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 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.

Dressed to kill? A review of why antiviral CD8 T lymphocytes fail to prevent progressive immunodeficiency in HIV-1 infection

Blood ◽  
2001 ◽  
Vol 98 (6) ◽  
pp. 1667-1677 ◽  
Author(s):  
Judy Lieberman ◽  
Premlata Shankar ◽  
N. Manjunath ◽  
Jan Andersson
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Interferon Γ ◽  
Short Term Exposure ◽  
Infected Cells ◽  
Hiv 1

Abstract CD8 T cells play an important role in protection and control of HIV-1 by direct cytolysis of infected cells and by suppression of viral replication by secreted factors. However, although HIV-1–infected individuals have a high frequency of HIV-1–specific CD8 T cells, viral reservoirs persist and progressive immunodeficiency generally ensues in the absence of continuous potent antiviral drugs. Freshly isolated HIV-specific CD8 T cells are often unable to lyse HIV-1–infected cells. Maturation into competent cytotoxic T lymphocytes may be blocked during the initial encounter with antigen because of defects in antigen presentation by interdigitating dendritic cells or HIV-infected macrophages. The molecular basis for impaired function is multifactorial, due to incomplete T-cell signaling and activation (in part related to CD3ζ and CD28 down-modulation), reduced perforin expression, and inefficient trafficking of HIV-specific CD8 T cells to lymphoid sites of infection. CD8 T-cell dysfunction can partially be corrected in vitro with short-term exposure to interleukin 2, suggesting that impaired HIV-specific CD4 T helper function may play a significant causal or exacerbating role. Functional defects are qualitatively different and more severe with advanced disease, when interferon γ production also becomes compromised.

scholarly journals Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes

2002 ◽  
Vol 76 (23) ◽  
pp. 12376-12380 ◽  
Author(s):  
Youichi Suzuki ◽  
Robert Craigie
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Preintegration Complex ◽  
Retroviral Integration ◽  
Target Dna ◽  
Initial Salt ◽  
Murine Leukemia

ABSTRACT Retroviral integration is mediated by a preintegration complex (PIC) which contains the viral DNA made by reverse transcription together with associated protein factors. Prior to association with target DNA, the PIC must avoid suicidal intramolecular integration of its viral DNA (autointegration). We have demonstrated that barrier-to-autointegration factor (BAF) blocks the autointegration of Moloney murine leukemia virus (MoMLV) PICs in vitro. In this study, we show that BAF is an authentic component of MoMLV. Analysis of the sedimentation properties of initial, salt-stripped, and BAF-reconstituted PICs reveals that the viral DNA within the PIC is reversibly compacted by BAF, consistent with the functional role of BAF in protecting the viral DNA from autointegration. Furthermore, we find that BAF can promote the association of PICs with target DNA. Thus, our data suggest that BAF plays critical roles in promoting preferential intermolecular integration by both blocking autointegration and stimulating the capture of target DNA.

scholarly journals Comparative Analysis of HIV-1 and Murine Leukemia Virus Three-Dimensional Nuclear Distributions

2016 ◽  
Vol 90 (10) ◽  
pp. 5205-5209 ◽  
Author(s):  
Valentina Quercioli ◽  
Cristina Di Primio ◽  
Antonio Casini ◽  
Lubbertus C. F. Mulder ◽  
Lenard S. Vranckx ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Murine Leukemia Virus ◽  
Fluorescent Protein ◽  
Leukemia Virus ◽  
Three Dimensional ◽  
Moloney Murine Leukemia Virus ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Hiv 1 ◽  
Murine Leukemia

Recent advances in fluorescence microscopy allow three-dimensional analysis of HIV-1 preintegration complexes in the nuclei of infected cells. To extend this investigation to gammaretroviruses, we engineered a fluorescent Moloney murine leukemia virus (MLV) system consisting of MLV-integrase fused to enhanced green fluorescent protein (MLV-IN-EGFP). A comparative analysis of lentiviral (HIV-1) and gammaretroviral (MLV) fluorescent complexes in the nuclei of infected cells revealed their different spatial distributions. This research tool has the potential to achieve new insight into the nuclear biology of these retroviruses.

scholarly journals tRNA Isoacceptor Preference prior to Retrovirus Gag-Pol Junction Links Primer Selection and Viral Translation

2007 ◽  
Vol 81 (9) ◽  
pp. 4397-4404 ◽  
Author(s):  
Matthew T. Palmer ◽  
Richard Kirkman ◽  
Barry R. Kosloff ◽  
Peter G. Eipers ◽  
Casey D. Morrow
Keyword(s):  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Primer Binding Site ◽  
Wild Type Virus ◽  
Viral Translation ◽  
Primer Selection ◽  
Codon Preference ◽  
Trna Primer ◽  
Hiv 1

