Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase

Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3′-processed–like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3′-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.

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Alternate Polypurine Tracts Affect Rous Sarcoma Virus Integration In Vivo

Journal of Virology ◽

10.1128/jvi.00361-06 ◽

2006 ◽

Vol 80 (20) ◽

pp. 10281-10284 ◽

Cited By ~ 10

Author(s):

Jangsuk Oh ◽

Kevin W. Chang ◽

W. Gregory Alvord ◽

Stephen H. Hughes

Keyword(s):

Long Terminal Repeat ◽

Rous Sarcoma Virus ◽

Terminal Repeat ◽

Host Genome ◽

Viral Dna ◽

Rous Sarcoma ◽

Polypurine Tract ◽

Sarcoma Virus ◽

Virus Integration

ABSTRACT When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.

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Sequence-specific recognition of the HIV-1 long terminal repeat by distamycin: a DNAase I footprinting study

Biochemical Journal ◽

10.1042/bj2990451 ◽

1994 ◽

Vol 299 (2) ◽

pp. 451-458 ◽

Cited By ~ 21

Author(s):

G Feriotto ◽

C Mischiati ◽

R Gambari

Keyword(s):

Long Terminal Repeat ◽

Dna Sequences ◽

Molecular Mechanisms ◽

Terminal Repeat ◽

Experimental Therapy ◽

Mobility Shift ◽

Target Dna ◽

Hiv 1

Pharmacological modulation of the interaction between transcription factors and target DNA sequences of cellular and viral genes could have important effects in the experimental therapy of a large variety of human pathologies. For instance, alteration of the DNA/protein interaction might be among the molecular mechanisms of action of DNA-binding drugs, leading to an inhibition of the expression of genes involved in the control of in vitro and in vivo growth of neoplastic cells and virus DNA replication. Natural oligopeptides, such as distamycin, are powerful inhibitors of the interaction between nuclear factors and target DNA sequences and, therefore, have been proposed as compounds retaining antibiotic, antineoplastic and antiviral properties. In this study we performed DNAase I footprinting analysis using a PCR product mimicking a region of the long terminal repeat (LTR) of the human immunodeficiency type 1 (HIV-1) retrovirus. The data obtained suggest that distamycin binds to different regions of the HIV-1 LTR depending on the DNA sequence. Electrophoretic mobility shift assays using both crude nuclear extracts from the Jurkat T-lymphoid cell line and the recombinant proteins transcription factor IID and Sp1 suggest that distamycin differentially inhibits the interaction of these two proteins with their specific DNA target sequences, in good agreement with the results obtained by DNAase I footprinting analysis.

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In vitro study of functional involvement of Sp1, NF-kappa B/Rel, and AP1 in phorbol 12-myristate 13-acetate-mediated HIV-1 long terminal repeat activation.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)43858-6 ◽

1994 ◽

Vol 269 (48) ◽

pp. 30616-30619

Author(s):

Y Li ◽

G Mak ◽

B R Franza

Keyword(s):

Long Terminal Repeat ◽

In Vitro Study ◽

Vitro Study ◽

Terminal Repeat ◽

Nf Kappa B ◽

Functional Involvement ◽

Hiv 1

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Binding of a cellular protein to the gibbon ape leukemia virus enhancer

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2735-2744.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2735-2744

Author(s):

J P Quinn ◽

N Holbrook ◽

D Levens

Keyword(s):

