Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Yasuyuki Miyazaki; Rossitza N. Irobalieva; Blanton S. Tolbert; Adjoa Smalls-Mantey; Kilali Iyalla; Kelsey Loeliger; Victoria D'Souza; Htet Khant; Michael F. Schmid; Eric L. Garcia; Alice Telesnitsky; Wah Chiu; Michael F. Summers

doi:10.1016/j.jmb.2010.09.009

Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Yasuyuki Miyazaki, Rossitza N. Irobalieva, Blanton S. Tolbert, Adjoa Smalls-Mantey, Kilali Iyalla, Kelsey Loeliger, Victoria D'Souza, Htet Khant, Michael F. Schmid, Eric L. Garcia, Alice Telesnitsky, Wah Chiu, Michael F. Summers

Microbiology, Immunology, and Molecular Genetics

Research output: Contribution to journal › Article › peer-review

59 Scopus citations

Abstract

The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (Ψ^CD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([Ψ^CD]₂, 132 nt, 42.8 kDa) using a ²H-edited NMR-spectroscopy-based approach. This approach enabled the detection of ¹H-¹H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional ¹H-¹H correlated and ¹H-¹³C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [Ψ^CD]₂ simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [Ψ^CD]₂ functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

Original language	English
Pages (from-to)	751-772
Number of pages	22
Journal	Journal of Molecular Biology
Volume	404
Issue number	5
DOIs	https://doi.org/10.1016/j.jmb.2010.09.009
State	Published - Dec 17 2010

Bibliographical note

Funding Information:
Support from the National Institutes of Health (NIH) ( GM42561 to M.F. Summers, CA069300 to A.T., and P41RR02250 to W.C.) is gratefully acknowledged. A.S.-M. was supported by NIH MARC U⁎Star ( GM08663 ) and Howard Hughes Medical Institute education grants, and R.I. was supported by NIH Training Grant No. 5T90DK070121-05 through the Gulf Coast Consortia. We are grateful to Alexander Grishaev (National Institute of Allergy and Infectious Diseases, NIH) for collecting SAXS data and assisting in its interpretation.

Keywords

Cryo-electron tomography
Genome packaging
NMR spectroscopy
RNA structure
Retrovirus

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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10.1016/j.jmb.2010.09.009

Cite this

Miyazaki, Y., Irobalieva, R. N., Tolbert, B. S., Smalls-Mantey, A., Iyalla, K., Loeliger, K., D'Souza, V., Khant, H., Schmid, M. F., Garcia, E. L., Telesnitsky, A., Chiu, W., & Summers, M. F. (2010). Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography. Journal of Molecular Biology, 404(5), 751-772. https://doi.org/10.1016/j.jmb.2010.09.009

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title = "Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography",

abstract = "The 5'-untranslated regions of all gammaretroviruses contain a conserved {"}double-hairpin motif{"} (ΨCD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming {"}kissing{"} interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([ΨCD]2, 132 nt, 42.8 kDa) using a 2H-edited NMR-spectroscopy-based approach. This approach enabled the detection of 1H-1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H-1H correlated and 1H-13C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 {\AA}×45 {\AA}×25 {\AA}). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [ΨCD]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [ΨCD]2 functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.",

keywords = "Cryo-electron tomography, Genome packaging, NMR spectroscopy, RNA structure, Retrovirus",

author = "Yasuyuki Miyazaki and Irobalieva, {Rossitza N.} and Tolbert, {Blanton S.} and Adjoa Smalls-Mantey and Kilali Iyalla and Kelsey Loeliger and Victoria D'Souza and Htet Khant and Schmid, {Michael F.} and Garcia, {Eric L.} and Alice Telesnitsky and Wah Chiu and Summers, {Michael F.}",

note = "Funding Information: Support from the National Institutes of Health (NIH) ( GM42561 to M.F. Summers, CA069300 to A.T., and P41RR02250 to W.C.) is gratefully acknowledged. A.S.-M. was supported by NIH MARC U⁎Star ( GM08663 ) and Howard Hughes Medical Institute education grants, and R.I. was supported by NIH Training Grant No. 5T90DK070121-05 through the Gulf Coast Consortia. We are grateful to Alexander Grishaev (National Institute of Allergy and Infectious Diseases, NIH) for collecting SAXS data and assisting in its interpretation. ",

