The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
David Harrich - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic elongation factor 1 complex subunits are critical hiv 1 Reverse Transcription cofactors
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Kylie Warren, Dongsheng Li, David Warrilow, Haran Sivakumaran, Ann Apolloni, Catherine M Abbott, Alun Jones, Jenny L Anderson, David HarrichAbstract:Cellular proteins have been implicated as important for HIV-1 Reverse Transcription, but whether any are Reverse Transcription complex (RTC) cofactors or affect Reverse Transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous Reverse Transcription assay to identify cellular proteins that stimulated late steps of Reverse Transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as Reverse Transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late Reverse Transcription in vitro. We observed that the p51 subunit of Reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with Reverse transcriptase following infection of cells. Reverse Transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of Reverse Transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of Reverse Transcription and trafficking of the RTC to the nucleus.
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Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro
PloS one, 2010Co-Authors: David Warrilow, Kylie Warren, David HarrichAbstract:Recent work suggests a role for multiple host factors in facilitating HIV-1 Reverse Transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 Reverse Transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor Reverse Transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of Reverse Transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late Reverse Transcription suggesting it stabilized the Reverse Transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous Reverse Transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded Reverse Transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, Reverse Transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve Reverse Transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the Reverse Transcription products, but it is unclear if this is related to improved DNA synthesis.
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Reverse Transcriptase and Cellular Factors: Regulators of HIV-1 Reverse Transcription
Viruses, 2009Co-Authors: Kylie Warren, David Warrilow, Luke W. Meredith, David HarrichAbstract:There is ample evidence that synthesis of HIV-1 proviral DNA from the viral RNA genome during Reverse Transcription requires host factors. However, only a few cellular proteins have been described in detail that affect Reverse Transcription and interact with Reverse transcriptase (RT). HIV-1 integrase is an RT binding protein and a number of IN-binding proteins including INI1, components of the Sin3a complex, and Gemin2 affect Reverse Transcription. In addition, recent studies implicate the cellular proteins HuR, AKAP149, and DNA topoisomerase I in Reverse Transcription through an interaction with RT. In this review we will consider interactions of Reverse Transcription complex with viral and cellular factors and how they affect the Reverse Transcription process.
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A U5 repressor of Reverse Transcription is required for optimal HIV-1 infectivity and replication
Retrovirology, 2009Co-Authors: Luke W. Meredith, Roland Marquet, Catherine Isel, Céline Ducloux, David HarrichAbstract:Here we provide strong evidence that a highly conserved stem loop structure in the U5 region of the HIV-1 RNA leader harbours a repressor of Reverse Transcription (RRT). We showed that two sequences in U5, at +143-145 and +151-153, are essential for RRT function. Mutation of either site strongly and unexpectedly increased endogenous Reverse Transcription, and cell infection assays showed that both mutations dramatically increased negative strand strong stop DNA synthesis. Early, late, 1-LTR and 2-LTR Reverse Transcription products were present proportionally, indicating that the downstream Reverse Transcription events were not affected. In vitro structural probing of the wild type and mutant RNA revealed an unexpected destabilization effect of the mutations on the whole U5 stem loop, which would explain the loss of regulation of Reverse Transcription. This functional effect was not observed in vitro, where, in the absence of viral proteins other than RT and cellular factors, all RNA performed similarly. These U5 mutations decreased virus replication in Jurkat and primary T-cells, which could be attributed to a marked defect in viral integration. Analysis of 1-LTR and 2-LTR circular DNA isolated from infected cells revealed that substantial deletions were present, indicating that the viral DNA was degraded by cellular nucleases. Together, our experiments suggest that regulated Reverse Transcription initiation is essential to allow synthesis of the viral DNA in a cellular environment that supports the assembly of a functional HIV-1 pre-integration complex, which also protects the proviral DNA from cellular degradation processes.
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Maturation of the HIV Reverse Transcription complex: putting the jigsaw together.
