scholarly journals SATB1-Binding Sequences and Alu-Like Motifs Define a Unique Chromatin Context in the Vicinity of Human Immunodeficiency Virus Type 1 Integration Sites

2007 ◽  
Vol 81 (11) ◽  
pp. 5617-5627 ◽  
Author(s):  
Pavan P. Kumar ◽  
Sameet Mehta ◽  
Prabhat Kumar Purbey ◽  
Dimple Notani ◽  
Ranveer S. Jayani ◽  
...  
Keyword(s):  
Site Selection ◽  
Binding Sites ◽  
Integration Site ◽  
Viral Integration ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Hiv 1 ◽  
Integration Site Selection

ABSTRACT Retroviral integration has recently been shown to be nonrandom, favoring transcriptionally active regions of chromatin. However, the mechanism for integration site selection by retroviruses is not clear. We show here the occurrence of Alu-like motifs in the sequences flanking the reported viral integration sites that are significantly different from those obtained from the randomly picked sequences from the human genome, suggesting that unique primary sequence features exist in the genomic regions targeted by human immunodeficiency virus type 1 (HIV-1). Additionally, these sequences were preferentially bound by SATB1, the T lineage-restricted chromatin organizer, in vitro and in vivo. Alu repeats make up nearly 10% of the human genome and have been implicated in the regulation of transcription. To specifically isolate sequences flanking the viral integration sites and also harboring both Alu-like repeats and SATB1-binding sites, we combined chromatin immunoprecipitation with sequential PCRs. The cloned sequences flanking HIV-1 integration sites were specifically immunoprecipitated and amplified from the pool of anti-SATB1-immunoprecipitated genomic DNA fragments isolated from HIV-1 NL4.3-infected Jurkat T-cell chromatin. Moreover, many of these sequences were preferentially partitioned in the DNA associated tightly with the nuclear matrix and not in the chromatin loops. Strikingly, many of these regions were disfavored for integration when SATB1 was silenced, providing unequivocal evidence for its role in HIV-1 integration site selection. We propose that definitive sequence features such as the Alu-like motifs and SATB1-binding sites provide a unique chromatin context in vivo which is preferentially targeted by the HIV-1 integration machinery.

scholarly journals Genomic Sites of Human Immunodeficiency Virus Type 2 (HIV-2) Integration: Similarities to HIV-1 In Vitro and Possible Differences In Vivo

2006 ◽  
Vol 80 (15) ◽  
pp. 7316-7321 ◽  
Author(s):  
Adam MacNeil ◽  
Jean-Louis Sankalé ◽  
Seema Thakore Meloni ◽  
Abdoulaye Dieng Sarr ◽  
Souleymane Mboup ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Site Selection ◽  
Integration Site ◽  
Proviral Integration ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Integration Site Selection

ABSTRACT Retroviruses have distinct preferences in integration site selection in the host cell genome during in vitro infection, with human immunodeficiency virus type 1 (HIV-1) integration strongly favoring transcriptional units. Additionally, studies with HIV-1 have shown that the genomic site of proviral integration may impact viral replication, with integration in heterochromatin associated with a block in viral transcription. HIV-2 is less pathogenic than HIV-1 and is believed to have a lower replication rate in vivo. Although differences in integration site selection between HIV-2 and HIV-1 could potentially explain the attenuated pathogenicity of HIV-2, no studies have characterized integration site selection by HIV-2. In this study, we mapped 202 HIV-2 integration sites during in vitro infection of peripheral blood mononuclear cells with a primary HIV-2 isolate. In addition, we assayed for in vivo proviral integration within heterochromatin in 21 HIV-1-infected subjects and 23 HIV-2-infected subjects, using an alphoid repeat PCR assay. During in vitro infection, HIV-2 displayed integration site preferences similar to those previously reported for HIV-1. Notably, 82% of HIV-2 integrations mapped to Refseq genes, and integration strongly favored regions of the genome with high gene density and high GC content. Though rare, the proportion of HIV-2 subjects with evidence of proviral integration within heterochromatin in vivo was higher than that of HIV-1-infected subjects. It is therefore possible that integration site selection may play a role in the differences in HIV-1 and HIV-2 in vivo pathogenesis.

scholarly journals Resting CD4+ T Cells from Human Immunodeficiency Virus Type 1 (HIV-1)-Infected Individuals Carry Integrated HIV-1 Genomes within Actively Transcribed Host Genes

