Regular article
Structure and dimerization of HIV-1 kissing loop aptamers1

https://doi.org/10.1006/jmbi.2001.4879Get rights and content

Abstract

Dimerization of two homologous strands of genomic RNA is an essential feature of the retroviral replication cycle. In HIV-1, genomic RNA dimerization is facilitated by a conserved stem-loop structure located near the 5′ end of the viral RNA called the dimerization initiation site (DIS). The DIS loop is comprised of nine nucleotides, six of which define an autocomplementary sequence flanked by three conserved purine residues. Base- pairing between the loop sequences of two copies of genomic RNA is necessary for efficient dimerization. We previously used in vitro evolution to investigate a possible structural basis for the marked sequence conservation of the DIS loop. In this study, chemical structure probing, measurements of the apparent dissociation constants, and computer structure analysis of dimerization-competent aptamers were used to analyze the dimers’ structure and binding. The selected aptamers were variants of the naturally occurring A and B subtypes. The data suggest that a sheared base-pair closing the loop of the DIS is important for dimerization in both subtypes. On the other hand, the open or closed state of the last base-pair in the stem differed in the two subtypes. This base-pair appeared closed in the subtype A DIS dimer and open in subtype B. Finally, evidence for a cross-talk between nucleotides 2, 5, and 6 was found in some, but not all, loop contexts, indicating some structural plasticity depending on loop sequence. Discriminating between the general rules governing dimer formation and the particular characteristics of individual DIS aptamers helps to explain the affinity and specificity of loop-loop interactions and could provide the basis for development of drugs targeted against the dimerization step during retroviral replication.

Introduction

Dimerization of retroviral RNA genomes prior to or concomitant with viral encapsidation and budding is a vital step in the retroviral replication cycle. An essential component of the dimerization process in HIV-1 is a stem-loop structure called the dimerization initiation site (DIS) located in the upstream leader sequence of the genomic RNA1, 2. The loop contains an autocomplementary hexanucleotide sequence comprised almost always of either GUGCAC (subtype A) or GCGCGC (subtype B) that, along with non-canonical interactions provided by conserved flanking purine nucleotides, is required for stable dimerization3. Stable dimerization proceeds through an RNA loop-loop kissing interaction whereby the loop autocomplementary sequences form intermolecular base-pairs. Deletion or mutation of this sequence results in mutant viruses with markedly diminished infectivity and replication kinetics, and specific defects have been demonstrated in genomic RNA dimerization, encapsidation, and proviral DNA synthesis4, 5, 6, 7, 8, 9.

Extensive chemical and enzymatic probing, site-directed mutagenesis, and molecular modeling have been used to construct a three-dimensional model of the subtype A kissing loop complex3, 10. Preliminary NMR data on subtype A model constructs provided several distance constraints consistent with this model, although a complete structure based solely upon the NMR data was not presented11. In addition, an NMR-derived model for the subtype B DIS dimer has been reported12. Interestingly, the NMR data of the subtype A and B dimers suggest significantly different conformations despite relatively modest sequence differences. In particular, the NMR model of the subtype B dimer suggests a melting of the last helical base-pair, and unpaired, but extensively stacked, adenines flanking the autocomplementary sequence. On the other hand, solution structure methods have suggested that the helical stems of subtype A dimers remain closed (i.e. base-paired) and that the purines flanking the autocomplementary sequence are involved in non-canonical base-pairing3, 10. Differences in the biochemical behavior of subtypes A and B dimers have been noted as well, and may be attributable in part to the presence or absence of a specific magnesium binding site among the two subtypes10. Knowledge of the detailed three-dimensional structure could ultimately lead to rational drug design targeted against specific features of the DIS stem-loop. In addition, detailed analysis of retroviral RNA dimerization could lend insight into other RNA loop-loop interactions, which are found in many other biological systems, such as in the control of replication of plasmids ColE1 and R113, 14, 15, multimerization of cellularly localized mRNA16, the organization of RNA structure within enterovirus and poliovirus genomes17, 18, and within the Neurospora ribozyme19. Understanding the mechanisms of this type of intermolecular recognition and binding event requires both structural and functional studies of the kissing loop complexes.

The basic mechanism underlying the kissing loop interaction involves a primary recognition event between one or several loop nucleotides followed by an extension of the base-pairing interaction to include the entire autocomplementary loop sequence20, 21. Under some circumstances, the loop-loop base-pairing advances farther to include part of or the entire stem sequence surrounding the loop to form an extended duplex (for example, see reference 22). In vitro studies on HIV-1 DIS RNA have shown that an extended duplex can form, depending on the presence or absence of nucleocapsid protein, incubation temperature, ionic conditions, and the sequence of the stems22, 23, 24, 25, 26, 27, 28. Whether or not the extended duplex occurs in vivo is unclear, as advantages associated with formation of an increasingly stable dimeric structure might be overcome by topological problems involved in twisting two very large RNAs around each other. In either event, the initial recognition between the two RNA molecules appears to be a loop-loop interaction, or kissing complex, and this is the subject of our current study.

