HIV-1 cDNA Integration: Requirement of HMG I(Y) Protein for Function of Preintegration Complexes In Vitro

Farnet, Chris M.; Bushman, Frederic D.

doi:10.1016/s0092-8674(00)81888-7

Cited by 338 publications

(280 citation statements)

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“…For instance, HMGI(Y) proteins are a prerequisite for HIV replication (28). Moreover, HMGI(Y) gene induction is a necessary step in adipocyte differentiation (R. M. Melillo, S.S., A. Stella, S. Battista, C. De Biasio, M.F., G. Vigietto, and A.F., unpublished results).…”

Section: Discussionmentioning

confidence: 92%

Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

Scala¹,

Portella²,

Fedele³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

104

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Section: Discussionmentioning

confidence: 92%

Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

Scala¹,

Portella²,

Fedele³

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

146

104

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show abstract

“…There also exists the possibility that the multimeric form of IN, as expressed in the context of the reducing E. coli environment, is labile, emphasizing a particular caution with regard to the disulfide bridges as shown by Petit et al (34). This potential lability also could explain large differences in the coupled joining reaction between recombinant IN and preintegration complexes directly purified from infected cells (35,36).…”

Section: Discussionmentioning

confidence: 99%

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

Deprez

Tauc

Leh

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Self-assembly of HIV-1 integrase (IN) in solution has been studied previously by time-resolved fluorescence, using tryptophan anisotropy decay. This approach provides information on the size of macromolecules via the determination of rotational correlation times ( ). We have shown that, at submicromolar concentration, IN is characterized by a long rotational correlation time ( 20°C ‫؍‬ 90 -100 ns) corresponding to a high-order oligomeric form, likely a tetramer. In the present work, we investigated the self-assembly properties of the DNA-bound IN by using three independent fluorophores. Under enzymatic assay conditions (10 ؊7 M IN, 2 ؋ 10 ؊8 M DNA), using either fluorescein-labeled or fluorescent guanosine analog-containing oligonucleotides that mimic a viral end long terminal repeat sequence, we found that the DNA- I ntegration of a DNA copy of the HIV-1 genome into the host genome is a critical step of the retrovirus life cycle. Integration is a multistep process including 3Ј processing, strand transfer, and DNA repair. Integrase (IN) is responsible for 3Ј processing and strand transfer and is sufficient to catalyze these two reactions in vitro, using short-length oligonucleotides (ODNs) that mimic the viral end long terminal repeat sequences. The repair step most likely is performed by a host system. IN is a member of the superfamily of polynucleotidyl transferases, and its catalytic core domain contains a triad of acidic residues that constitute the so-called D,D-35-E motif. This motif is strictly required for catalysis (1, 2) and is involved in the coordination of the metal cation cofactor (3, 4). Catalysis can be performed efficiently in vitro by using either Mn 2ϩ or Mg 2ϩ , but several investigations suggest that these two divalent cations have different effects on the reaction specificity. Specific protein-DNA contacts occur preferentially in the presence of Mg 2ϩ (5), and more nonspecific cleavage is detected in the presence of Mn 2ϩ (6). The transfer pattern on the target DNA also depends on the nature of the cation (7). Consequently, the efficiency of inhibitors can be different by using either Mg 2ϩ or Mn 2ϩ in assays (8). In each case, the Mg 2ϩ -dependent activity may be more reliable because it is more physiologically relevant.Specific recognition between IN and the substrate viral DNA is a prerequisite for the cleavage of two nucleotides at the 3Ј end of the viral DNA. The catalytic core domain establishes specific contacts with the viral DNA, and it has been determined that the terminal 13-base region of the long terminal repeat plays a significant role for Mg 2ϩ -dependent processing in terms of specificity (5, 9). The C-terminal domain also mediates viral DNA-IN interactions, but in a nonspecific fashion, and more likely contributes to complex stabilization (10-12). Furthermore, it is strongly believed that self-association of IN plays a key role in governing the enzyme function (13-16). Recently, we have shown that the capability of IN to use Mg 2ϩ is related to the protein self-association ...

