scholarly journals Retroviral cDNA Integration: Stimulation by HMG I Family Proteins

2000 ◽  
Vol 74 (23) ◽  
pp. 10965-10974 ◽  
Author(s):  
Ling Li ◽  
Kristine Yoder ◽  
Mark S. T. Hansen ◽  
Jennifer Olvera ◽  
Michael D. Miller ◽  
...  
Keyword(s):  
Dna Binding ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Infected Cells ◽  
High Concentrations ◽  
Cdna Integration ◽  
Hiv 1

ABSTRACT To replicate, a retrovirus must synthesize a cDNA copy of the viral RNA genome and integrate that cDNA into a chromosome of the host. We have investigated the role of a host cell cofactor, HMG I(Y) protein, in integration of human immunodeficiency virus type 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) cDNA. Previously we reported that HMG I(Y) cofractionates with HIV-1 preintegration complexes (PICs) isolated from freshly infected cells. PICs depleted of required components by treatment with high concentrations of salt could be reconstituted by addition of purified HMG I(Y) in vitro. Here we report studies using immunoprecipitation that indicate that HMG I(Y) is associated with MoMLV preintegration complexes. In mechanistic studies, we show for both HIV-1 and MoMLV that each HMG I(Y) monomer must contain multiple DNA binding domains to stimulate integration by HMG I(Y)-depleted PICs. We also find that HMG I(Y) can condense model HIV-1 or MoMLV cDNA in vitro as measured by stimulation of intermolecular ligation. This reaction, like reconstitution of integration, depends on the presence of multiple DNA binding domains in each HMG I(Y) monomer. These data suggest that binding of multivalent HMG I(Y) monomers to multiple cDNA sites compacts retroviral cDNA, thereby promoting formation of active integrase-cDNA complexes.

scholarly journals Barrier-to-Autointegration Factor BAF Binds p55 Gagand Matrix and Is a Host Component of Human ImmunodeficiencyVirus Type 1Virions

2003 ◽  
Vol 77 (24) ◽  
pp. 13084-13092 ◽  
Author(s):  
Malini Mansharamani ◽  
David R. M. Graham ◽  
Daphne Monie ◽  
Kenneth K. Lee ◽  
James E. K. Hildreth ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Blood Leukocytes ◽  
Target Dna ◽  
Direct Binding ◽  
Infected Cells ◽  
Hiv 1 ◽  
Host Component

ABSTRACT Barrier-to-autointegration factor (BAF) is a conserved human chromatin protein exploited by retroviruses. Previous investigators showed that BAF binds double-stranded DNA nonspecifically and is a host component of preintegration complexes (PICs) isolated from cells infected with human immunodeficiency virus type 1 (HIV-1) or Moloney murine leukemia virus. BAF protects PIC structure and stimulates the integration of salt-stripped PICs into target DNA in vitro. PICs are thought to acquire BAF from the cytoplasm during infection. However, we identified two human tissues (of 16 tested) in which BAF mRNA was not detected: thymus and peripheral blood leukocytes, which are enriched in CD4+ T lymphocytes and macrophage precursors, respectively. BAF protein was detected in activated but not resting CD4+ T lymphocytes; thus, if BAF were essential for PIC function, we hypothesized that virions might “bring their own BAF.” Supporting this model, BAF copurified with HIV-1 virions that were digested with subtilisin to remove microvesicle contaminants, and BAF was present in approximately zero to three copies per virion. In three independent assays, BAF bound directly to both p55 Gag (the structural precursor of HIV-1 virions) and its cleaved product, matrix. Using lysates from cells overexpressing Gag, endogenous BAF and Gag were coimmunoprecipitated by antibodies against Gag. Purified recombinant BAF had low micromolar affinities (1.1 to 1.4μ M) for recombinant Gag and matrix. We conclude that BAF is present at low levels in incoming virions, in addition to being acquired from the cytoplasm of newly infected cells. We further conclude that BAF might contribute to the assembly or activity of HIV-1 PICs through direct binding to matrix, as well as DNA.

scholarly journals Modulation of Activity of Moloney Murine Leukemia Virus Preintegration Complexes by Host Factors In Vitro

