Chromatin function regulates retroviral gene expression

Synopsis: experimental work described in this Thesis has been mainly focused on the study of the regulation of viral gene expression, in two different members of the Retroviridae family, namely the lentivirus HIV-1 and the gammaretrovirus MoMLV. The results are divided into two parts, Part A and Part B. The work reported in Part A gives critical clues for the comprehension of the still obscure mechanisms that regulate function of the HIV-1 transactivator Tat in the initiation and elongation of HIV-1 transcription. In particular, we exploited a proteomic screening aimed at characterizing novel Tat partners. This approach allowed us to identify the cellular histone chaperone NAP-1 (Nucleosome Assembly Protein-1) as a Tat-binding protein. We found that this interaction exerts a positive role on Tat-driven LTR transactivation and on HIV-1 infection. To our knowledge, this is the first demonstration of an interaction between Tat and a cellular histone chaperone. We propose a mechanism by which Tat benefits from this class of proteins to relieve the repression imposed by chromatin conformation on proviral transcription. The findings described in Part B are part of an ongoing project, that we derived from our previous observation that HIV-1 transcription is also regulated by long-range chromatin interactions. In particular, we originally found that a gene loop structure is imposed on the provirus upon transcriptional activation. We now show that MoMLV also adopts a transcription-dependent LTR-LTR gene loop conformation. This observation leads us to hypothesize that gene looping might be a general hallmark of retroviral transcription. Moreover, we also demonstrate that an aberrant loop might form between the retroviral MoMLV LTRs and regulatory regions of the host cell genome. We suggest that this event might be at the basis of the phenomenon of insertional mutagenesis observed in some of the gene therapy clinical trials that so far have exploited members of the gammaretroviridae family of viruses

Intragenic transcriptional cis-activation of the human immunodeficiency virus 1 does not result in allele-specific inhibition of the endogenous gene

<p>Abstract</p> <p>Background</p> <p>The human immunodeficiency virus type 1 (HIV-1) favors integration in active genes of host chromatin. It is believed that transcriptional interference of the viral promoter over the endogenous gene or vice versa might occur with implications in HIV-1 post-integrative transcriptional latency.</p> <p>Results</p> <p>In this work a cell line has been transduced with a HIV-based vector and selected for Tat-inducible expression. These cells were found to carry a single silent integration in sense orientation within the second intron of the <it>HMBOX1 </it>gene. The HIV-1 Tat transactivator induced the viral LTR and repressed <it>HMBOX1 </it>expression independently of vector integration. Instead, single-cell quantitative <it>in situ </it>hybridization revealed that allele-specific transcription of <it>HMBOX1 </it>carrying the integrated provirus was not affected by the transactivation of the viral LTR in <it>cis</it>.</p> <p>Conclusion</p> <p>A major observation of the work is that the HIV-1 genome has inserted in genes that are also repressed by Tat and this could be an advantage for the virus during transcriptional reactivation. In addition, it has also been observed that transcription of the provirus and of the endogenous gene in which it is integrated may coexist at the same time in the same genomic location.</p

Inhibition of Non Canonical HIV-1 Tat Secretion Through the Cellular Na+,K+-ATPase Blocks HIV-1 Infection

Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na+,K+-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity

The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription

<p>Abstract</p> <p>Background</p> <p>Despite the large amount of data available on the molecular mechanisms that regulate HIV-1 transcription, crucial information is still lacking about the interplay between chromatin conformation and the events that regulate initiation and elongation of viral transcription. During transcriptional activation, histone acetyltransferases and ATP-dependent chromatin remodeling complexes cooperate with histone chaperones in altering chromatin structure. In particular, human Nucleosome Assembly Protein-1 (hNAP-1) is known to act as a histone chaperone that shuttles histones H2A/H2B into the nucleus, assembles nucleosomes and promotes chromatin fluidity, thereby affecting transcription of several cellular genes.</p> <p>Results</p> <p>Using a proteomic screening, we identified hNAP-1 as a novel cellular protein interacting with HIV-1 Tat. We observed that Tat specifically binds hNAP1, but not other members of the same family of factors. Binding between the two proteins required the integrity of the basic domain of Tat and of two separable domains of hNAP-1 (aa 162–290 and 290–391). Overexpression of hNAP-1 significantly enhanced Tat-mediated activation of the LTR. Conversely, silencing of the protein decreased viral promoter activity. To explore the effects of hNAP-1 on viral infection, a reporter HIV-1 virus was used to infect cells in which hNAP-1 had been either overexpressed or knocked-down. Consistent with the gene expression results, these two treatments were found to increase and inhibit viral infection, respectively. Finally, we also observed that the overexpression of p300, a known co-activator of both Tat and hNAP-1, enhanced hNAP-1-mediated transcriptional activation as well as its interaction with Tat.</p> <p>Conclusion</p> <p>Our study reveals that HIV-1 Tat binds the histone chaperone hNAP-1 both in vitro and in vivo and shows that this interaction participates in the regulation of Tat-mediated activation of viral gene expression.</p

