RNA flexibility in the dimerization domain of a gamma retrovirus

Abstract

Retroviruses are the causative agents of serious diseases, such as acquired immunodeficiency syndromes and several cancers, and are also useful gene therapy vectors. Retroviruses contain two sense-strand RNA genomes, which become linked at their 5′ ends to form an RNA dimer. Understanding the molecular basis for dimerization may yield new approaches for controlling viral infectivity. Because this RNA domain is highly conserved within retrovirus groups, it has not been possible to define a consensus structure for the 5′ dimerization domain by comparative sequence analysis. Here, we defined a 170-nucleotide minimal dimerization active sequence (MiDAS) for a representative gamma retrovirus, the Moloney murine sarcoma virus, by stringent competitive dimerization. We then analyzed the structure at every nucleotide in the MiDAS monomeric starting state with quantitative selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry. Notably, SHAPE analysis demonstrated that the RNA monomer contains an extensive flexible domain spanning 50 nucleotides. These findings support a structural model in which RNA flexibility directly facilitates retroviral genome dimerization by reducing the energetic cost of disrupting pre-existing base pairings in the monomer.

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Figure 1: 5′-untranslated region of MuSV.
Figure 2: Competitive-dimerization assay for stringent definition of RNA structures essential for dimerization.
Figure 3: The MiDAS for MuSV defined by competitive dimerization.
Figure 4: SHAPE analysis of the MuSV MiDAS RNA and of the PALSTB and Δ289–300 mutants.
Figure 5: Secondary structure model of the MuSV MiDAS RNA.
Figure 6: Quantitative difference maps for the effects of mutations on MiDAS structure.
Figure 7: Structural model for overall flexibility in the 231–315 domain.

References

  1. 1

    Murti, K.G., Bondurant, M. & Tereba, A. Secondary structural features in the 70S RNAs of Moloney murine leukemia and Rous sarcoma viruses as observed by electron microscopy. J. Virol. 37, 411–419 (1981).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Darlix, J.L., Lapadat-Tapolski, M., de Rocquigny, H. & Roques, B.P. First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. J. Mol. Biol. 254, 523–537 (1995).

    CAS  Article  Google Scholar 

  3. 3

    Paillart, J. 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. 70, 8348–8354 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Laughrea, M. et al. Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and proviral DNA synthesis. J. Virol. 71, 3397–3406 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Hibbert, C.S., Mirro, J. & Rein, A. mRNA molecules containing murine leukemia virus packaging signals are encapsidated as dimers. J. Virol. 78, 10927–10938 (2004).

    CAS  Article  Google Scholar 

  6. 6

    Mikkelsen, J.G., Lund, A.H., Duch, M. & Pedersen, F.S. Recombination in the 5′ leader of murine leukemia virus is accurate and influenced by sequence identity with a strong bias toward the kissing-loop dimerization region. J. Virol. 72, 6967–6978 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Mikkelsen, J.G., Lund, A.H., Duch, M. & Pedersen, F.S. Mutations of the kissing-loop dimerization sequence influence the site specificity of murine leukemia virus recombination in vivo. J. Virol. 74, 600–610 (2000).

    CAS  Article  Google Scholar 

  8. 8

    Tounekti, N. et al. Effect of dimerization on the conformation of the encapsidation psi domain of the Moloney murine leukemia virus. J. Mol. Biol. 223, 205–220 (1992).

    CAS  Article  Google Scholar 

  9. 9

    Konings, D.A.M., Nash, M.A., Maizel, J.V. & Arlinghaus, R.B. Novel GACG-hairpin pair motif in the 5′ untranslated region of type C retroviruses related to murine leukemia virus. J. Virol. 66, 632–640 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Paillart, J., Marquet, R., Skripkin, E., Ehresmann, C. & Ehresmann, B. Dimerization of retroviral genomic RNAs: structural and functional implications. Biochimie 78, 639–653 (1996).

    CAS  Article  Google Scholar 

  11. 11

    D'Souza, V., Dey, A., Habib, D. & Summers, M.F. NMR Structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus. J. Mol. Biol. 337, 427–442 (2004).

    CAS  Article  Google Scholar 

  12. 12

    Michel, F. & Westhof, E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J. Mol. Biol. 216, 585–610 (1990).

    CAS  Article  Google Scholar 

  13. 13

    Frank, D.N. & Pace, N.R. Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu. Rev. Biochem. 67, 153–180 (1998).

    CAS  Article  Google Scholar 

  14. 14

    Gutell, R.R., Lee, J.C. & Cannone, J.J. The accuracy of ribosomal RNA comparative structure models. Curr. Opin. Struct. Biol. 12, 301–310 (2002).

    CAS  Article  Google Scholar 

  15. 15

    Mathews, D.H. et al. Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc. Natl. Acad. Sci. USA 101, 7287–7292 (2004).

    CAS  Article  Google Scholar 

  16. 16

    Dowell, R.D. & Eddy, S.R. Evaluation of several lightweight stochastic context-free grammars for RNA secondary structure prediction. BMC Bioinformatics 5, 71 (2004).

    Article  Google Scholar 

  17. 17

    Oroudjev, E.M., Kang, P.C.E. & Kohlstaedt, L.A. An additional dimer linkage structure in Moloney murine leukemia virus RNA. J. Mol. Biol. 291, 603–613 (1999).