1998 ◽  
Vol 72 (3) ◽  
pp. 2125-2131 ◽  
Author(s):  
Ling Li ◽  
Chris M. Farnet ◽  
W. French Anderson ◽  
Frederic D. Bushman
Keyword(s):  
Dna Binding ◽  
Murine Leukemia Virus ◽  
Binding Proteins ◽  
Leukemia Virus ◽  
Dna Binding Proteins ◽  
Host Factor ◽  
Moloney Murine Leukemia Virus ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT We have explored the requirements for host proteins in the integration of Moloney murine leukemia virus (MoMuLV) cDNA in vitro. Following infection, it is possible to lyse cells and obtain preintegration complexes (PICs) capable of integrating the MoMuLV cDNA into an added target DNA in vitro (intermolecular integration). PICs can be stripped of required proteins by gel filtration in high-salt buffers (600 mM KCl), allowing the nature of the removed factors to be investigated by in vitro reconstitution. In a previous study of human immunodeficiency virus type 1 (HIV-1) PICs, the host protein HMG I(Y) was found to be able to restore activity to salt-stripped PICs. In contrast, salt stripping and reconstitution of MoMuLV PICs led to the proposal that a host factor is important for a different activity, blocking integration into the cDNA itself (autointegration). In this report, we investigated reconstitution of salt-stripped MoMuLV PICs and found that addition of cellular extract from uninfected NIH 3T3 cells could block autointegration and also restore intermolecular integration. Isolation of the intermolecular integration-complementing activity yielded HMG I(Y), as in the HIV-1 case. However, HMG I(Y) could not block autointegration, implicating a different host factor in this process. Additionally, when MoMuLV PICs were partially purified but not salt stripped, the intermolecular integration activity was reduced but could be stimulated by the addition of any of several purified DNA binding proteins. In summary, three activities were detected: (i) the intermolecular integration cofactor HMG I(Y), (ii) an autointegration barrier protein, and (iii) stimulatory DNA binding proteins.

scholarly journals Role of Papillomavirus E1 Initiator Dimerization in DNA Replication

2005 ◽  
Vol 79 (13) ◽  
pp. 8661-8664 ◽  
Author(s):  
Stephen Schuck ◽  
Arne Stenlund
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Severe Effect ◽  
Origin Of Dna Replication ◽  
Initiation Of Dna Replication

ABSTRACT Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro. We conclude that the dimer interaction is primarily required for initial recognition of ori.

N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65

1993 ◽  
Vol 13 (2) ◽  
pp. 852-860
Author(s):  
M B Toledano ◽  
D Ghosh ◽  
F Trinh ◽  
W J Leonard
Keyword(s):  
Dna Binding ◽  
Point Mutations ◽  
Terminal Region ◽  
Sequence Motif ◽  
Structural Determinants ◽  
Nf Kappa B ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Dna Binding Specificity

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.

scholarly journals Fused protein domains inhibit DNA binding by LexA.

1992 ◽  
Vol 12 (7) ◽  
pp. 3006-3014 ◽  
Author(s):  
E A Golemis ◽  
R Brent
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
In Vitro Assays ◽  
Dimerization Domain ◽  
Activation Domains ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

scholarly journals RNA polymerase II transcription factors H4TF-1 and H4TF-2 require metal to bind specific DNA sequences.

1987 ◽  
Vol 7 (12) ◽  
pp. 4582-4584 ◽  
Author(s):  
L Dailey ◽  
S B Roberts ◽  
N Heintz
Keyword(s):  
Dna Binding ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Chelating Agents ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Dna Binding Domains ◽  
Binding Domains ◽  
Rna Polymerase Ii Transcription

Specific DNA-binding and in vitro transcription activities of H4TF-1 and H4TF-2 are inactivated by chelating agents. Binding activity is restored by addition of Zn2+, and H4TF-2 is also reactivated by Fe2+. In contrast, preformed factor-DNA complexes are resistant to chelators. Therefore, metal ions are a required component of the H4TF-1 and H4TF-2 DNA-binding domains.

Cooperative interactions between paired domain and homeodomain

Development ◽  
1996 ◽  
Vol 122 (9) ◽  
pp. 2639-2650 ◽  
Author(s):  
S. Jun ◽  
C. Desplan
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Target Genes ◽  
Cooperative Interaction ◽  
Paired Domain ◽  
Cooperative Interactions ◽  
Dna Binding Domains ◽  
Binding Domains

The Pax proteins are a family of transcriptional regulators involved in many developmental processes in all higher eukaryotes. They are characterized by the presence of a paired domain (PD), a bipartite DNA binding domain composed of two helix-turn-helix (HTH) motifs, the PAI and RED domains. The PD is also often associated with a homeodomain (HD) which is itself able to form homo- and hetero-dimers on DNA. Many of these proteins therefore contain three HTH motifs each able to recognize DNA. However, all PDs recognize highly related DNA sequences, and most HDs also recognize almost identical sites. We show here that different Pax proteins use multiple combinations of their HTHs to recognize several types of target sites. For instance, the Drosophila Paired protein can bind, in vitro, exclusively through its PAI domain, or through a dimer of its HD, or through cooperative interaction between PAI domain and HD. However, prd function in vivo requires the synergistic action of both the PAI domain and the HD. Pax proteins with only a PD appear to require both PAI and RED domains, while a Pax-6 isoform and a new Pax protein, Lune, may rely on the RED domain and HD. We propose a model by which Pax proteins recognize different target genes in vivo through various combinations of their DNA binding domains, thus expanding their recognition repertoire.

