Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Tosoni, Elena; Frasson, Ilaria; Scalabrin, Matteo; Perrone, Rosalba; Butovskaya, Elena; Nadai, Matteo; Palù, Giorgio; Fabris, Dan; Richter, Sara N.

doi:10.1093/nar/gkv897

Cited by 123 publications

(155 citation statements)

References 83 publications

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“…We have shown that the HIV-1 LTR promoter can fold into three mutually exclusive G4s and that nucleolin, the most abundant nucleolar protein, can bind, induce and stabilize the LTR G4s22. We reasoned that other proteins may exist that regulate the G4/ds equilibrium at the LTR: we thus looked for additional proteins by pull-down assay of 293T nuclear cell extracts against the whole region in the HIV-1 LTR that can fold into G4, i.e.…”

Section: Resultsmentioning

confidence: 99%

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The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

Scalabrin

Frasson

Ruggiero

et al. 2017

Sci Rep

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G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

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Section: Resultsmentioning

confidence: 99%

“…When the HIV-1 G4s were stabilized by G4 ligands, antiviral effects, mainly dependent on inhibition of LTR-mediated transcription, were observed182021. Furthermore, the cellular protein nucleolin has been shown to stabilize the HIV-1 LTR G4s and induce potent inhibition of viral transcription22.…”

mentioning

confidence: 99%

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

Scalabrin

Frasson

Ruggiero

et al. 2017

Sci Rep

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“…These proteins include Nucleolin that interacts with p55 Gag and it has been recently demonstrated to affect viral transcription (46,47). Moreover, Hsp70 proteins mediate Tat activation and viral transcription, and are released in HIV-1 virions (48–50).…”

Section: Discussionmentioning

confidence: 99%

ADAR1 restricts LINE-1 retrotransposition

et al. 2016

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Adenosine deaminases acting on RNA (ADARs) are involved in RNA editing that converts adenosines to inosines in double-stranded RNAs. ADAR1 was demonstrated to be functional on different viruses exerting either antiviral or proviral effects. Concerning HIV-1, several studies showed that ADAR1 favors viral replication. The aim of this study was to investigate the composition of the ADAR1 ribonucleoprotein complex during HIV-1 expression. By using a dual-tag affinity purification procedure in cells expressing HIV-1 followed by mass spectrometry analysis, we identified 14 non-ribosomal ADAR1-interacting proteins, most of which are novel. A significant fraction of these proteins were previously demonstrated to be associated to the Long INterspersed Element 1 (LINE1 or L1) ribonucleoparticles and to regulate the life cycle of L1 retrotransposons that continuously re-enter host-genome.Hence, we investigated the function of ADAR1 in the regulation of L1 activity.By using different cell-culture based retrotransposition assays in HeLa cells, we demonstrated a novel function of ADAR1 as suppressor of L1 retrotransposition. Apparently, this inhibitory mechanism does not occur through ADAR1 editing activity. Furthermore, we showed that ADAR1 binds the basal L1 RNP complex. Overall, these data support the role of ADAR1 as regulator of L1 life cycle.

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“…hTelScra 5′-GGATGTGAGTGTGAGTGTGAGG-3′; LTRScra 5′-TTTTTGGAGCGTGTGTGCGCGAGAGCGTGCGCGTGGCGAGCGTTGAGTGGTTTTT-3′; rnd 5′-AAAAACTACTGCACGCTCGCTACGACGACACTGTCGCGCATACAAGCTGCAAAAA-3′ ( Random in (23)); T9 5′-TTTTTTTTT-3′; T30 5′-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3′; hp 5′-CGCAGCGTGGCTTTGTTTGCCACGCTGCG-3′; dsDNA: hTelScra+complementary sequence; NRAS RNA 5′-GGGAGGGGCGGGUCUGGG-3′; U3-III HIV RNA 5′-GGGAGGCGUGGCCUGGGCGGGACUGGGG-3′; U3-IV HIV RNA 5′-GGGCGGGACUGGGGAGUGG-3′; hTel 2 RNA 5′-UAGGGUUAGGGU-3′; TAR HIV RNA 5′-GGCAGAUCUGAGCCUGGGAGCUCUCUGCC-3′; rnd RNA 5′-GAGCGUGCGCGUGCGAGCGUGAGUGAGCGUGGG-3′. To allow the correct orientation of 1H6 binding site on the chip, 1H6 was incubated for 1 h at rt in the presence of the G4-folded un3 .…”

Section: Methodsmentioning

confidence: 99%

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

et al. 2016

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We have previously shown that clusters of guanine quadruplex (G4) structures can form in the human herpes simplex-1 (HSV-1) genome. Here we used immunofluorescence and immune-electron microscopy with a G4-specific monoclonal antibody to visualize G4 structures in HSV-1 infected cells. We found that G4 formation and localization within the cells was virus cycle dependent: viral G4s peaked at the time of viral DNA replication in the cell nucleus, moved to the nuclear membrane at the time of virus nuclear egress and were later found in HSV-1 immature virions released from the cell nucleus. Colocalization of G4s with ICP8, a viral DNA processing protein, was observed in viral replication compartments. G4s were lost upon treatment with DNAse and inhibitors of HSV-1 DNA replication. The notable increase in G4s upon HSV-1 infection suggests a key role of these structures in the HSV-1 biology and indicates new targets to control both the lytic and latent infection.

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Nucleolin stabilizes G-quadruplex structures folded by the LTR promoter and silences HIV-1 viral transcription

Cited by 123 publications

References 83 publications

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

ADAR1 restricts LINE-1 retrotransposition

Visualization of DNA G-quadruplexes in herpes simplex virus 1-infected cells

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