Dimerization of short RNA fragments from avian leukosis virus as revealed with 2-aminopurine fluorescence

The dimerization of genomic retroviral RNA is well studied for several groups of viruses, the dimerization of human immunodeficiency (HIV) RNA being investigated in more detail. Regions of dimerization apparently involve the short sequences RNA which are directly responsible for the formation of two type dimers: kissing loop-loop (KD) and linear (LD). The 5'-end sequences from RNA avian viruses, where the dimers are basically formed, considerably differ from those of HIV. However, as it was described earlier, the mechanism of dimerization of RNA from human immunodeficiency and from avian leukosis viruses are identical. The fluorescence of adenine analogue 2-aminopurine (2-AP) incorporated into loop sequence of short fragments RNA ALV was used for analysis of dimers formation. Using the temperature dependence of fluorescence intensity 2-AP we have determined RNA melting temperature under various conditions for KD RNA ALV formed by two strands. Effects of magnesium and aminoglycoside antibiotic paromomycin on stabilization of kissing loop-loop dimer RNA have been studied. Under the experimental conditions KD RNA ALV was found to have the stability at the magnesium concentration higher than 1 mM and at paromomycin concentration higher than 2.5 mkM.     

Interaction of the dimeric form of retroviral RNA with paromonycin and magnesium as revealed using 2-aminopurine fluorescence

Studying the dimeric RNA structural organization is a step toward the understanding of retroviral genomic RNA dimerization. A kissing loop dimer is rearranged into an extended dimer during maturation of the virus particle. The extended dimer formation may be inhibited by ligands interacting with the RNA kissing loop dimer. A study was made of the interaction of dimeric RNA with paromomycin and magnesium ions. RNA dimers were formed from two hairpin RNAs having complementary sequences in the loop. The structural features of RNA dimers and the influence of the ligands were inferred from the fluorescence of 2-aminopurine (2-AP) incorporated in one of the two RNA hairpin sequences. As dimeric RNA interacted with paromomycin, 2-AP fluorescence increased. The increase was explained by a flipping of the fluorescent base out of the RNA structure. The binding constants and stoichiometry were estimated for dimeric RNA binding with paromomycin. An RNA dimer was found to interact with two paromomycin molecules; the binding constant was approximately the same (about 3 × 10⁵ M⁻¹) for both types of dimers. It was observed that the antibiotic and Mg²⁺ ions compete for binding to the hairpin RNA dimer and that one paromomycin molecule is displaced by one Mg²⁺ ion.     

2-aminopurine fluorescence: discrimination between specific and unspecific ligand binding to the kissing-loop dimer of the HIV-1 RNA

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.     

2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA

Abstract

The fluorescent 2-aminopurine probe (2-AP) incorporated into the loop of 23-mer RNA hairpin of HIV-1 genome dimerization initiation site (DIS) was used for discrimination of specific and unspecific binding of paromomycin and spermine to the kissing loop dimer (KD) formed in solution. While both ligands stabilized the KD RNA structure, only paromomycin binding resulted in significant increase of 2-AP fluorescence. These observations suggest that the 2-AP fluorescent RNA construct might be useful for selecting ligands specifically binding the HIV-1 kissing loop RNA dimer.     

Characteristic of the two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two copies of genomic RNA is a necessary step of retroviral replication. In the case of human immunodeficiency virus type 1 (HIV-1) the process is explored in many details. It is proved that conserved stem-loop structure is an essential element in RNA dimerization. Similar model of two-step dimerization mechanism can be considered for avian sarcoma and leukosis virus group (ASLV) in spite of the absence of homology between dimer initiation site (DIS) of ASLV and that of HIV-1. In this paper, short RNA fragments of two viruses: avian sarcoma virus CT-10 and avian leukosis virus HPRS-103 have been chosen in order to investigate the structural requirements of dimerization process and compare them to that of HIV-1. The rate of spontaneous transition from loose to tight dimer was studied as a function of stem length and temperature. Although both types of dimers were observed for both avian retroviruses chosen, fragments of CT-10 requires much higher RNA concentration to form loose dimer. In spite of identical sequence of the loops (5'-A-CUGCAG-3') avian sarcoma virus CT-10 RNA fragments dimerization was greatly impaired. The differences can be explained by deletion of adenine 271 in avian sarcoma virus CT-10 in the stem and by resulting shortening of the self-complementary loop.     