Reviews in medical virology, 2009Co-Authors: David Warrilow, Gilda Tachedjian, David HarrichAbstract:Upon HIV attachment, fusion and entry into the host cell cytoplasm, the viral core undergoes rearrangement to become the mature Reverse Transcription complex (RTC). Reduced infectivity of viral deletion mutants of the core proteins, capsid and negative factor (Nef), can be complemented by vesicular stomatitis virus (VSV) pseudotyping suggesting a role for these viral proteins in a common event immediately post-entry. This event may be necessary for correct trafficking of the early complex. Enzymatic activation of the complex occurs either before or during RTC maturation, and may be dependent on the presence of deoxynucleotides in the host cell. The RTC initially becomes enlarged immediately after entry, which is followed by a decrease in its sedimentation rate consistent with core uncoating. Several HIV proteins associated with the RTC and recently identified host-cell proteins are important for Reverse Transcription while genome-wide siRNA knockdown studies have identified additional host cell factors that may be required for Reverse Transcription. Determining precisely how these proteins assist the RTC function needs to be addressed. Copyright © 2009 John Wiley & Sons, Ltd.
Jean-luc Darlix - One of the best experts on this subject based on the ideXlab platform.
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HIV-1 Reverse Transcription
Methods in Molecular Biology, 2014Co-Authors: Andrea Cimarelli, Jean-luc DarlixAbstract:Reverse Transcription is an obligatory step in retrovirus replication in the course of which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded positive sense RNA genome to synthesize the double-stranded viral DNA. At the same time the RT-associated RNaseH activity degrades the genomic RNA template, which has just been copied. The viral nucleocapsid protein NCp7 is an obligatory partner of RT, chaperoning synthesis of the complete viral DNA flanked by the two long-terminal repeats (LTR), required for viral DNA integration into the host genome and its expression. Here we describe assays for in vitro and ex vivo monitoring of Reverse Transcription and the chaperoning role of the nucleocapsid protein (NC).
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The specificity and flexibility of l1 Reverse Transcription priming at imperfect T-tracts.
PLoS genetics, 2013Co-Authors: Clément Monot, Jean-luc Darlix, Monika Kuciak, Sébastien Viollet, Ashfaq Ali Mir, Caroline Gabus, Gaël CristofariAbstract:L1 retrotransposons have a prominent role in reshaping mammalian genomes. To replicate, the L1 ribonucleoprotein particle (RNP) first uses its endonuclease (EN) to nick the genomic DNA. The newly generated DNA end is subsequently used as a primer to initiate Reverse Transcription within the L1 RNA poly(A) tail, a process known as target-primed Reverse Transcription (TPRT). Prior studies demonstrated that most L1 insertions occur into sequences related to the L1 EN consensus sequence (degenerate 5′-TTTT/A-3′ sites) and frequently preceded by imperfect T-tracts. However, it is currently unclear whether—and to which degree—the liberated 3′-hydroxyl extremity on the genomic DNA needs to be accessible and complementary to the poly(A) tail of the L1 RNA for efficient priming of Reverse Transcription. Here, we employed a direct assay for the initiation of L1 Reverse Transcription to define the molecular rules that guide this process. First, efficient priming is detected with as few as 4 matching nucleotides at the primer 3′ end. Second, L1 RNP can tolerate terminal mismatches if they are compensated within the 10 last bases of the primer by an increased number of matching nucleotides. All terminal mismatches are not equally detrimental to DNA extension, a C being extended at higher levels than an A or a G. Third, efficient priming in the context of duplex DNA requires a 3′ overhang. This suggests the possible existence of additional DNA processing steps, which generate a single-stranded 3′ end to allow L1 Reverse Transcription. Based on these data we propose that the specificity of L1 Reverse Transcription initiation contributes, together with the specificity of the initial EN cleavage, to the distribution of new L1 insertions within the human genome.
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Implications of the nucleocapsid and the microenvironment in retroviral Reverse Transcription.
Viruses, 2010Co-Authors: Marylène Mougel, Andrea Cimarelli, Jean-luc DarlixAbstract:This mini-review summarizes the process of Reverse-Transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. The hybridization of complementary viral sequences by the nucleocapsid protein (NC) receives a special focus, since it acts to chaperone the strand transfers obligatory for synthesis of the complete viral DNA and flanking long terminal repeats (LTR). Since the physiological microenvironment can impact on Reverse-Transcription, this mini-review also focuses on factors present in the intra-cellular or extra-cellular milieu that can drastically influence both the timing and the activity of Reverse-Transcription and hence virus infectivity.
