, 41), and the time point of reverse transcription in the viral life cycle (35, 58) are unique among retroviruses and show functional analogies to hepadnaviruses (37). PFVs have been suggested as good candidates from which to develop viral vectors, and several vectors are currently under construction for gene transfer purposes (9, 15, 21, 26, 38, 45, 47, 55, 56). A retroviral vector system consists of the vector proper, which harbors all elements in cis required for genome packaging, reverse transcription, integration, and transgene expression, and a packaging cell line, which supplies in trans the viral structural and enzymatic Proteins required to execute these functions (34). Ideally, there is little genetic overlap between vector and packaging construct sequences to reduce the problem of generating replication-competent virus following recombination events (34). In general, retroviruses harbor a cis-acting sequence (psi in murine retroviruses) in the 5′ untranslated region (UTR) of the genome which facilitates the dominant package of genomic viral RNA (8, 32). This sequence can usually be deleted from the packaging constructs expressing the Gag and Pol Proteins (25, 34). In PFVs the Pol Protein is expressed from a spliced RNA which uses the major splice donor (SD) located at position +53 following the start of transcription (+1) and a splice acceptor located in the gag gene (+1,850) (27, 57). Therefore, the deletion of the SD site from PFV packaging constructs and expression of Pol Protein from a separate construct may disturb a balanced expression of Gag and Pol Proteins, provided this is essential for optimal vector production. Furthermore, it has recently been shown that PFV vectors essentially require, besides a sequence in the 5′ region of the (pre-)genomic RNA (cis-acting sequence I [CASI]), essentially a second sequence located in the 3′ Pol gene (CASII) in order to transfer successfully marker genes (15, 21, 55). Deletion of CASII from vector constructs largely reduced the viral RNA content of particles and also influenced the function of the Pol-encoded protease to cleave the Gag precursor Protein (21). This suggested complex interactions between the Gag and Pol Proteins and the (pre-) genomic RNA in forming a functional PFV capsid. Since CASII is located in a coding region which cannot be deleted from packaging constructs, we aimed to investigate deletions in CASI to achieve separation of vector sequences from packaging sequences. To do this, we used molecular clones of an isolate that was reported to be obtained from human material (1). Since there is a lack of evidence for authentic human foamy viruses (2, 44, 49), the origin of this isolate most probably related to an incidental trans-species transmission from chimpanzees harboring virtually identical PFVs (22, 23, 48).

S J Lo - One of the best experts on this subject based on the ideXlab platform.

  • Translational regulation of hepatitis B viral gene expression
    Journal of Gastroenterology and Hepatology, 1993
    Co-Authors: S J Lo, S. Y. Sheu
    Abstract:

    At least five species of unspliced mRNA of the hepatitis B virus (HBV), which are 3.5, 3.4, 2.4, 2.1 and 0.8 kb, have been identified. To study this amount of HBV Proteins they were translationally regulated and both in vitro and in vivo experiments performed. The four in vitro synthesized RNA corresponding to the 2.1, 2.4, 3.4, and 3.5 kb mRNA, respectively, were translated in a rabbit reticulocyte lysate. In conjunction with in vivo transfection experiments, it was demonstrated that: (i) a preferential translational initiation is involved in the differential amount of surface antigens; (ii) the 2.1 kb mRNA is the template for the synthesis of the middle and major surface antigens in a ratio of about 1 : 4; (iii) the 2.4 kb mRNA, although containing sequences for coding the large, middle and major surface antigen, is primarily used for the synthesis of large surface antigens; (iv) both the core and Pol Proteins can be independently synthesized from the 3.4 kb mRNA (pregenome RNA) and the synthesis of Pol Protein is favourably carried out via a leaky scanning mechanism; (v) the 3.5 kb mRNA (pre-C mRNA) is the template for the production of precore Protein (the precursor of hepatitis B e antigen) but not for the synthesis of both core and Pol Proteins. Taken together, the results indicate that: (i) the translational initiation context of each HBV-encoded Protein plays a key role in determining the level of Protein synthesis; (ii) the occurrence of bifunctional mRNA of pregenome RNA and 2.1 kb mRNA provides another alternative means for minimization of the viral genome size; (iii) the leaky scanning mechanism is involved in the expression of the second Protein in bifunctional mRNA.

  • expression and stability of the hepatitis b Pol Protein in escherichia coli
    Journal of Gastroenterology and Hepatology, 1993
    Co-Authors: S J Lo
    Abstract:

    The pGEX plasmid containing the gene of glutathione S-transferase (GST) was employed to express various lengths of the hepatitis B virus Pol Protein in a form of fusion Protein in Escherichia coli. Results of SDS-PAGE and Western blot analyses indicated that: (i) expression of GST-Pol fusion Proteins varied from undetectable to 30% of total Protein in different clones; (ii) presence of the carboxyl terminus of the Pol Protein apparently lowered the expression amount of fusion Proteins; (iii) some distinct Pol Protein bands of lower molecular weight were constantly detected in several clones. The presence of the smaller Pol Protein therefore raises a possibility that the Pol Protein contains protease cutting sites.