The Role of Structural Elements in Dimerization of RNA Fragments in Avian Retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop–loop interaction of two hairpins, or kissing hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

Effect of aminoglycoside antibiotics on the conformation of the unpaired loop adenine of avian leucosis virus RNA as revealed using 2-aminopurine fluorescence

The fluorescent properties of 2-aminopurine (2-AP) incorporated in an RNA sequence are used to study the structural dynamics and local changes of the retroviral RNA structure. Using 2-AP, the conformational states of the unpaired loop adenine in avian leucosis virus RNA were studied upon its interaction with aminoglycoside antibiotics. The intensity of 2-AP fluorescence in the monomeric RNA hairpin was higher than in both RNA dimers. The intensity of fluorescence in the extended dimer was significantly lower than in the kissing loop dimer. The finding was be explained by the fact that stacking contacts in the extended dimer produce a more compact loop structure than in the kissing loop dimer. When the binding of aminogycoside antibiotics with the kissing loop dimer RNA was analyzed, only tobramycin increased the intensity of 2-AP fluorescence almost threefold. The results showed that 2-AP fluorescence is suitable for detecting local changes in complexes of retroviral RNA with ligands.     

Elements located upstream and downstream of the major splice donor site influence the ability of HIV-2 leader RNA to dimerize in vitro

An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during budding and maturation of the viral particle. In HIV-1, a 5' leader region site termed stem-loop 1 (SL1) promotes RNA dimerization in vitro and influences dimerization in vivo. In HIV-2, two sequences promote dimerization of RNA fragments in vitro: the 5'-end of the primer-binding site (PBS) and a stem-loop region homologous to the HIV-1 SL1 sequence. Because HIV-2 RNA constructs of different lengths use these two dimerization signals disproportionately, we hypothesized that other sequences could modulate their relative utilization. Here, we characterized the influence of sequences upstream and downstream of the major splice donor site on the formation of HIV-2 RNA dimers in vitro using a variety of RNA constructs and dimerization and electrophoresis protocols. We first assayed the formation of loose or tight dimers for 1-444 and 1-561 model RNAs. Although both RNAs could form PBS-dependent loose dimers, the 1-561 RNA was unable to make SL1-dependent tight dimers. Using RNAs truncated at their 5'- and/or 3'-ends and by making compensatory base substitutions, we found that two elements interfere with the formation of SL1-dependent tight dimers. The cores of these elements are located at nucleotides 189-196 and 543-550. Our results suggest that base pairing between these sequences prevents the formation of SL1-dependent tight dimers, probably by sequestering SL1 in a stable intramolecular arrangement. Moreover, we found that nucleotides downstream of SL1 decreased the rate of tight dimerization. Interestingly, dimerization at 37 degrees C in the presence of nucleocapsid protein increased the yield of SL1-mediated tight dimerization in vitro, even in the presence of the two interfering elements, suggesting a relationship between the nucleocapsid protein and activation of the SL1 dimerization signal in vivo.     

Characteristics of two-step RNA dimerization in avian sarcoma and leukosis viruses

Dimerization of two genomic RNA copies is essential for the assembly of retrovirus particles. This process has been studied in detail, and a two-step mechanism has been proposed for the human immunodeficiency virus type 1 (HIV-1). A similar model can be assumed for avian sarcoma and leukosis viruses (ASLV), despite the lack of homology between the dimerization initiation site (DIS) of ASLV and that of HIV-1. The structural features of the ASLV DIS were studied with the examples of the avian leukosis virus HPRS-103 and the avian sarcoma virus CT-10. The rate of spontaneous transition from loose to tight dimers at a higher temperature was studied as dependent on the stem length in the DIS hairpin. Dimers of both types were formed by the selected RNA fragments of the two viruses. The conditions of loose dimer formation differed considerably, although the two viruses had identical sequences (5-A-CUGCAG-3) of the hairpin loop. Dimerization of CT-10 RNA fragments required an RNA concentration at least an order of magnitude higher than in the case of HPRS-103. The difference was explained by deletion of an adenine from the hairpin stem of C-10.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 147–154.Original Russian Text Copyright © 2005 by Beniaminov, Samokhin, Ulyanov, Minyat.     

A unique,thermostable dimer linkage structure of RD114 retroviral RNA

Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem–loop (psl) sequences at the 5′ end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55°C–70°C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87°C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 5′ region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 5′ R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 3′ R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 3′ end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.     

Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.     