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When is it time for Reverse Transcription to start and go
Retrovirology, 2009Co-Authors: Marylène Mougel, Laurent Houzet, Jean-luc DarlixAbstract:Upon cell infection by a retrovirus, the viral DNA polymerase, called Reverse transcriptase (RT), copies the genomic RNA to generate the proviral DNA flanked by two long terminal repeats (LTR). A discovery twenty years ago demonstrated that the structural viral nucleocapsid protein (NC) encoded by Gag is an essential cofactor of Reverse Transcription, chaperoning RT during viral DNA synthesis. However, it is only recently that NC was found to exert a control on the timing of Reverse Transcription, in a spatio-temporal manner. This brief review summarizes findings on the timing of Reverse Transcription in wild type HIV-1 and in nucleopcapsid (NC) mutants where virions contain a large amount of newly made viral DNA. This brief review also proposes some explanations of how NC may control late Reverse Transcription during Gag assembly in virus producer cells.
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When is it time for Reverse Transcription to start and go?
Retrovirology, 2009Co-Authors: Marylène Mougel, Laurent Houzet, Jean-luc DarlixAbstract:Upon cell infection by a retrovirus, the viral DNA polymerase, called Reverse transcriptase (RT), copies the genomic RNA to generate the proviral DNA flanked by two long terminal repeats (LTR). A discovery twenty years ago demonstrated that the structural viral nucleocapsid protein (NC) encoded by Gag is an essential cofactor of Reverse Transcription, chaperoning RT during viral DNA synthesis. However, it is only recently that NC was found to exert a control on the timing of Reverse Transcription, in a spatio-temporal manner. This brief review summarizes findings on the timing of Reverse Transcription in wild type HIV-1 and in nucleopcapsid (NC) mutants where virions contain a large amount of newly made viral DNA. This brief review also proposes some explanations of how NC may control late Reverse Transcription during Gag assembly in virus producer cells.
Marylène Mougel - One of the best experts on this subject based on the ideXlab platform.
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Implications of the nucleocapsid and the microenvironment in retroviral Reverse Transcription.
Viruses, 2010Co-Authors: Marylène Mougel, Andrea Cimarelli, Jean-luc DarlixAbstract:This mini-review summarizes the process of Reverse-Transcription, an obligatory step in retrovirus replication during which the retroviral RNA/DNA-dependent DNA polymerase (RT) copies the single-stranded genomic RNA to generate the double-stranded viral DNA while degrading the genomic RNA via its associated RNase H activity. The hybridization of complementary viral sequences by the nucleocapsid protein (NC) receives a special focus, since it acts to chaperone the strand transfers obligatory for synthesis of the complete viral DNA and flanking long terminal repeats (LTR). Since the physiological microenvironment can impact on Reverse-Transcription, this mini-review also focuses on factors present in the intra-cellular or extra-cellular milieu that can drastically influence both the timing and the activity of Reverse-Transcription and hence virus infectivity.
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When is it time for Reverse Transcription to start and go
Retrovirology, 2009Co-Authors: Marylène Mougel, Laurent Houzet, Jean-luc DarlixAbstract:Upon cell infection by a retrovirus, the viral DNA polymerase, called Reverse transcriptase (RT), copies the genomic RNA to generate the proviral DNA flanked by two long terminal repeats (LTR). A discovery twenty years ago demonstrated that the structural viral nucleocapsid protein (NC) encoded by Gag is an essential cofactor of Reverse Transcription, chaperoning RT during viral DNA synthesis. However, it is only recently that NC was found to exert a control on the timing of Reverse Transcription, in a spatio-temporal manner. This brief review summarizes findings on the timing of Reverse Transcription in wild type HIV-1 and in nucleopcapsid (NC) mutants where virions contain a large amount of newly made viral DNA. This brief review also proposes some explanations of how NC may control late Reverse Transcription during Gag assembly in virus producer cells.
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When is it time for Reverse Transcription to start and go?
