2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity

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Science Advances  15 Apr 2020:
Vol. 6, no. 16, eaay6410
DOI: 10.1126/sciadv.aay6410
  • Fig. 1 Cryo-EM single-particle reconstruction and atomic model of VP3.

    (A) The cryo-EM structure of the VP3 tetramer [Electron Microscopy Data Bank (EMDB) ID: EMD-0632) is shown along the twofold axes of the D2 symmetry. Individual subunits chain A (aqua blue) and chain B (lemon) and their twofold related symmetry pairs chain A′ (sky blue) and chain B′ (honey dew) are shown in shades of blue and yellow. The top panel shows a dimeric subunit with one twofold axis, and the bottom panel shows second twofold axis. (B) Ribbon representation of the atomic model of VP3 tetramer relative to the orientation in the top panel of (A). Each individual dimer is formed by two antiparallel monomers. Individual domains are shown in separate colors: kinase-like (KL) domain (red), guanine-N7 MTase (N7-MTase) domain (green), 2′-O-MTase domain (orange), GTase domain (cyan), and PDE domain (magenta). The same coloring scheme is used in other figures. (C) Domain demarcation along the VP3 peptide chain sequence based on the structural annotation. (D) Modular domain organization of VP3 monomer structure. (E) A representative sample of the 2.7 Å cryo-EM map showing side-chain densities (from the 2′-O-MTase domain) along with the modeled structure. See movies S1 to S3.

  • Fig. 2 VP3 domain organization and structural comparisons.

    (A) Structural comparison of the GTase domains in RV VP3 (cyan) and BTV VP4 (light gray) showing similar structural folds. GTase domain of VP3 additionally participates in the interaction with the KL domain (helices 165 to 178, shown in red) from the neighboring subunit at the dimeric interface (circled). (B) Electron density for the GMP observed at the catalytic center of GTase domain in the x-ray structure. Conserved catalytic residues interacting with the GMP are denoted. (C) Structural comparison of the guanine N7-MTase (green) and (D) 2′-O-MTase (orange) domains in RV VP3 with those in BTV VP4 (light gray). Note that the substrate binding region shows ~50% conservation in amino acid sequences. The 2′-O-MTase domain also contains the well-characterized catalytic KDKE tetrad motif (in asterisks), which is a conserved feature of MTase domains of other capping enzymes. N- and C-terminal residues of the MTase domains in RV VP3 (L181 to L560 for N7-MTase and I256 to D440 for 2′-O-MTase) and BTV VP4 (R109 to D509 for N7-MTase and T175 to D377 for 2′-O-MTase) are indicated (domain colors as in previous figures).

  • Fig. 3 VP3 exhibits RTPase and RNA helicase activity.

    (A) An autoradiograph showing a VP3 or PDE domain concentration-dependent decrease in the 5′-end [γ-32P] GTP-labeled RNA band intensity confirms that VP3 exhibits RTPase activity and that this activity is associated with the PDE domain. RV NSP2, which was previously shown to have RTPase activity, is used as a positive control. The RTPase activity of VP3 is inhibited in the presence of EDTA indicating that the RTPase activity of VP3 is metal dependent. (B) Low-resolution cryo-EM single-particle reconstruction of VP3 in complex with 8-mer single-stranded RNA (ssRNA), suggests that the RNA binds near a cleft in the PDE domain (black arrow). The difference map was obtained using low-resolution cryo-EM map from apo-VP3 and cryo-EM map from VP3-ssRNA complex in Chimera. (C and D) Biolayer interferometry (BLI) analysis of RV VP3 and PDE domain binding with ssRNA (8-mer) shows that both full-length VP3 and PDE domain bind with ssRNA with similar nanomolar affinity. The two–binding site model fits the data with excellent R2 (~0.99) and χ2 (~0) values. (E and F) Fluorescence-based helicase assay. The 6-fluorescin amidite (6-FAM)–labeled RNA strand is quenched by a complementary quencher [Black Hole Quencher (BHQ)–labeled] strand in dsRNA. Increased fluorescence intensity with increasing concentrations of VP3 indicates the strand separation confirming the dsRNA helicase activity of VP3. Helicase activity requires the full-length VP3; the PDE domain alone does not show any significant increase in fluorescence intensity. (F) The fluorescence intensity is also modulated by adenosine triphosphate (ATP) concentrations.

  • Fig. 4 Proposed model for mRNA capping during endogenous transcription.

    (A) The VP3 tetramer is positioned inside the core particle between two VP1 molecules such that the PDE domains on either side of the VP3 tetramer face the transcript exit channels in the two neighboring VP1 molecules at each fivefold axis. Note that the length of the VP3 tetramer (~126 Å) matches the distance between the two VP1 exit channels (~130 Å). Similar placement of VP3 tetramers between the other five nonredundant VP1 pairs inside the core particle is envisioned. The VP6 and VP2 layers of the DLP are shown in blue and green shades, respectively, along with VP1 molecules (in gray) attached to VP2 at the fivefold axes. In this arrangement of VP3, two simultaneous capping activities can take place using the VP3 oligomeric assembly. The blue arrows indicate the possible path for the nascent transcript during the capping process. (B) Cartoon representation of a possible path for the nascent transcript emerging from VP1 through the PDE and GTase domains of one VP3 subunit and the MTase domains in the twofold related VP3 subunit, followed by its exit through the closest exit channel at the fivefold axes (denoted by a pentagon) of the DLP. Before capping, the RNA duplex formed by the nascent transcript and the template strand needs to be separated. The dsRNA helicase activity exhibited by VP3 suggests such a role for VP3 in RNA duplex separation with subsequent 5′-end capping.

Supplementary Materials

  • Supplementary Materials

    2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity

    Dilip Kumar, Xinzhe Yu, Sue E. Crawford, Rodolfo Moreno, Joanita Jakana, Banumathi Sankaran, Ramakrishnan Anish, Soni Kaundal, Liya Hu, Mary K Estes, Zhao Wang, B. V. Venkataram Prasad

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    • Figs. S1 to S9
    • Tables S1 to S3
    • Legends for movies S1 to S3

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