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“…Ty3 introduces its strand transfers at nucleotides Ϫ5 and Ϫ1 (thus inserting Ty3 sequence beginning at bp Ϫ6), adjacent to the downstream end of DNA-bound Brf (42), and within the domain of the transcription bubble (39). Retroelements preferentially mediate integration into regions of DNA containing bends or kinks (47)(48)(49)(50). The work that is presented here suggests that nucleophilic attack mediated by Ty3 integrase resembles the process mediated by retroviral integrase proteins in the sense that it favors positions with distorted base pairing or stacking but that, in the case of Ty3, protein-protein interactions play a dominant role, perhaps by converting a basal activity for nonspecific insertion to locationspecific function.…”

Section: Discussionmentioning

confidence: 99%

The Brf and TATA-binding Protein Subunits of the RNA Polymerase III Transcription Factor IIIB Mediate Position-specific Integration of the Gypsy-like Element, Ty3

Yieh

Kassavetis

Geiduschek

et al. 2000

Journal of Biological Chemistry

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Ty3 integrates into the transcription initiation sites of genes transcribed by RNA polymerase III. It is known that transcription factors (TF) IIIB and IIIC are important for recruiting Ty3 to its sites of integration upstream of tRNA genes, but that RNA polymerase III is not required. In order to investigate the respective roles of TFIIIB and TFIIIC, we have developed an in vitro integration assay in which Ty3 is targeted to the U6 small nuclear RNA gene, SNR6. Because TFIIIB can bind to the TATA box upstream of the U6 gene through contacts mediated by TATA-binding protein (TBP), TFIIIC is dispensable for in vitro transcription. Thus, this system offers an opportunity to test the role of TFIIIB independent of a requirement of TFIIIC. We demonstrate that the recombinant Brf and TBP subunits of TFIIIB, which interact over the SNR6 TATA box, direct integration at the SNR6 transcription initiation site in the absence of detectable TFIIIC or TFIIIB subunit B؆. These findings suggest that the minimal requirements for pol III transcription and Ty3 integration are very similar.Integration site selection is a key step in the retroelement life cycle, potentially influencing both the effect of the insertion on the host genome and the expression of the element itself. Similar to retroviruses, yeast Ty elements transpose through reverse transcription of an almost full-length RNA copy into a full-length DNA copy, which is integrated into the host genome. Despite very similar molecular mechanisms of integration, budding yeast elements (Tys), both gypsy-like (Ty3) and copialike (Ty1, 2, 4, and 5), differ from retroviruses in that they exhibit dramatic global integration site preferences (1-4). Ty1 elements, for example, integrate preferentially within a window of 750 bp, 1 upstream of genes transcribed by RNA polymerase III (pol III). Analysis of the yeast genome sequence shows that Ty2 and Ty4 also occupy this region upstream of a fraction of tRNA genes. Ty5 integrates into regions of silenced DNA, including the silent mating type loci and telomeres. Hence Ty1, Ty2, Ty4, and Ty5 exhibit regional integration specificity. Despite similarities with these other elements, Ty3 differs in that it integrates within a highly defined window, one or two base pairs (bp) upstream of pol III transcription initiation sites.Targeting of integration appears to be directed by the cooperative actions of Ty3 element-and cell-encoded factors. For example, in addition to the element-encoded integrase protein, Ty3 and Ty1 targeting requires the presence of a transcriptionally competent pol III promoter (5, 6), and Ty5 targeting requires factors involved in the establishment of silent chromatin (7).Class III genes, including plasmid-borne U6, 5S, and tRNA genes, are used in vivo for position-specific Ty3 integration. Comparison of integration sites suggests that Ty3 integration preference is not a direct function of specific local sequences. Each class of pol III-transcribed genes differs from the others in composition of promoter elements (8) and d...

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HIV-1 cDNA Integration: Requirement of HMG I(Y) Protein for Function of Preintegration Complexes In Vitro

Cited by 338 publications

References 58 publications

Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

Adenovirus-mediated suppression of HMGI(Y) protein synthesis as potential therapy of human malignant neoplasias

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

The Brf and TATA-binding Protein Subunits of the RNA Polymerase III Transcription Factor IIIB Mediate Position-specific Integration of the Gypsy-like Element, Ty3

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