Journal of Molecular Biology
Regular articleStructure and dimerization of HIV-1 kissing loop aptamers1
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 NNN or NNN, 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)
- et al.
Non-canonical interactions in a kissing loop complexthe dimerization initiation site of HIV-1 genomic RNA
J. Mol. Biol.
(1997) - et al.
Evidence that a kissing loop structure facilitates genomic RNA dimerisation in HIV-1
J. Mol. Biol.
(1996) Control of ColE1 plasmid replicationthe process of binding of RNA I to the primer transcript
Cell
(1984)- et al.
Complexes formed by complementary RNA stem-loops. Their formation, structures and interaction with ColE1 Rom protein
J. Mol. Biol.
(1991) - et al.
Dimerization of HIV-1Lai RNA at low ionic strength. An autocomplementary sequence in the 5′ leader region is evidenced by an antisense oligonucleotide
J. Biol. Chem.
(1995) - et al.
NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer
J. Biol. Chem.
(1996) - et al.
NMR structure of the mature dimer initiation complex of HIV-1 genomic RNA
FEBS Letters
(1999) - et al.
Dimerization of retroviral genomic RNAsstructural and functional implications
Biochimie
(1996) - et al.
Functional sites in the 5′ region of human immunodeficiency virus type 1 RNA form defined structural domains
J. Mol. Biol.
(1993) - et al.
The crystal structure of the dimerization initiation site of genomic HIV-1 RNA reveals an extended duplex with two adenine bulges
Structure Fold. Des.
(1999)
Is a closing “GA pair” a rule for stable loop-loop RNA complexes?
J. Biol. Chem.
Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro
Proc. Natl Acad. Sci. USA
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
Role of the DIS hairpin in replication of human immunodeficiency virus type 1
J. Virol.
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.
Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation
J. Virol.
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.
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.
Dimerization of HIV-1 genomic RNA of subtypes A and BRNA loop structure and magnesium binding
RNA
Solution studies of the dimerization initiation site of HIV-1 genomic RNA
Nucl. Acids Res.
Structure of the dimer initiation complex of HIV-1 genomic RNA
Nature Struct. Biol.
Antisense RNA
Annu. Rev. Biochem.
Antisense RNA control in bacteria, phages, and plasmids
Annu. Rev. Microbiol.
RNA-RNA interaction is required for the formation of specific bicoid mRNA 3′ UTR-STAUFEN ribonucleoprotein particles
EMBO J.
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2022, Virus ResearchCitation 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.
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Edited by D. E. Draper