The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription-4

On vector for HA-hNAP-1 or with a control vector, and then infected with VSG-luciferase HIV-1 vector. Luciferase activity was measured after 24 h post-infection. The mean ± s.d. of at least three different experiments is shown. The panel on the right side shows anti-HA western blottings to assess HA-hNAP-1 expression in a representative experiment. Silencing of hNAP-1 impairs LTR transcription upon HIV-1 infection. HeLa cells were treated with an siRNA directed against hNAP-1 or a control siRNA. Forty-eight hours after the beginning of siRNA treatment, cells were infected with the luciferase reported virus, and luciferase assays were performed on cell lysates 24 hours later. The mean ± s.d. of at least three different experiments is shown. The panel on the right side shows anti-hNAP-1 western blottings to assess the levels of endogenous hNAP-1 and tubulin expression in a representative experiment.<p><b>Copyright information:</b></p><p>Taken from "The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription"</p><p>http://www.retrovirology.com/content/5/1/8</p><p>Retrovirology 2008;5():8-8.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266780.</p><p></p

The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription-3

Aining the HIV-1 LTR upstream of the luciferase gene, and with vectors for HA-tagged hNAP-1 (100 ng) and HIV-1 Tat (5 and 25 ng), as indicated. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The co-expression of hNAP-1 significantly increased Tat transactivation of the LTR promoter. The western blot at the bottom shows the levels of transfected hNAP-1 protein in a representative experiment. hNAP-1 knock down decreases Tat transactivation. HeLa cells were transfected with a specific siRNA against hNAP-1 or a control siRNA, and then transfected with the LTR-luciferase reporter together with Tat (5 and 25 ng). The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. The western blot at the bottom shows the levels of endogenous hNAP-1 protein and of tubulin as a control in a representative experiment. hNAP-1, Tat and the acetyltransferase p300 synergistically activate viral transcription. HeLa cells were transfected with LTR-luciferase reporter plasmid and with vectors for HIV-1 Tat (5 ng), HA-hNAP-1 (100 ng) and p300 (100 ng), as indicated. After 24 h from transfection, luciferase assays were performed. The histogram shows mean ± s.d. of at least three independent experiments; the results are shown as fold transactivation over LTR-luciferase reporter alone. p300 enhances Tat-hNAP-1 interaction in vivo. The plasmids indicated on top of the figure were transfected into HEK 293T cells. The upper panel shows western blots with the indicated antibodies after immunoprecipitation using an anti-Flag antibody; the lower three panels show western blotting controls from whole cell lysates (WCL) from transfected cells to show the levels of expression of the transfected proteins.<p><b>Copyright information:</b></p><p>Taken from "The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription"</p><p>http://www.retrovirology.com/content/5/1/8</p><p>Retrovirology 2008;5():8-8.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266780.</p><p></p

The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription-0

DS-PAGE gel, followed by silver staining. Protein bands present exclusively in the sample transfected with Tat-Flag were excised from the gel and their identification attempted by ESI-MS/MS. The identified proteins, in addition to hNAP-1 and Tat-Flag, are indicated (1: B23/nucleophosmin; 2: pre-mRNA splicing factor SF2p32 – Tat-associated protein TAP; 3: ribosomal protein S4). Amino acid sequence of the human NAP-1 protein (locus NP_631946) – 391 aa. The underlined amino acid sequences correspond to peptides obtained from MS/MS analysis of three independent preparations (P = 7.8 × 10).<p><b>Copyright information:</b></p><p>Taken from "The histone chaperone protein Nucleosome Assembly Protein-1 (hNAP-1) binds HIV-1 Tat and promotes viral transcription"</p><p>http://www.retrovirology.com/content/5/1/8</p><p>Retrovirology 2008;5():8-8.</p><p>Published online 28 Jan 2008</p><p>PMCID:PMC2266780.</p><p></p