Retrovirology, 2009Co-Authors: Marylène Mougel, Laurent Houzet, Jean-luc DarlixAbstract:Upon cell infection by a retrovirus, the viral DNA polymerase, called Reverse transcriptase (RT), copies the genomic RNA to generate the proviral DNA flanked by two long terminal repeats (LTR). A discovery twenty years ago demonstrated that the structural viral nucleocapsid protein (NC) encoded by Gag is an essential cofactor of Reverse Transcription, chaperoning RT during viral DNA synthesis. However, it is only recently that NC was found to exert a control on the timing of Reverse Transcription, in a spatio-temporal manner. This brief review summarizes findings on the timing of Reverse Transcription in wild type HIV-1 and in nucleopcapsid (NC) mutants where virions contain a large amount of newly made viral DNA. This brief review also proposes some explanations of how NC may control late Reverse Transcription during Gag assembly in virus producer cells.
David Warrilow - One of the best experts on this subject based on the ideXlab platform.
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eukaryotic elongation factor 1 complex subunits are critical hiv 1 Reverse Transcription cofactors
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Kylie Warren, Dongsheng Li, David Warrilow, Haran Sivakumaran, Ann Apolloni, Catherine M Abbott, Alun Jones, Jenny L Anderson, David HarrichAbstract:Cellular proteins have been implicated as important for HIV-1 Reverse Transcription, but whether any are Reverse Transcription complex (RTC) cofactors or affect Reverse Transcription indirectly is unclear. Here we used protein fractionation combined with an endogenous Reverse Transcription assay to identify cellular proteins that stimulated late steps of Reverse Transcription in vitro. We identified 25 cellular proteins in an active protein fraction, and here we show that the eEF1A and eEF1G subunits of eukaryotic elongation factor 1 (eEF1) are important components of the HIV-1 RTC. eEF1A and eEF1G were identified in fractionated human T-cell lysates as Reverse Transcription cofactors, as their removal ablated the ability of active protein fractions to stimulate late Reverse Transcription in vitro. We observed that the p51 subunit of Reverse transcriptase and integrase, two subunits of the RTC, coimmunoprecipitated with eEF1A and eEF1G. Moreover eEF1A and eEF1G associated with purified RTCs and colocalized with Reverse transcriptase following infection of cells. Reverse Transcription in cells was sharply down-regulated when eEF1A or eEF1G levels were reduced by siRNA treatment as a result of reduced levels of RTCs in treated cells. The combined evidence indicates that these eEF1 subunits are critical RTC stability cofactors required for efficient completion of Reverse Transcription. The identification of eEF1 subunits as unique RTC components provides a basis for further investigations of Reverse Transcription and trafficking of the RTC to the nucleus.
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Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro
PloS one, 2010Co-Authors: David Warrilow, Kylie Warren, David HarrichAbstract:Recent work suggests a role for multiple host factors in facilitating HIV-1 Reverse Transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 Reverse Transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor Reverse Transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of Reverse Transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late Reverse Transcription suggesting it stabilized the Reverse Transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous Reverse Transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded Reverse Transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, Reverse Transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve Reverse Transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the Reverse Transcription products, but it is unclear if this is related to improved DNA synthesis.
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Reverse Transcriptase and Cellular Factors: Regulators of HIV-1 Reverse Transcription
Viruses, 2009Co-Authors: Kylie Warren, David Warrilow, Luke W. Meredith, David HarrichAbstract:There is ample evidence that synthesis of HIV-1 proviral DNA from the viral RNA genome during Reverse Transcription requires host factors. However, only a few cellular proteins have been described in detail that affect Reverse Transcription and interact with Reverse transcriptase (RT). HIV-1 integrase is an RT binding protein and a number of IN-binding proteins including INI1, components of the Sin3a complex, and Gemin2 affect Reverse Transcription. In addition, recent studies implicate the cellular proteins HuR, AKAP149, and DNA topoisomerase I in Reverse Transcription through an interaction with RT. In this review we will consider interactions of Reverse Transcription complex with viral and cellular factors and how they affect the Reverse Transcription process.
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Maturation of the HIV Reverse Transcription complex: putting the jigsaw together.