The role of structural elements in dimerization of RNA fragments in avian retroviruses

The slipped loop structure, earlier identified as an unusual DNA structure, was found to be a possible element of the RNA folding. In order to experimentally test this suggestion, model oligoribonucleotides capable of forming the SLS were synthesized. Treatment of the oligoribonucleotides with nuclease S1 and RNases specific for single- and double-stranded RNA demonstrated the steric possibility of SLS formation. To determine the possible functional role of SLS-RNA, various naturally occurring RNAs were screened in silico. Among the most interesting findings were dimerization initiation sites of avian retroviral genomic RNAs. Analysis of RNA from 31 viruses showed that formation of the intermolecular SLS during RNA dimerization is theoretically possible, competing with the formation of an alternative hairpin structure. Identification of the secondary structure of selected RNA dimers employing nuclease digestion techniques as well as covariance analysis of the retroviral RNA dimerization initiation site sequences were used to show that the alternative conformation (loop-loop interaction of two hairpins) is the most preferred. Alternative structures and conformational transitions in RNA dimerization mechanisms in avian retroviruses are discussed.     

HIV-1 RNA dimerization initiation site is structurally similar to the ribosomal A site and binds aminoglycoside antibiotics

Human immunodeficiency virus (HIV) genomic RNA is packaged into virions as a dimer. The first step of dimerization is the formation of a kissing-loop complex at the so-called dimerization initiation site (DIS). We found an unexpected and fortuitous resemblance between the HIV-1 DIS kissing-loop complex and the eubacterial 16 S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. Similarities exist not only at the primary and secondary structure level but also at the tertiary structure level, as revealed by comparison of the respective DIS and A site crystal structures. Gel shift, inhibition of lead-induced cleavage, and footprinting experiments showed that paromomycin and neomycin specifically bind to the kissing-loop complex formed by the DIS, with an affinity and a geometry similar to that observed for the A site. Modeling of the aminoglycoside-DIS complex allowed us to identify antibiotic modifications likely to increase the affinity and/or the specificity for the DIS. This could be a starting point for designing antiviral drugs against HIV-1 RNA dimerization.     

Human immunodeficiency virus type 1 RNA outside the primary encapsidation and dimer linkage region affects RNA dimer stability in vivo.

To characterize the cis-acting determinants that function in RNA dimer formation and maintenance, we examined the stability of RNA dimers isolated from virus particles containing mutations in the encapsidation region of human immunodeficiency virus type 1 (HIV-1). The genomic RNAs of all mutants containing lesions in elements required for in vitro dimerization exhibited thermal stability similar to that of wild-type (WT) HIV-1. These data indicate that the eventual formation of stable dimeric RNA in vivo is not absolutely dependent on the elements that promote dimer formation in vitro. Surprisingly, mutants that lacked a large segment of the middle portion of the genome, outside the likely primary dimer linkage region, formed RNA dimers that were measurably more stable than WT. In addition, the insertion of one or multiple copies of a foreign gene, which resulted in a series of vectors that approached RNA length similar to that of WT RNA, still exhibited augmented dimer stability. These results suggest that there are regions in the HIV-1 genome outside the primary dimer initiation and dimer linkage regions that can negatively affect dimer stability.     

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.     

Comparison of an Avian Osteopetrosis Virus with an Avian Lymphomatosis Virus by RNA-DNA Hybridization

Myeloblastosis-associated virus (MAV)-2(0), a virus which was derived from avian myeloblastosis virus and induced a high incidence of osteopetrosis, was compared with avian lymphomatosis virus 5938, a recent field isolate which induced a high incidence of lymphomatosis. The following information was obtained. (i) MAV-2(0) induced osteopetrosis, nephroblastoma, and a very low incidence of hepatocellular carcinoma. No difference was seen in the oncogenic spectrum of end point and plaque-purified MAV-2(0). (ii) ¹²⁵I-labeled RNA sequences from MAV-2(0) formed hybrids with DNA extracted from osteopetrotic bone at a rate suggesting five proviral copies per haploid cell genome. The extent of hybridization of MAV-2(0) RNA with DNA from osteopetrotic tissue was more extensive (87%) than was observed in reactions with DNA from uninfected chicken embryos (52%). (iii) Competition of unlabeled viral RNA in hybridization reactions between the radioactive RNA from the two viruses and their respective proviral sequences present in tumor tissues showed that 15 to 20% of the viral sequences detected in these reactions were unshared. In contrast, no differences were detected in competition analyses of RNA sequences from the two viruses detected in DNA of normal chicken cells. (iv) MAV-2(0) 35S RNA was indistinguishable in size from avian lymphomatosis virus 5938 35S RNA by polyacrylamide gel electrophoresis.