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doi = "10.1016/j.jmb.2010.09.009",

language = "English",

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Miyazaki, Y, Irobalieva, RN, Tolbert, BS, Smalls-Mantey, A, Iyalla, K, Loeliger, K, D'Souza, V, Khant, H, Schmid, MF, Garcia, EL, Telesnitsky, A, Chiu, W & Summers, MF 2010, 'Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography', Journal of Molecular Biology, vol. 404, no. 5, pp. 751-772. https://doi.org/10.1016/j.jmb.2010.09.009

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T1 - Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

AU - Miyazaki, Yasuyuki

AU - Irobalieva, Rossitza N.

AU - Tolbert, Blanton S.

AU - Smalls-Mantey, Adjoa

AU - Iyalla, Kilali

AU - Loeliger, Kelsey

AU - D'Souza, Victoria

AU - Khant, Htet

AU - Schmid, Michael F.

AU - Garcia, Eric L.

AU - Telesnitsky, Alice

AU - Chiu, Wah

AU - Summers, Michael F.

N1 - Funding Information: Support from the National Institutes of Health (NIH) ( GM42561 to M.F. Summers, CA069300 to A.T., and P41RR02250 to W.C.) is gratefully acknowledged. A.S.-M. was supported by NIH MARC U⁎Star ( GM08663 ) and Howard Hughes Medical Institute education grants, and R.I. was supported by NIH Training Grant No. 5T90DK070121-05 through the Gulf Coast Consortia. We are grateful to Alexander Grishaev (National Institute of Allergy and Infectious Diseases, NIH) for collecting SAXS data and assisting in its interpretation.

PY - 2010/12/17

Y1 - 2010/12/17

N2 - The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (ΨCD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([ΨCD]2, 132 nt, 42.8 kDa) using a 2H-edited NMR-spectroscopy-based approach. This approach enabled the detection of 1H-1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H-1H correlated and 1H-13C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [ΨCD]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [ΨCD]2 functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

AB - The 5'-untranslated regions of all gammaretroviruses contain a conserved "double-hairpin motif" (ΨCD) that is required for genome packaging. Both hairpins (SL-C and SL-D) contain GACG tetraloops that, in isolated RNAs, are capable of forming "kissing" interactions stabilized by two intermolecular G-C base pairs. We have determined the three-dimensional structure of the double hairpin from the Moloney murine leukemia virus ([ΨCD]2, 132 nt, 42.8 kDa) using a 2H-edited NMR-spectroscopy-based approach. This approach enabled the detection of 1H-1H dipolar interactions that were not observed in previous studies of isolated SL-C and SL-D hairpin RNAs using traditional 1H-1H correlated and 1H-13C-edited NMR methods. The hairpins participate in intermolecular cross-kissing interactions (SL-C to SL-D' and SLC' to SL-D) and stack in an end-to-end manner (SL-C to SL-D and SL-C' to SL-D') that gives rise to an elongated overall shape (ca 95 Å×45 Å×25 Å). The global structure was confirmed by cryo-electron tomography (cryo-ET), making [ΨCD]2 simultaneously the smallest RNA to be structurally characterized to date by cryo-ET and among the largest to be determined by NMR. Our findings suggest that, in addition to promoting dimerization, [ΨCD]2 functions as a scaffold that helps initiate virus assembly by exposing a cluster of conserved UCUG elements for binding to the cognate nucleocapsid domains of assembling viral Gag proteins.

KW - Cryo-electron tomography

KW - Genome packaging

KW - NMR spectroscopy

KW - RNA structure

KW - Retrovirus

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Structure of a Conserved Retroviral RNA Packaging Element by NMR Spectroscopy and Cryo-Electron Tomography

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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