2004 ◽  
Vol 78 (12) ◽  
pp. 6122-6133 ◽  
Author(s):  
Yefei Han ◽  
Kara Lassen ◽  
Daphne Monie ◽  
Ahmad R. Sedaghat ◽  
Shino Shimoji ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Cell Populations ◽  
Rt Pcr ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Hiv 1 ◽  
Host Genes

ABSTRACT Resting CD4+ T-cell populations from human immunodeficiency virus type 1 (HIV-1)-infected individuals include cells with integrated HIV-1 DNA. In individuals showing suppression of viremia during highly active antiretroviral therapy (HAART), resting CD4+ T-cell populations do not produce virus without cellular activation. To determine whether the nonproductive nature of the infection in resting CD4+ T cells is due to retroviral integration into chromosomal regions that are repressive for transcription, we used inverse PCR to characterize the HIV-1 integration sites in vivo in resting CD4+ T cells from patients on HAART. Of 74 integration sites from 16 patients, 93% resided within transcription units, usually within introns. Integration was random with respect to transcriptional orientation relative to the host gene and with respect to position within the host gene. Of integration sites within well-characterized genes, 91% (51 of 56) were in genes that were actively expressed in resting CD4+ T cells, as directly demonstrated by reverse transcriptase PCR (RT-PCR). These results predict that HIV-1 sequences may be included in the primary transcripts of host genes as part of rapidly degraded introns. RT-PCR experiments confirmed the presence of HIV-1 sequences within transcripts initiating upstream of the HIV-1 transcription start site. Taken together, these results demonstrate that HIV-1 genomes reside within actively transcribed host genes in resting CD4+ T cells in vivo.

scholarly journals A High-Throughput Method for Cloning and Sequencing Human Immunodeficiency Virus Type 1 Integration Sites

2006 ◽  
Vol 80 (22) ◽  
pp. 11313-11321 ◽  
Author(s):  
Sanggu Kim ◽  
Yein Kim ◽  
Teresa Liang ◽  
Janet S. Sinsheimer ◽  
Samson A. Chow
Keyword(s):  
High Throughput ◽  
Integration Site ◽  
Target Site ◽  
Viral Dna ◽  
Target Site Selection ◽  
Immunodeficiency Virus ◽  
High Throughput Method ◽  
Integration Sites ◽  
Hiv 1

ABSTRACT Integration of retroviral DNA is nonspecific and can occur at many sites throughout chromosomes. However, the process is not uniformly distributed, and both hot and cold spots for integration exist. The mechanism that determines target site specificity is not well understood. Because of the nonspecific and widespread nature of integration, studies analyzing the mechanism and factors that control target site selection require the collection and analysis of a large library of human immunodeficiency virus type 1 (HIV-1) proviral clones. Such analyses are time-consuming and labor-intensive using conventional means. We have developed an efficient and high-throughput method of sequencing and mapping a large number of independent integration sites in the absence of any selection or bias. The new assay involves the use of a modified HIV-1 (NL-Mme) containing a type IIS restriction site, MmeI, at the right end of viral DNA. Digestion of genomic DNA from NL-Mme-infected cells generated viral DNA-containing fragments of a discrete size. Subsequent ligation-mediated PCR yielded short integration site fragments termed Int-tags, which were concatemerized for determining multiple integration sites in a single sequencing reaction. Analysis of chromosomal features and sequence preference associated with integration events confirmed the validity of the new high-throughput assay. The assay will aid the effort in understanding the mechanisms of target site selection during HIV-1 DNA integration, and the described methodology can be adapted easily to integration site studies involving other retroviruses and transposons.

scholarly journals Chromosome Structure and Human Immunodeficiency Virus Type 1 cDNA Integration: Centromeric Alphoid Repeats Are a Disfavored Target

1998 ◽  
Vol 72 (5) ◽  
pp. 4005-4014 ◽  
Author(s):  
Sandrine Carteau ◽  
Christopher Hoffmann ◽  
Frederic Bushman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Naked Dna ◽  
Specific Pcr ◽  
Immunodeficiency Virus ◽  
Integration Sites ◽  
Cdna Integration ◽  
Hiv 1