    CAS  Article  Google Scholar 

  18. 18

    Ly, H. & Parslow, T.G. Bipartite signal for genomic RNA dimerization in Moloney murine leukemia virus. J. Virol. 76, 3135–3144 (2002).

    CAS  Article  Google Scholar 

  19. 19

    D'Souza, V. et al. Identification of a high affinity nucleocapsid protein binding element within the Moloney murine leukemia virus Ψ-RNA packaging signal: implications for genome recognition. J. Mol. Biol. 314, 217–232 (2001).

    CAS  Article  Google Scholar 

  20. 20

    De Tapia, M., Metzler, V., Mougel, M., Ehresmann, B. & Ehresmann, C. Dimerization of the Moloney murine leukemia virus genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278–303) and new roles for stem-loops H2 (310–352) and H3 (355–374). Biochemistry 37, 6077–6085 (1998).

    CAS  Article  Google Scholar 

  21. 21

    Kim, C. & Tinoco, I. A retroviral RNA kissing complex containing only two GC base pairs. Proc. Natl. Acad. Sci. USA 97, 9396–9401 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Rein, A., Harvin, D.P., Mirro, J., Ernst, S.M. & Gorelick, R.J. Evidence that a central domain of nuclecapsid protein is required for RNA packaging in murine leukemia virus. J. Virol. 68, 6124–6129 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Merino, E.J., Wilkinson, K.A., Coughlan, J.L. & Weeks, K.M. RNA structure analysis at single nucleotide resolution by selective 2′-hydroxyl acylation and primer extension (SHAPE). J. Am. Chem. Soc. 127, 4223–4231 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Wilkinson, K.A., Merino, E.J. & Weeks, K.M. RNA SHAPE chemistry reveals non-hierarchical interactions dominate equilibrium structural transitions in tRNAAsp transcripts. J. Am. Chem. Soc. 127, 4659–4667 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Girard, P.M., Bonnet-Mathoniere, B., Muriaux, D. & Paoletti, J. A short autocomplementary sequence in the 5′ leader region is responsible for dimerization of MoMuLV genomic RNA. Biochemistry 34, 9785–9794 (1995).

    CAS  Article  Google Scholar 

  26. 26

    Ly, H., Nierlich, D., Olsen, J. & Kaplan, A. Moloney murine sarcoma virus genomic RNAs dimerize via a two-step process: a concentration-dependent kissing-loop interaction is driven by initial contact between consecutive guanosines. J. Virol. 73, 7255–7261 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Ly, H., Nierlich, D., Olsen, J. & Kaplan, A. Functional characterization of the dimer linkage structure RNA of Moloney murine sarcoma virus. J. Virol. 74, 9937–9945 (2000).

    CAS  Article  Google Scholar 

  28. 28

    Rasmussen, S.V., Mikkelsen, J.G. & Pedersen, F.S. Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences. J. Mol. Biol. 323, 613–628 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Aagaard, L., Rasmussen, S.V., Mikkelsen, J.G. & Pedersen, F.S. Efficient replication of full-length murine leukemia viruses modified at the dimer initiation site regions. Virology 318, 360–370 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Holbrook, S.R., Cheong, C., Tinoco, I., Jr . & Kim, S.H. Crystal structure of an RNA double helix incorporating a track of non-Watson-Crick base pairs. Nature 353, 579–581 (1991).

    CAS  Article  Google Scholar 

  31. 31

    Wild, K., Weichenrieder, O., Leonard, G.A. & Cusack, S. The 2 Å structure of helix 6 of the human signal recognition particle RNA. Struct. Fold. Des. 7, 1345–1352 (1999).

    CAS  Article  Google Scholar 

  32. 32

    D'Souza, V. & Summers, M.F. Structural basis for packaging the dimeric genome of Moloney murine leukemia virus. Nature 431, 586–590 (2004).

    CAS  Article  Google Scholar 

  33. 33

    Monie, T.P. et al. Identification and visualization of the dimerization initiation site of the prototype lentivirus, Maedi Visna virus: a potential GACG tetraloop displays structural homology with the α- and γ-retroviruses. Biochemistry 44, 294–302 (2005).

    CAS  Article  Google Scholar 

  34. 34

    Chamberlin, S.I., Merino, E.J. & Weeks, K.M. Catalysis of amide synthesis by RNA phosphodiester and hydroxyl groups. Proc. Natl. Acad. Sci. USA 99, 14688–14693 (2002).

    CAS  Article  Google Scholar 

  35. 35

    Das, R., Laederach, A., Pearlman, S.M., Herschlag, D. & Altman, R.B. SAFA: Semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344–354 (2005).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was supported by a grant from the US National Institutes of Health (GM64803 to K.M.W. and A. Kaplan). We are indebted to A. Kaplan, C. Gherghe and A. Rein for many helpful discussions; to E. Merino and K. Wilkinson for assistance with SHAPE chemistry; and to D. Mathews for extensive advice with the RNAStructure program.

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Correspondence to Kevin M Weeks.

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Supplementary information

Supplementary Fig. 1

Dimerization activity of 3′ and 5′ truncation mutants. (PDF 398 kb)

Supplementary Fig. 2

Proposed secondary structures for MuLV and HaSV dimerization domains. (PDF 242 kb)

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Badorrek, C., Weeks, K. RNA flexibility in the dimerization domain of a gamma retrovirus. Nat Chem Biol 1, 104–111 (2005). https://doi.org/10.1038/nchembio712

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