Abstract
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.
Highlights
The presence of G4s in viruses and their involvement in key steps of viral infection has been provided[6]
We have shown that the human immunodeficiency virus-1 (HIV-1) long terminal repeat (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 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. LTR-II + III +IV
Summary
The presence of G4s in viruses and their involvement in key steps of viral infection has been provided[6]. We have shown that the herpes simplex virus 1 possesses several repeats of sequences forming stable G4s, which were visualized in infected cells by a G4-specific antibody[14]; stabilization of these tetraplex structures by a G4 ligand inhibited viral DNA replication[15]. The largest body of evidence of G4-mediated regulation of viruses has been provided for the human immunodeficiency virus-1 (HIV-1), the etiologic agent of the acquired immune deficiency syndrome (AIDS). The cellular protein nucleolin has been shown to stabilize the HIV-1 LTR G4s and induce potent inhibition of viral transcription[22]. By means of mass spectrometry, surface plasmon resonance (SPR), fluorescence energy transfer (FRET), Taq polymerase stop and reporter assays, we here identified and characterized G4-selectivity and function of the hnRNP A2/B1, the first protein shown to unfold G4s in the HIV-1 LTR promoter
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