ABSTRACT Integration of retroviral cDNA into host chromosomal DNA is an essential and distinctive step in viral replication. Despite considerable study, the host determinants of sites for integration have not been fully clarified. To investigate integration site selection in vivo, we used two approaches. (i) We have analyzed the host sequences flanking 61 human immunodeficiency virus type 1 (HIV-1) integration sites made by experimental infection and compared them to a library of 104 control sequences. (ii) We have also analyzed HIV-1 integration frequencies near several human repeated-sequence DNA families, using a repeat-specific PCR-based assay. At odds with previous reports from smaller-scale studies, we found no strong biases either for or against integration near repetitive sequences such as Alu or LINE-1 elements. We also did not find a clear bias for integration in transcription units as proposed previously, although transcription units were found somewhat more frequently near integration sites than near controls. However, we did find that centromeric alphoid repeats were selectively absent at integration sites. The repeat-specific PCR-based assay also indicated that alphoid repeats were disfavored for integration in vivo but not as naked DNA in vitro. Evidently the distinctive DNA organization at centromeres disfavors cDNA integration. We also found a weak consensus sequence for host DNA at integration sites, and assays of integration in vitro indicated that this sequence is favored as naked DNA, revealing in addition an influence of target primary sequence.

scholarly journals Allogeneic leukocytes but not therapeutic blood elements induce reactivation and dissemination of latent human immunodeficiency virus type 1 infection: implications for transfusion support of infected patients

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2128-2135 ◽  
Author(s):  
MP Busch ◽  
TH Lee ◽  
J Heitman
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Blood Components ◽  
Route Of Transmission ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Donor Lymphocytes

Abstract Various immunologic stimuli and heterologous viral regulatory elements have been shown to increase susceptibility to, and replication of, human immunodeficiency virus type 1 (HIV-1) in lymphocytes and monocytes in vitro. Transfusion of allogeneic blood components from heterologous donors constitutes a profound immunologic stimulus to the recipient, in addition to being a potential route of transmission of lymphotropic viral infections. To investigate the hypothesis that transfusions, and particularly those containing leukocytes, activate HIV-1 replication in infected recipient cells, we cocultured peripheral blood mononuclear cells (PBMC) from three anti-HIV-1-positive individuals with allogeneic donor PBMC, as well as partially purified populations of donor lymphocytes, monocytes, granulocytes, platelets, and red blood cells (RBC) and allogeneic cell-free plasma. Allogeneic PBMC induced a dose-related activation of HIV-1 expression in in vivo infected cells, followed by dissemination of HIV-1 to previously uninfected patient cells. Activation of HIV-1 replication was observed with donor lymphocytes, monocytes, and granulocytes, whereas no effect was seen with leukocyte-depleted RBC, platelets, or plasma (ie, therapeutic blood constituents). Allogeneic donor PBMC were also shown to upregulate HIV-1 expression in a “latently” infected cell line, and to increase susceptibility of heterologous donor PBMC to acute HIV-1 infection. Studies should be performed to evaluate whether transfusions of leukocyte-containing blood components accelerate HIV-1 dissemination and disease progression in vivo. If so, HIV-1-infected patients should be transfused as infrequently as possible and leukocyte-depleted (filtered) blood components should be used to avoid this complication.

scholarly journals The Interaction of Vpr with Uracil DNA Glycosylase Modulates the Human Immunodeficiency Virus Type 1 In Vivo Mutation Rate

2000 ◽  
Vol 74 (15) ◽  
pp. 7039-7047 ◽  
Author(s):  
Louis M. Mansky ◽  
Sandra Preveral ◽  
Luc Selig ◽  
Richard Benarous ◽  
Serge Benichou
Keyword(s):  
Human Immunodeficiency Virus ◽  
Mutation Rate ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Dna Glycosylase ◽  
Uracil Dna Glycosylase ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT The Vpr protein of human immunodeficiency virus type 1 (HIV-1) influences the in vivo mutation rate of the virus. Since Vpr interacts with a cellular protein implicated in the DNA repair process, uracil DNA glycosylase (UNG), we have explored the contribution of this interaction to the mutation rate of HIV-1. Single-amino-acid variants of Vpr were characterized for their differential UNG-binding properties and used to trans complement vpr null mutant HIV-1. A striking correlation was established between the abilities of Vpr to interact with UNG and to influence the HIV-1 mutation rate. We demonstrate that Vpr incorporation into virus particles is required to influence the in vivo mutation rate and to mediate virion packaging of the nuclear form of UNG. The recruitment of UNG into virions indicates a mechanism for how Vpr can influence reverse transcription accuracy. Our data suggest that distinct mechanisms evolved in primate and nonprimate lentiviruses to reconcile uracil misincorporation into lentiviral DNA.