Reviews in medical virology, 2009Co-Authors: David Warrilow, Gilda Tachedjian, David HarrichAbstract:Upon HIV attachment, fusion and entry into the host cell cytoplasm, the viral core undergoes rearrangement to become the mature Reverse Transcription complex (RTC). Reduced infectivity of viral deletion mutants of the core proteins, capsid and negative factor (Nef), can be complemented by vesicular stomatitis virus (VSV) pseudotyping suggesting a role for these viral proteins in a common event immediately post-entry. This event may be necessary for correct trafficking of the early complex. Enzymatic activation of the complex occurs either before or during RTC maturation, and may be dependent on the presence of deoxynucleotides in the host cell. The RTC initially becomes enlarged immediately after entry, which is followed by a decrease in its sedimentation rate consistent with core uncoating. Several HIV proteins associated with the RTC and recently identified host-cell proteins are important for Reverse Transcription while genome-wide siRNA knockdown studies have identified additional host cell factors that may be required for Reverse Transcription. Determining precisely how these proteins assist the RTC function needs to be addressed. Copyright © 2009 John Wiley & Sons, Ltd.
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Cell factors stimulate human immunodeficiency virus Type 1 Reverse Transcription in vitro
Journal of virology, 2007Co-Authors: David Warrilow, Adam Davis, Christopher J. Burrell, Luke W. Meredith, David HarrichAbstract:After fusion of the human immunodeficiency virus type 1 (HIV-1) envelope with the host cell membrane, the HIV-1 core enters the cell cytoplasm. Core components are then restructured to form the Reverse Transcription complex (RTC); the biochemical details of this process are currently unclear. To investigate early RTC formation, we characterized the endogenous Reverse Transcription activity of virions, which was less efficient than Reverse Transcription during cell infection and suggested a requirement for a cell factor. The addition of detergent to virions released Reverse transcriptase and capsid, and Reverse Transcription products became susceptible to the action of exogenous nucleases, indicating virion disruption. Disruption was coincident with the loss of the endogenous Reverse Transcription activity of virions, particularly late Reverse Transcription products. Consistent with this observation, the use of a modified “spin thru” method, which uses brief detergent exposure, also disrupted virions. The addition of lysates made from mammalian cell lines (Jurkat, HEK293T, and NIH 3T3 cells) to virions delipidated by detergent stimulated late Reverse Transcription efficiency. A complex with Reverse Transcription activity that was slower sedimenting than virions on a velocity gradient was greatly stimulated to generate full-length Reverse Transcription products and was associated with only relatively small amounts of capsid. These experiments suggest that cell factors are required for efficient Reverse Transcription of HIV-1.
Stephen P. Goff - One of the best experts on this subject based on the ideXlab platform.
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characterization of intracellular Reverse Transcription complexes of human immunodeficiency virus type 1
Journal of Virology, 2001Co-Authors: Ariberto Fassati, Stephen P. GoffAbstract:To examine the early events of the life cycle of human immunodeficiency virus type 1 (HIV-1), we analyzed the intracellular complexes mediating Reverse Transcription isolated from acutely infected cells. Partial purification of the Reverse Transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that two species of RTCs are formed but only one species is able to synthesize DNA. Most of the capsid, matrix, and Reverse transcriptase (RT) proteins dissociate from the complex soon after cell infection, but Vpr remains associated with the RTC. The RTCs isolated 1, 4, and 7 h after infection are competent for Reverse Transcription in vitro, indicating that a small proportion of RT remains associated with them. HIV RTCs isolated early after infection have a sedimentation velocity of approximately 560S. Later, different species with a sedimentation velocity ranging from 350S to 100S appear. Nuclear-associated RTCs have a sedimentation velocity of 80S. Shortly after initiation of Reverse Transcription, the viral strong-stop DNA within the RTC is sensitive to nuclease digestion and becomes protected when Reverse Transcription is almost completed.
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Characterization of intracellular Reverse Transcription complexes of Moloney murine leukemia virus.
Journal of virology, 1999Co-Authors: Ariberto Fassati, Stephen P. GoffAbstract:To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating Reverse Transcription. Partial purification of the Reverse Transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete Reverse Transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and Reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of Reverse Transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during Reverse Transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further.