Dirk Lindemann - One of the best experts on this subject based on the ideXlab platform.

  • Purification of foamy viral particles.
    Virology, 2017
    Co-Authors: Ralf Spannaus, Dirk Lindemann, Christina Miller, Jochen Bodem
    Abstract:

    Abstract Foamy viruses are non-pathogenic retroviruses and represent a tool for vector development. For gene therapy applications and for analyses of viral Protein composition infectious particles need to be purified, which has been difficult for foamy viruses in the past. Here, we describe a novel, simple, and fast purification method for prototype foamy viruses with high purity using size exclusion and affinity chromatography. More than 99,9% of the contaminating Proteins were removed. The purified viruses were used to determine the amount of the incorporated Pol Protein relative to Gag. The determined Gag to Pol PR-RT ratio of 30:1 confirmed previous studies suggesting FV virions encapsidate fewer number of Pol molecules than orthoretroviruses.

  • Orthoretroviral-like prototype foamy virus gag-Pol expression is compatible with viral replication
    Retrovirology, 2011
    Co-Authors: Anka Swiersy, Constanze Wiek, Hanswalter Zentgraf, Dirk Lindemann
    Abstract:

    Background Foamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor Protein and not as a Gag-Pol fusion Protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol Protein interactions, ensures Pol particle encapsidation.

  • Orthoretroviral-like prototype foamy virus gag-Pol expression is compatible with viral replication
    Retrovirology, 2011
    Co-Authors: Anka Swiersy, Constanze Wiek, Hanswalter Zentgraf, Dirk Lindemann
    Abstract:

    Background Foamy viruses (FVs) unlike orthoretroviruses express Pol as a separate precursor Protein and not as a Gag-Pol fusion Protein. A unique packaging strategy, involving recognition of briding viral RNA by both Pol precursor and Gag as well as potential Gag-Pol Protein interactions, ensures Pol particle encapsidation. Results Several Prototype FV (PFV) Gag-Pol fusion Protein constructs were generated to examine whether PFV replication is compatible with an orthoretroviral-like Pol expression. During their analysis, non-particle-associated secreted Pol precursor Protein was discovered in extracellular wild type PFV particle preparations of different origin, copurifying in simple virion enrichment protocols. Different analysis methods suggest that extracellular wild type PFV particles contain predominantly mature p85^PR-RT and p40^IN Pol subunits. Characterization of various PFV Gag-Pol fusion constructs revealed that PFV Pol expression in an orthoretroviral manner is compatible with PFV replication as long as a proteolytic processing between Gag and Pol Proteins is possible. PFV Gag-Pol translation by a HIV-1 like ribosomal frameshift signal resulted in production of replication-competent virions, although cell- and particle-associated Pol levels were reduced in comparison to wild type. In-frame fusion of PFV Gag and Pol ORFs led to increased cellular Pol levels, but particle incorporation was only marginally elevated. Unlike that reported for similar orthoretroviral constructs, a full-length in-frame PFV Gag-Pol fusion construct showed wildtype-like particle release and infectivity characteristics. In contrast, in-frame PFV Gag-Pol fusion with C-terminal Gag ORF truncations or non-removable Gag peptide addition to Pol displayed wildtype particle release, but reduced particle infectivity. PFV Gag-Pol precursor fusion Proteins with inactivated protease were highly deficient in regular particle release, although coexpression of p71^Gag resulted in a significant copackaging of these Proteins. Conclusions Non-particle associated PFV Pol appears to be naturally released from infected cells by a yet unknown mechanism. The absence of particle-associated Pol precursor suggests its rapid processing upon particle incorporation. Analysis of different PFV Gag-Pol fusion constructs demonstrates that orthoretroviral-like Pol expression is compatible with FV replication in principal as long as fusion Protein processing is possible. Furthermore, unlike orthoretroviruses, PFV particle release and infectivity tolerate larger differences in relative cellular Gag/Pol levels.

  • Determination of the relative amounts of Gag and Pol Proteins in foamy virus particles.
    Retrovirology, 2005
    Co-Authors: Marc Cartellieri, Dirk Lindemann, Wolfram W. Rudolph, Ottmar Herchenröder, Axel Rethwilm
    Abstract:

    We determined the relative ratios of Gag and Pol molecules in highly purified virions of spumaretroviruses or foamy viruses (FVs) using monoclonal antibodies and bacterially expressed reference Proteins. We found that the cleaved p68Gag moiety dominates in infectious FVs. Furthermore, approximate mean ratios in FV are 16:1 (pr71Gag plus p68Gag:p85RT),12:1 (p68Gag:p85RT), and 10:1 (pr71Gag plus p68Gag:p40IN). Thus, the results indicate that FVs have found a way to incorporate approximately as much Pol Protein into their capsids as orthoretroviruses, despite a completely different Pol expression strategy.

Dana L Jackson - One of the best experts on this subject based on the ideXlab platform.