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A unique, thermostable dimer linkage structure of RD114 retroviral RNA.

ABSTRACT: 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 degrees C-70 degrees C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87 degrees 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.

SUBMITTER: Kharytonchyk S

PROVIDER: S-EPMC2822922 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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A unique, thermostable dimer linkage structure of RD114 retroviral RNA.

Kharytonchyk Sergei S Pedersen Finn Skou FS

RNA (New York, N.Y.) 20100114 3

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 degrees C-70 degrees C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87 degrees C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple ...[more]

PMID: 20075164

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Project description:Retroviral particles contain a dimeric RNA genome, which serves as template for the generation of double-stranded DNA by reverse transcription. Transfer between RNA strands during DNA synthesis is governed by both sequence similarity between templates and structural features of the dimeric RNA. A kissing hairpin, believed to facilitate intermolecular recognition and dimer formation, was previously found to be a preferred site for recombination. To investigate if hairpin loop-loop-complementarity is the primary determinant for this recombination preference, we have devised a novel 5' leader recombination assay based upon co-packaging of two wild-type or loop-modified murine leukemia virus vector RNAs. We found that insertion of an alternative palindromic loop in one of the two vectors disrupted site-directed template switching, whereas site-specificity was restored between vectors with complementary non-wild-type palindromes. By pairing vector RNAs that contained identical non-palindromic loop motifs and that were unlikely to interact by loop-loop kissing, we found no preference for recombination at the kissing hairpin site. Of vector pairs designed to interact through base pairing of non-palindromic loop motifs, we could in one case restore hairpin-directed template switching, in spite of the reduced sequence identity, whereas another pair failed to support hairpin- directed recombination. However, analyses of in vitro RNA dimerization of all studied vector combinations showed a good correlation between efficient dimer formation between loop-modified viral RNAs and in vivo cDNA transfer at the kissing hairpin. Our findings demonstrate that complementarity between wild-type or non-wild-type hairpin kissing loops is essential but not sufficient for site-specific 5' leader recombination and lend further support to the hypothesis that a specific 'kissing' loop-loop interaction is guided by complementary sequences and maintained within the mature dimeric RNA of retroviruses.

Project description:Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255-7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.

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A unique, thermostable dimer linkage structure of RD114 retroviral RNA.

Publications

A unique, thermostable dimer linkage structure of RD114 retroviral RNA.

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