Long Terminal Repeat ◽

Specific Binding ◽

Leukemia Virus ◽

Cellular Protein ◽

Terminal Repeat ◽

Enhancer Activity ◽

Repeated Element ◽

Gibbon Ape Leukemia Virus

The gibbon ape leukemia virus (GALV) contains enhancer activity within its long terminal repeat. In the GALV Seato strain this activity resides in a 48-base-pair (bp) repeated element. We demonstrate the existence of a cellular protein which binds in this region of the Seato strain. A sensitive method for enriching protein-DNA complexes from crude extracts coupled with exonuclease and DNase footprint analysis revealed the specific binding of this protein to a 21-bp region within each repeated element. A 22-bp oligonucleotide fragment defined solely by the 21-bp footprint binds a protein in vitro and displays enhancer activity in vivo, suggesting that this protein is a major determinant of GALV enhancer activity. The protein is present in three cell lines which are positive for enhancer activity and is not detected in Jurkat cells, which are negative for enhancer activity. Only GALV long-terminal-repeat variants which support high levels of enhancer activity in vivo compete with this protein for specific binding in vitro, suggesting a potential role for the protein in determining enhancer activity. This protein binding is not inhibited by competition with heterologous retroviral enhancers, demonstrating that it is not a ubiquitous retroviral enhancer binding protein.

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Nature, Position, and Frequency of Mutations Made in a Single Cycle of HIV-1 Replication

Journal of Virology ◽

10.1128/jvi.00915-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 9864-9878 ◽

Cited By ~ 148

Author(s):

Michael E. Abram ◽

Andrea L. Ferris ◽

Wei Shao ◽

W. Gregory Alvord ◽

Stephen H. Hughes

Keyword(s):

Viral Replication ◽

Mutation Rate ◽

Hot Spots ◽

High Rate ◽

Published Data ◽

Single Cycle ◽

Efficient System ◽

Hiv 1

ABSTRACT There is considerable HIV-1 variation in patients. The extent of the variation is due to the high rate of viral replication, the high viral load, and the errors made during viral replication. Mutations can arise from errors made either by host DNA-dependent RNA polymerase II or by HIV-1 reverse transcriptase (RT), but the relative contributions of these two enzymes to the mutation rate are unknown. In addition, mutations in RT can affect its fidelity, but the effect of mutations in RT on the nature of the mutations that arise in vivo is poorly understood. We have developed an efficient system, based on existing technology, to analyze the mutations that arise in an HIV-1 vector in a single cycle of replication. A lacZα reporter gene is used to identify viral DNAs that contain mutations which are analyzed by DNA sequencing. The forward mutation rate in this system is 1.4 × 10−5 mutations/bp/cycle, equivalent to the retroviral average. This rate is about 3-fold lower than previously reported for HIV-1 in vivo and is much lower than what has been reported for purified HIV-1 RT in vitro. Although the mutation rate was not affected by the orientation of lacZα, the sites favored for mutations (hot spots) in lacZα depended on which strand of lacZα was present in the viral RNA. The pattern of hot spots seen in lacZα in vivo did not match any of the published data obtained when purified RT was used to copy lacZα in vitro.

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HIV-1 and HIV-2 produce different amounts of 2-long terminal repeat circular DNA in vitro

AIDS ◽

10.1097/qad.0b013e328319edab ◽

2008 ◽

Vol 22 (18) ◽

pp. 2543-2545 ◽

Cited By ~ 2

Author(s):

Marie Gueudin ◽

Joséphine Braun ◽

Jean-Christophe Plantier ◽

François Simon

Keyword(s):

Long Terminal Repeat ◽

Terminal Repeat ◽

Circular Dna ◽

Hiv 1

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Evolution of the Equine Infectious Anemia Virus Long Terminal Repeat during the Alteration of Cell Tropism

Journal of Virology ◽

10.1128/jvi.79.9.5653-5664.2005 ◽

2005 ◽

Vol 79 (9) ◽

pp. 5653-5664 ◽

Cited By ~ 17

Author(s):

Wendy Maury ◽

Robert J. Thompson ◽

Quentin Jones ◽

Sarahann Bradley ◽

Tara Denke ◽

...