We previously performed an in vitro selection/evolution study on model DIS stem-loops randomized at some or all of the loop positions in an effort to understand the sequence and structural constraints on this conserved motif29. Results of that study revealed some fundamental sequence and structural requirements for loop-loop interactions in general and for dimerization of HIV-1 RNA in particular. Specifically, constraints were identified for loop size, autocomplementary sequence identity, autocomplementary sequence size, and non-canonical interactions involving the nucleotides flanking the autocomplementary sequence. Interestingly, there were very few autocomplementary sequences capable of promoting homodimerization. On the other hand, numerous families of aptamers were isolated that were incapable of homodimerization because their loops did not contain perfect autocomplementary sequences. However, these species were capable of efficiently dimerizing with appropriate complementary partner species isolated from the same pool.

In this study, we have measured the apparent dimerization equilibrium constant of individual dimers derived from subtypes A and B, and have applied chemical probing techniques to investigate commonalties and differences in the structures of dimers of different sequences. Using our library of aptamers possessing similar dimerization activity yet different sequences, we were able to determine the contributions of particular nucleotides to dimer structure and stability. Chemical structure probing is in agreement with the proposed sheared base-pair between positions 1 and 9 of the loop and showed protections consistent with a base triple interaction in some loop contexts. Surprisingly, we also found the status of the last base-pair in the stem was dependent upon the loop sequence.

Section snippets

Isolation of dimerization-competent clonal RNAs

The library of dimerization-competent model DIS RNA aptamers used in this study was isolated by in vitro evolution29. Briefly, positions 1, 2, 5, 6, and 9 of the DIS loop sequences derived from HIV-1 subtypes A or B were randomized (yielding pre-selection “randomer” pools with loop sequences NNGUNNACN or NNGCNNGCN, respectively; the hexanucleotide sequences involved in intermolecular Watson-Crick base- pairing are underlined throughout). RNAs were selected and amplified according to their

Discussion

The conserved DIS sequence of HIV-1 has been shown to be a functionally important motif inasmuch as mutations or deletions in this region cause defects in infectivity and replication kinetics4, 5, 6, 7, 8, 42. Because this region of the viral RNA is conserved and functionally important, it is of interest to study its structure in some detail. However, high resolution structural studies of the DIS have been hampered somewhat by technical difficulties. NMR analysis of DIS model constructs based

Generation of pure clonal RNAs

The RNAs used in this study were selected from randomized pools for their ability to dimerize as described previously29. Individual pUC18 plasmid clones containing unique aptamer sequences were propagated in DH5α cells. Individual sequenced plasmids were prepared and used as substrates for T7 RNA polymerase transcription after digestion with Sma I. RNAs (53 nt) were gel purified on 8 % (w/v) denaturing polyacrylamide gels prior to use in dimerization and structure probing experiments.

Where

Acknowledgements

Delphine Mignot is acknowledged for skillful technical assistance. This work was supported by a grant from the Agence Nationale de Recherches sur le SIDA (ANRS). J.S.L. was a fellow of the ANRS.

References (48)

  • F. Duconge et al.

    Is a closing “GA pair” a rule for stable loop-loop RNA complexes?

    J. Biol. Chem.

    (2000)
  • E. Skripkin et al.

    Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro

    Proc. Natl Acad. Sci. USA

    (1994)
  • M. Laughrea et al.

    A 19-nucleotide sequence upstream of the 5′ major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA

    Biochemistry

    (1994)
  • B. Berkhout et al.

    Role of the DIS hairpin in replication of human immunodeficiency virus type 1

    J. Virol.

    (1996)
  • J.C. Paillart et al.

    A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis

    J. Virol.

    (1996)
  • J.L. Clever et al.

    Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation

    J. Virol.

    (1997)
  • M. Laughrea et al.

    Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization

    J. Virol.

    (1997)
  • N. Shen et al.

    Impact of human immunodeficiency virus type 1 RNA dimerization on viral infectivity and of stem-loop B on RNA dimerization and reverse transcription and dissociation of dimerization from packaging

    J. Virol.

    (2000)
  • F. Jossinet et al.

    Dimerization of HIV-1 genomic RNA of subtypes A and BRNA loop structure and magnesium binding

    RNA

    (1999)
  • F. Dardel et al.

    Solution studies of the dimerization initiation site of HIV-1 genomic RNA

    Nucl. Acids Res.

    (1998)
  • A. Mujeeb et al.

    Structure of the dimer initiation complex of HIV-1 genomic RNA

    Nature Struct. Biol.

    (1998)
  • Y. Eguchi et al.

    Antisense RNA

    Annu. Rev. Biochem.

    (1991)
  • E.G. Wagner et al.

    Antisense RNA control in bacteria, phages, and plasmids

    Annu. Rev. Microbiol.

    (1994)
  • D. Ferrandon et al.

    RNA-RNA interaction is required for the formation of specific bicoid mRNA 3′ UTR-STAUFEN ribonucleoprotein particles

    EMBO J.

    (1997)
  • Cited by (37)

    • Genome sequence analysis suggests coevolution of the DIS, SD, and Psi hairpins in HIV-1 genomes

      2022, Virus Research
      Citation Excerpt :

      Such base changes as U2C, G4A and others disturbed DIS lower part. The second fact can be related to the requirement for the formation of a stable kissing complex, that is highly dependent on the apical loop sequence, nature of the loop-closing base pair and the stem sequence (Chu et al., 2017; Durand et al., 2016; Kieken et al., 2002; Lodmell et al., 2000, 2001). Certain mutations in DIS hairpins can meet the requirement for structural compatibility between the stem conformation and the apical loop conformation in HIV-1 genomes of some dimerization groups but not of other groups.

    View all citing articles on Scopus
    1

    Edited by D. E. Draper

    View full text