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characterization of intracellular Reverse Transcription complexes of moloney murine leukemia virus
Journal of Virology, 1999Co-Authors: Ariberto Fassati, Stephen P. GoffAbstract:To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating Reverse Transcription. Partial purification of the Reverse Transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete Reverse Transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and Reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of Reverse Transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during Reverse Transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further. All retroviruses synthesize a double-stranded DNA copy of their RNA genome that is subsequently integrated into the host cell chromosomal DNA. The process of Reverse Transcription of the RNA genome into DNA is carried out in the cytoplasm soon after viral penetration into the cell and is generally completed within 8 to 12 h (30). Little is known about the structure and the protein composition of the intracellular complex in which Reverse Transcription occurs, particularly during the early steps after virus internalization. In the murine leukemia virus (MLV), the intracellular viral structure that contains the fully Reverse transcribed viral DNA (also called the preintegration complex [PIC]) retains components of the virion core (including CA protein), has a relatively large size, sedimenting at 160S, and is competent to integrate the DNA in vitro (5). The PIC of human immunodeficiency virus type 1 (HIV-1) appears to have a different organization since it contains no capsid proteins, but it contains at least Reverse transcriptase (RT), integrase (IN), and a portion of matrix (MA) proteins (6, 9, 16, 23). In addition, two cellular proteins have been found to associate with the PIC. They increase the efficiency of integration of the viral genome in vitro (10) and/or prevent self-integration of the viral DNA, which would result in an nonproductive infection (17). MLVs are widely used as vectors for gene therapy because of their relatively simple genome organization and their ability to infect a wide variety of cell types and integrate DNAs into the host cell genome (21). However, a major limitation of MLVbased vectors is their inability to infect nondividing cells (18, 22, 28), as opposed to vectors based on lentiviruses (24). The reasons for the inability of MLV-based vectors to infect nondividing cells are uncertain; the large size of their PIC or the lack of appropriate nuclear targeting signals may be responsible. A more detailed knowledge of the organization of the intracellular viral complex in which Reverse Transcription occurs would improve our understanding of the interactions between the incoming virus and the infected cells. It may also allow the design of new MLV-based vectors in which the Reverse Transcription complex (RTC) is targeted to the nucleus of nondividing cells. To characterize the dynamics of the early steps of virus life cycle, we have analyzed detergent-free cytoplasmic and nuclear extracts at various time points after acute infection. These studies revealed the existence of three distinct species of intracellular complexes. Two species are incompetent for Reverse Transcription in vivo, while a third species starts and completes Reverse Transcription in the cell cytoplasm. MATERIALS AND METHODS
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4 Strong-stop Strand Transfer during Reverse Transcription
Cold Spring Harbor Monograph Archive, 1993Co-Authors: Alice Telesnitsky, Stephen P. GoffAbstract:Reverse Transcription of retroviral genomic RNA is, in a sense, a discontinuous process. The production of each strand of integration-competent retroviral DNA begins with the synthesis of short, discrete DNA products that can be elongated only after they are moved to secondary template locations in a process called strand transfer. Both strand transfers during Reverse Transcription involve the translocation of short DNA products from their sites of synthesis at one end of the genome to acceptor template regions at the other end of the genome, where DNA synthesis resumes. Because of the strand-transfer reactions, the final product of Reverse Transcription is a linear double-stranded DNA molecule that is longer at each end than the RNA from which it was duplicated. The termini of the completed viral DNA consist of identical sequences termed long terminal repeats (LTRs), which are formed when information present near the ends of the genomic RNA is duplicated at the tips of the preintegrative DNA. The conservation of the two-LTR structure in all retroviruses and among other retroelements whose life cycles involve an integrated DNA form, such as yeast Tys and Drosophila copia elements, suggests that the process of strand transfer is as conserved a feature of these elements’ replication as the process of Reverse Transcription itself. Strand transfer, as a specialized form of template switching, is mechanistically related to some models for recombination during Reverse Transcription. Thus, the mechanisms for strong-stop strand transfer and for the generation of retroviral genetic diversity through recombination and transduction of...