ABSTRACT An essential step in the replication of all retroviruses is the capture of a cellular tRNA that is used as the primer for reverse transcription. The 3′-terminal 18 nucleotides of the tRNA are complementary to the primer binding site (PBS). Moloney murine leukemia virus (MuLV) preferentially captures tRNAPro. To investigate the specificity of primer selection, the PBS of MuLV was altered to be complementary to different tRNAs. Analysis of the infectivity of the virus and stability of the PBS following in vitro replication revealed that MuLV prefers to select tRNAPro, tRNAGly, or tRNAArg. Previous studies from our laboratory have suggested that tRNA primer capture is coordinated with translation. Coincidentally, a cluster of proline, arginine, and glycine precedes the Gag-Pol junction of MuLV. Human immunodeficiency virus type 1 (HIV-1), which prefers \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{3}^{\mathrm{Lys}}\) \end{document} as the primer, can be forced to utilize tRNAMet, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} , tRNAHis, or tRNAGlu, although these viruses replicate poorly. Codons for methionine, lysine, histidine, or glutamic acid are found prior to the Gag-Pol frameshift site. HIV-1 was mutated so that the 5 lysine codons prior to the Gag-Pol frameshift region were specific for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} . HIV-1 forced to use \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} as the primer, with the mutation of codons specific for \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{tRNA}_{1,2}^{\mathrm{Lys}}\) \end{document} prior to the Gag-Pol junction, had enhanced infectivity and replicated similarly to the wild-type virus. The results demonstrate that codon preference prior to the Gag-Pol junction influences primer selection and suggest a coordination of Gag-Pol synthesis and acquisition of the tRNA primer required for retrovirus replication.

scholarly journals Comparative clonal analysis of human immunodeficiency virus type 1 (HIV-1)-specific CD4+ and CD8+ cytolytic T lymphocytes isolated from seronegative humans immunized with candidate HIV-1 vaccines.

1992 ◽  
Vol 176 (6) ◽  
pp. 1531-1542 ◽  
Author(s):  
S A Hammond ◽  
R C Bollinger ◽  
P E Stanhope ◽  
T C Quinn ◽  
D Schwartz ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Lymphocytes ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cytolytic T Lymphocytes ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Hiv 1

The lysis of infected host cells by virus-specific cytolytic T lymphocytes (CTL) is an important factor in host resistance to viral infection. An optimal vaccine against human immunodeficiency virus type 1 (HIV-1) would elicit virus-specific CTL as well as neutralizing antibodies. The induction by a vaccine of HIV-1-specific CD8+ CTL in humans has not been previously reported. In this study, CTL responses were evaluated in HIV-1-seronegative human volunteers participating in a phase I acquired immune deficiency syndrome (AIDS) vaccine trial involving a novel vaccine regimen. Volunteers received an initial immunization with a live recombinant vaccinia virus vector carrying the HIV-1 env gene and a subsequent boost with purified env protein. An exceptionally strong env-specific CTL response was detected in one of two vaccine recipients, while modest but significant env-specific CTL activity was present in the second vaccinee. Cloning of the responding CTL gave both CD4+ and CD8+ env-specific CTL clones, permitting a detailed comparison of critical functional properties of these two types of CTL. In particular, the potential antiviral effects of these CTL were evaluated in an in vitro system involving HIV-1 infection of cultures of normal autologous CD4+ lymphoblasts. At extremely low effector-to-target ratios, vaccine-induced CD8+ CTL clones lysed productively infected cells present within these cultures. When tested for lytic activity against target cells expressing the HIV-1 env gene, CD8+ CTL were 3-10-fold more active on a per cell basis than CD4+ CTL. However, when tested against autologous CD4+ lymphoblasts acutely infected with HIV-1, CD4+ clones lysed a much higher fraction of the target cell population than did CD8+ CTL. CD4+ CTL were shown to recognize not only the infected cells within these acutely infected cultures but also noninfected CD4+ T cells that had passively taken up gp120 shed from infected cells and/or free virions. These results were confirmed in studies in which CD4+ lymphoblasts were exposed to recombinant gp120 and used as targets for gp120-specific CD4+ and CD8+ CTL clones. gp120-pulsed, noninfected targets were lysed in an antigen-specific fashion by CD4+ but not CD8+ CTL clones. Taken together, these observations demonstrate that in an in vitro HIV-1 infection, sufficient amounts of gp120 antigen are produced and shed by infected cells to enable uptake by cells that are not yet infected, resulting in the lysis of these noninfected cells by gp120-specific, CD4+ CTL.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Assembly and Catalysis of Concerted Two-End Integration Events by Moloney Murine Leukemia Virus Integrase

2001 ◽  
Vol 75 (20) ◽  
pp. 9561-9570 ◽  
Author(s):  
Fan Yang ◽  
Monica J. Roth
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Strand Transfer ◽  
Viral Dna ◽  
Retroviral Integration ◽  
Target Dna ◽  
The Individual ◽  
Murine Leukemia

ABSTRACT Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.

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 Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

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