The NF-kappa B and Sp1 motifs of the human immunodeficiency virus type 1 long terminal repeat function as novel thyroid hormone response elements

1993 ◽  
Vol 13 (8) ◽  
pp. 5057-5069
Author(s):  
V Desai-Yajnik ◽  
H H Samuels
Keyword(s):  
Human Immunodeficiency Virus ◽  
Thyroid Hormone ◽  
Long Terminal Repeat ◽  
Human Immunodeficiency Virus Type ◽  
Binding Sites ◽  
Nf Kappa B ◽  
Response Elements ◽  
Immunodeficiency Virus ◽  
Hiv 1

We report that thyroid hormone (T3) receptor (T3R) can activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Purified chick T3R-alpha 1 (cT3R-alpha 1) binds as monomers and homodimers to a region in the LTR (nucleotides -104 to -75 [-104/-75]) which contains two tandem NF-kappa B binding sites and to a region (-80/-45) which contains three Sp1 binding sites. In contrast, human retinoic acid receptor alpha (RAR-alpha) and mouse retinoid X receptor beta (RXR-beta) do not bind to these elements. However, RXR-beta binds to these elements as heterodimers with cT3R-alpha 1 and to a lesser extent with RAR-alpha. Gel mobility shift assays also revealed that purified NF-kappa B p50/65 or p50/50 can bind to one but not both NF-kappa B sites simultaneously. Although the binding sites for p50/65, p50/50, and T3R, or Sp1 and T3R, overlap, their binding is mutually exclusive, and with the inclusion of RXR-beta, the major complex is the RXR-beta-cT3R-alpha 1 heterodimer. The NF-kappa B region of the LTR and the NF-kappa B elements from the kappa light chain enhancer both function as T3 response elements (TREs) when linked to a heterologous promoter. The TREs in the HIV-1 NF-kappa B sites appear to be organized as a direct repeat with an 8- or 10-bp gap between the half-sites. Mutations within the NF-kappa B motifs which eliminate binding of cT3R-alpha 1 also abolish stimulation by T3, indicating that cT3R-alpha 1 binding to the Sp1 region does not independently mediate activation by T3. The Sp1 region, however, is converted to a functionally strong TRE by the viral tat factor. These studies indicate that the HIV-1 LTR contains both tat-dependent and tat-independent TREs and reveal the potential for T3R to modulate other genes containing NF-kappa B- and Sp1-like elements. Furthermore, they indicate the importance of other transcription factors in determining whether certain T3R DNA binding sequences can function as an active TRE.

scholarly journals Novel TRIM5 Isoforms Expressed by Macaca nemestrina

2007 ◽  
Vol 81 (22) ◽  
pp. 12210-12217 ◽  
Author(s):  
Greg Brennan ◽  
Yury Kozyrev ◽  
Toshiaki Kodama ◽  
Shiu-Lok Hu
Keyword(s):  
Coiled Coil ◽  
Macaca Nemestrina ◽  
Cdna Clones ◽  
Reading Frame ◽  
Spry Domain ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
Hiv 1

ABSTRACT The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5α isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5α-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (Δ2) in the transcript at the 5′ terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5θ. This deletion is likely due to a single nucleotide polymorphism that alters the 3′ splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (Δexon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5η. There are 18 amino acid differences between M. nemestrina TRIM5η and Macaca mulatta TRIM5α, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5α-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5η or TRIM5θ show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo.

scholarly journals In vivo characteristics of human immunodeficiency virus type 1 intersubtype recombination: determination of hot spots and correlation with sequence similarity

2003 ◽  
Vol 84 (10) ◽  
pp. 2715-2722 ◽  
Author(s):  
Gkikas Magiorkinis ◽  
Dimitrios Paraskevis ◽  
Anne-Mieke Vandamme ◽  
Emmanouil Magiorkinis ◽  
Vana Sypsa ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Recombination Frequency ◽  
Sequence Similarity ◽  
Virus Species ◽  
Immunodeficiency Virus ◽  
Hiv 1

Recombination plays a pivotal role in the evolutionary process of many different virus species, including retroviruses. Analysis of all human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants revealed that they are more complex than described initially. Recombination frequency is higher within certain genomic regions, such as partial reverse transcriptase (RT), vif/vpr, the first exons of tat/rev, vpu and gp41. A direct correlation was observed between recombination frequency and sequence similarity across the HIV-1 genome, indicating that sufficient sequence similarity is required upstream of the recombination breakpoint. This finding suggests that recombination in vivo may occur preferentially during reverse transcription through the strand displacement-assimilation model rather than the copy-choice model.

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