An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation

1994 ◽  
Vol 14 (10) ◽  
pp. 6570-6583 ◽  
Author(s):  
N D Perkins ◽  
A B Agranoff ◽  
E Pascal ◽  
G J Nabel
Keyword(s):  
Human Immunodeficiency Virus ◽  
Transcription Factors ◽  
Dna Binding ◽  
Binding Sites ◽  
Nf Kappa B ◽  
Dna Binding Domains ◽  
Amino Terminal ◽  
Binding Domains ◽  
Immunodeficiency Virus ◽  
Hiv 1

Induction of human immunodeficiency virus type 1 (HIV-1) gene expression in stimulated T cells has been attributed to the activation of the transcription factor NF-kappa B. The twice-repeated kappa B sites within the HIV-1 long terminal repeat are in close proximity to three binding sites for Sp1. We have previously shown that a cooperative interaction of NF-kappa B with Sp1 is required for the efficient stimulation of HIV-1 transcription. In this report, we define the domains of each protein responsible for this effect. Although the transactivation domains seemed likely to mediate this interaction, we find, surprisingly, that this interaction occurs through the putative DNA-binding domains of both proteins. Sp1 specifically interacted with the amino-terminal region of RelA(p65). Similarly, RelA bound directly to the zinc finger region of Sp1. This interaction was specific and resulted in cooperative DNA binding to the kappa B and Sp1 sites in the HIV-1 long terminal repeat. Furthermore, the amino-terminal region of RelA did not associate with several other transcription factors, including MyoD, E12, or Kox15, another zinc finger protein. These findings suggest that the juxtaposition of DNA-binding sites promotes a specific protein interaction between the DNA-binding regions of these transcription factors. This interaction is required for HIV transcriptional activation and may provide a mechanism to allow for selective activation of kappa B-regulated genes.

scholarly journals Purification and Characterization of the AAA+ Domain of Sinorhizobium meliloti DctD, a σ54-Dependent Transcriptional Activator

2004 ◽  
Vol 186 (11) ◽  
pp. 3499-3507 ◽  
Author(s):  
Hao Xu ◽  
Baohua Gu ◽  
B. Tracy Nixon ◽  
Timothy R. Hoover
Keyword(s):  
Dna Binding ◽  
Rna Polymerase ◽  
Sinorhizobium Meliloti ◽  
Atp Hydrolysis ◽  
Binding Domain ◽  
Stable Complex ◽  
Dna Binding Domain ◽  
Dna Binding Domains ◽  
Binding Domains

ABSTRACT Activators of σ54-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open complex between the promoter and RNA polymerase. These activators are modular, consisting of an N-terminal regulatory domain, a C-terminal DNA-binding domain, and a central activation domain belonging to the AAA+ superfamily of ATPases. The AAA+ domain of Sinorhizobium meliloti C4-dicarboxylic acid transport protein D (DctD) is sufficient to activate transcription. Deletion analysis of the 3′ end of dctD identified the minimal functional C-terminal boundary of the AAA+ domain of DctD as being located between Gly-381 and Ala-384. Histidine-tagged versions of the DctD AAA+ domain were purified and characterized. The DctD AAA+ domain was significantly more soluble than DctD( Δ 1-142), a truncated DctD protein consisting of the AAA+ and DNA-binding domains. In addition, the DctD AAA+ domain was more homogeneous than DctD( Δ 1-142) when analyzed by native gel electrophoresis, migrating predominantly as a single high-molecular-weight species, while DctD( Δ 1-142) displayed multiple species. The DctD AAA+ domain, but not DctD( Δ 1-142), formed a stable complex with σ54 in the presence of the ATP transition state analogue ADP-aluminum fluoride. The DctD AAA+ domain activated transcription in vitro, but many of the transcripts appeared to terminate prematurely, suggesting that the DctD AAA+ domain interfered with transcription elongation. Thus, the DNA-binding domain of DctD appears to have roles in controlling the oligomerization of the AAA+ domain and modulating interactions with σ54 in addition to its role in recognition of upstream activation sequences.

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