Keyword(s):

Long Terminal Repeat ◽

Equine Infectious Anemia Virus ◽

Terminal Repeat ◽

Binding Motif ◽

Cell Tropism ◽

Transcription Factor Binding Motif ◽

Equine Infectious Anemia ◽

Anemia Virus

ABSTRACT Equine infectious anemia virus (EIAV) is a lentivirus with in vivo cell tropism primarily for tissue macrophages; however, in vitro the virus can be adapted to fibroblasts and other cell types. Tropism adaptation is associated with both envelope and long terminal repeat (LTR) changes, and findings strongly suggest that these regions of the genome influence cell tropism and virulence. Furthermore, high levels of genetic variation have been well documented in both of these genomic regions. However, specific EIAV nucleotide or amino acid changes that are responsible for cell tropism changes have not been identified. A study was undertaken with the highly virulent, macrophage-tropic strain of virus EIAVwyo to identify LTR changes associated with alterations in cell tropism. We found the stepwise generation of a new transcription factor binding motif within the enhancer that was associated with adaptation of EIAV to endothelial cells and fibroblasts. An LTR that contained the new motif had enhanced transcriptional activity in fibroblasts, whereas the new site did not alter LTR activity in a macrophage cell line. This finding supports a previous prediction that selection for new LTR genetic variants may be a consequence of cell-specific selective pressures. Additional investigations of the EIAVwyo LTR were performed in vivo to determine if LTR evolution could be detected over the course of a 3-year infection. Consistent with previous in vivo findings, we observed no changes in the enhancer region of the LTR over that time period, indicating that the EIAVwyo LTR was evolutionarily stable in vivo.

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The 5′ Untranslated Region and Gag product of Idefix, a Long Terminal Repeat-Retrotransposon from Drosophila melanogaster, Act Together To Initiate a Switch between Translated and Untranslated States of the Genomic mRNA

Molecular and Cellular Biology ◽

10.1128/mcb.23.22.8246-8254.2003 ◽

2003 ◽

Vol 23 (22) ◽

pp. 8246-8254 ◽

Cited By ~ 14

Author(s):

Carine Meignin ◽

Jean-Luc Bailly ◽

Frédérick Arnaud ◽

Bernard Dastugue ◽

Chantal Vaury

Keyword(s):

Drosophila Melanogaster ◽

Long Terminal Repeat ◽

Untranslated Region ◽

Terminal Repeat ◽

Entry Site ◽

Independent Manner ◽

Gag Protein ◽

Genomic Arrangement

ABSTRACT Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5′ untranslated region (5′UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5′UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5′UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

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Construction and In Vitro Characterization of Attenuated Feline Immunodeficiency Virus Long Terminal Repeat Mutant Viruses

Journal of Virology ◽

10.1128/jvi.75.2.1054-1060.2001 ◽

2001 ◽

Vol 75 (2) ◽

pp. 1054-1060 ◽

Cited By ~ 12

Author(s):

Luisa Bigornia ◽

Kristen M. Lockridge ◽

Ellen E. Sparger

Keyword(s):

Long Terminal Repeat ◽

Virus Replication ◽

Feline Immunodeficiency Virus ◽

Terminal Repeat ◽

Immunodeficiency Virus ◽

Activation Pathways ◽

Virus Expression ◽

Transcriptional Elements

ABSTRACT AP-1- and ATF-binding sites are cis-acting transcriptional elements within the U3 domain of the feline immunodeficiency virus (FIV) long terminal repeat (LTR) that serve as targets for cellular activation pathways and may regulate virus replication. We report that FIV LTR mutant proviruses encoding U3 deletions of the ATF-binding sequence exhibited restricted virus expression and replication in both feline lymphocytes and macrophages. In contrast, deletion of the AP-1 site had negligible effects on virus expression and replication. FIV LTR mutant proviruses encoding deletions of both the AP-1 and ATF sites or a 72-bp deletion encompassing the AP-1 site, duplicated C/EBP sites, and ATF sites were severely restricted for virus expression. These results demonstrate that deletion of either the ATF-binding site or multiplecis-acting transcriptional elements attenuates FIV. These attenuated FIV mutants provide opportunities to characterize the role of cis-acting elements in virus replication in vivo and to test LTR mutants as attenuated virus vaccines.

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