Biomedical subjects

H Huthoff

Publications and source records attributed to H Huthoff.

4 recordsLinked to original sources

PubMed2001

Mutations in the TAR hairpin affect the equilibrium between alternative conformations of the HIV-1 leader RNA.

The HIV-1 untranslated leader RNA can adopt two mutually exclusive conformations that represent alternative secondary structures. This leader RNA can fold either an extended duplex through long-distance base pairing or a branched conformation in which the RNA locally folds into hairpin structures. Both leader RNA conformations have the TAR hairpin in common, which forms the extreme 5' end of all HIV-1 transcripts. We report that truncation of the TAR hairpin shifts the equilibrium between the two RNA conformations away from the thermodynamically favored long-distance interaction. However, the equilibrium is partially restored in response to the cations Na(+) and Mg(2+). The transcripts with mutant TAR structures allowed us to investigate conditions affecting the competition between the alternative conformations of the HIV-1 leader RNA. We also demonstrate that the change in conformation of the leader RNA due to TAR truncations severely affects formation of the HIV-1 RNA dimer.

Base Pairing↗

PubMed2001

The dimer initiation site hairpin mediates dimerization of the human immunodeficiency virus, type 2 RNA genome.

The untranslated leader of retroviral RNA genomes encodes multiple structural signals that are critical for virus replication. In the human immunodeficiency virus, type 1 (HIV-1) leader, a hairpin structure with a palindrome-containing loop is termed the dimer initiation site (DIS), because it triggers in vitro RNA dimerization through base pairing of the loop-exposed palindromes (kissing loops). Controversy remains regarding the region responsible for HIV-2 RNA dimerization. Different studies have suggested the involvement of the transactivation region, the primer binding site, and a hairpin structure that is the equivalent of the HIV-1 DIS hairpin. We have performed a detailed mutational analysis of the HIV-2 leader RNA, and we also used antisense oligonucleotides to probe the regions involved in dimerization. Our results unequivocally demonstrate that the DIS hairpin is the main determinant for HIV-2 RNA dimerization. The 6-mer palindrome sequence in the DIS loop is essential for dimer formation. Although the sequence can be replaced by other 6-mer palindromes, motifs that form more than two A/U base pairs do not dimerize efficiently. The inability to form stable kissing-loop complexes precludes formation of dimers with more extended base pairing. Structure probing of the DIS hairpin in the context of the complete HIV-2 leader RNA suggests a 5-base pair elongation of the DIS stem as it is proposed in current RNA secondary structure models. This structure is supported by phylogenetic analysis of leader RNA sequences from different viral isolates, indicating that RNA genome dimerization occurs by a similar mechanism for all members of the human and simian immunodeficiency viruses.

Base Sequence↗

PubMed2001

Two alternating structures of the HIV-1 leader RNA.

In this study we demonstrate that the HIV-1 leader RNA exists in two alternative conformations, a branched structure consisting of several well-known hairpin motifs and a more stable structure that is formed by extensive long-distance base pairing. The latter conformation was first identified as a compactly folded RNA that migrates unusually fast in nondenaturing gels. The minimally required domains for formation of this conformer were determined by mutational analysis. The poly(A) and DIS regions of the leader are the major determinants of this RNA conformation. Further biochemical characterization of this conformer revealed that both hairpins are disrupted to allow extensive long-distance base pairing. As the DIS hairpin is known to be instrumental for formation of the HIV-1 RNA dimer, the interplay between formation of the conformer and dimerization was addressed. Formation of the conformer and the RNA dimer are mutually exclusive. Consequently, the conformer must rearrange into a branched structure that exposes the dimer initiation signal (DIS) hairpin, thus triggering formation of the RNA dimer. This structural rearrangement is facilitated by the viral nucleocapsid protein NC. We propose that this structural polymorphism of the HIV-1 leader RNA acts as a molecular switch in the viral replication cycle.

Alternative Splicing↗

PubMed2001

Structural features in the HIV-1 repeat region facilitate strand transfer during reverse transcription.

Two obligatory DNA strand transfers take place during reverse transcription of a retroviral RNA genome. The first strand transfer is facilitated by terminal repeat (R) elements in the viral genome. This strand-transfer reaction depends on base pairing between the cDNA of the 5'R and the 3'R. There is accumulating evidence that retroviral R regions contain features other than sequence complementarity that stimulate this critical nucleic acid hybridization step. The R region of the human immunodeficiency virus type 1 (HIV-1) is relatively extended (97 nt) and encodes two well-conserved stem-loop structures, the TAR and poly(A) hairpins. The role of these motifs was studied in an in vitro strand-transfer assay with two separate templates, the 5'R donor and the 3'R acceptor, and mutants thereof. The results indicate that the upper part of the TAR hairpin structure in the 5'R donor is critical for efficient strand transfer. This seems to pose a paradox, as the 5'R template is degraded by RNase H before strand transfer occurs. We propose that it is not the RNA hairpin motif in the 5'R donor, but rather the antisense motif in the ssDNA copy, which can also fold a hairpin structure, that is critical for strand transfer. Mutation of the loop sequence in the TAR hairpin of the donor RNA, which is copied in the loop of the cDNA hairpin, reduces the transfer efficiency more than fivefold. It is proposed that the natural strand-transfer reaction is enhanced by interaction of the anti-TAR ssDNA hairpin with the TAR hairpin in the 3'R acceptor. Base pairing can occur between the complementary loops ("loop-loop kissing"), and strand transfer is completed by the subsequent formation of an extended RNA-cDNA duplex.

Base Sequence↗