Regulatory RNA domains
Retroviral transcripts are flanked by untranslated leader and trailer sequences that contain important regulatory RNA motifs. These RNA signals are crucial for both gene-expression and virion-associated functions. For example, the 5’ leader of HIV-1 contains RNA domains that mediate transcription, splicing and translation (‘early’ events), as well as RNA dimerization, packaging and initiation of reverse transcription (‘late’ events). The obligatory strand transfer during reverse transcription is mediated by a terminally redundant repeat (R) present at the 5’ and 3’ extremities of HIV-1 transcripts. Efficient polyadenylation occurs at the 3’ end of HIV-1 transcripts. The observed overlap of multiple regulatory RNA signals within the leader of HIV associates this RNA domain with enormous biological complexity. In the Berkhout lab, structure-function relationships and the dissection of distinct regulatory RNA domains are an ongoing effort.

Enlarged view

NC mediated rearrangement of the HIV-1 leader RNA secondary structure. Disruption of the elongated ground state conformation leads to exposure of the DIS hairpin and subsequent dimerization.

Multiple conformations of the HIV-1 leader RNA
We observed that the HIV-1 leader RNA can adopt two mutually exclusive conformations of different thermodynamic stability. The most stable conformation traps the DIS hairpin (Dimer Initiation Signal) in a long-distance basepairing interaction with upstream sequences, such that RNA dimerization is down-regulated. We have also demonstrated that the viral NC protein (a nucleic acid chaperone) disrupts this RNA conformation, leading to folding and exposure of the DIS hairpin and subsequent RNA dimerization. Dimerization of HIV-1 transcripts is closely related to packaging of genomic RNA into virion particles. Thus, we are currently investigating the possibility that the HIV-1 leader RNA may act as a molecular switch in the viral replication cycle through adopting alternative conformations. This project aims at resolving the structure and function of these RNA conformations through a combination of phylogenetic, biochemical and virological approaches. Collaborations exist with the laboratories of Jørgen Kjems (University of Århus, Denmark) and Cees Pleij (University of Leiden).

Relevant publications

Abbink TE, Berkhout B.
A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon. J Biol Chem. 2003 Mar 28;278(13):11601-11.

Berkhout B, Ooms M, Beerens N, Huthoff H, Southern E, Verhoef K.
In vitro evidence that the untranslated leader of the HIV-1 genome is an RNA checkpoint that regulates multiple functions through conformational changes. J Biol Chem. 2002 May 31;277(22):19967-75.

Dirac AM, Huthoff H, Kjems J, Berkhout B.
Regulated HIV-2 RNA dimerization by means of alternative RNA conformations. Nucleic Acids Res. 2002 Jun 15;30(12):2647-55.

Huthoff H, Berkhout B.
Multiple secondary structure rearrangements during HIV-1 RNA dimerization. Biochemistry. 2002 Aug 20;41(33):10439-45.

Dirac AM, Huthoff H, Kjems J, Berkhout B.
Requirements for RNA heterodimerization of the human immunodeficiency virus type 1 (HIV-1) and HIV-2 genomes. J Gen Virol. 2002 Oct;83(Pt 10):2533-42.

Dirac AM, Huthoff H, Kjems J, Berkhout B.
The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome. J Biol Chem. 2001 Aug 24;276(34):32345-52.

Huthoff H, Berkhout B.
Two alternating structures of the HIV-1 leader RNA. RNA. 2001 Jan;7(1):143-57.

Berkhout B, van Wamel JLB. 2000. The leader of the HIV-1 RNA genome forms a compactly folded tertiary structure. RNA 6:282-295.

Berkhout B. Multiple biological roles associated with the repeat (R) region of the HIV-1 RNA genome. 2000. Adv Pharmacol 48:29-73.

Das AT, Klaver B, Berkhout B. 1999. A hairpin structure in the R region of the Human Immunodeficiency Virus type 1 RNA genome is instrumental in polyadenylation site selection. J Virol 73:81-91.

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