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. 2011 Sep 30;412(4):737-50.
doi: 10.1016/j.jmb.2011.07.053. Epub 2011 Aug 3.

Interstitial contacts in an RNA-dependent RNA polymerase lattice

Andres B Tellez  1 , Jing Wang, Elizabeth J Tanner, Jeannie F Spagnolo, Karla Kirkegaard, Esther Bullitt
Affiliations

Interstitial contacts in an RNA-dependent RNA polymerase lattice

Andres B Tellez et al. J Mol Biol. .

Abstract

Catalytic activities can be facilitated by ordered enzymatic arrays that co-localize and orient enzymes and their substrates. The purified RNA-dependent RNA polymerase from poliovirus self-assembles to form two-dimensional lattices, possibly facilitating the assembly of viral RNA replication complexes on the cytoplasmic face of intracellular membranes. Creation of a two-dimensional lattice requires at least two different molecular contacts between polymerase molecules. One set of polymerase contacts, between the "thumb" domain of one polymerase and the back of the "palm" domain of another, has been previously defined. To identify the second interface needed for lattice formation and to test its function in viral RNA synthesis, we used a hybrid approach of electron microscopic and biochemical evaluation of both wild-type and mutant viral polymerases to evaluate computationally generated models of this second interface. A unique solution satisfied all constraints and predicted a two-dimensional structure formed from antiparallel arrays of polymerase fibers that use contacts from the flexible amino-terminal region of the protein. Enzymes that contained mutations in this newly defined interface did not form lattices and altered the structure of wild-type lattices. When reconstructed into virus, mutations that disrupt lattice assembly exhibited growth defects, synthetic lethality or both, supporting the function of the oligomeric lattice in infected cells. Understanding the structure of polymerase lattices within the multimeric RNA-dependent RNA polymerase complex should facilitate antiviral drug design and provide a precedent for other positive-strand RNA viruses.

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Figures

Figure 1
Figure 1. Structure and oligomerization of poliovirus 3D polymerase
(a) The domain structure of the 461-amino acid poliovirus 3D polymerase is shown, with domains based on the three-dimensional structure of the full-length polymerase . (b) Two-dimensional lattices formed by purified wild-type polymerase, as shown by negative staining followed by electron microscopy. Magnification bar: 10 nm for enlarged image, and 100 nm for inset. (c) The polymerase-polymerase interactions shown along Interface I, a set of polymerase-polymerase interaction surfaces observed in a crystal form and tested for functionality by site-directed mutagenesis ; ; ; . Domains of the first two polymerases on the left are color-coded as in panel a. Amino acid Leu446, integral to Interface I, is shown as a red space-filling model. A single polymerase molecule is approximately 5 nm in length, width and height.
Figure 2
Figure 2. Lattice formation and wavelength-dependent turbidity of L446A mutant polymerase
Electron microscopy of purified wild-type (a) and L446A (b) polymerase following incubation for 24 hours and negative staining. Magnification Bar 500 Å. (c) Time courses of the acquisition of turbidity as a function of wavelength in preparations of wild-type and L446A mutant polymerase, which contains a mutation lethal to the virus and predicted to disrupt Interface I (Fig. 1c), following dilution from high-salt, high-glycerol storage buffer to a concentration of 6 μM. All times from 0 to 60 minutes were super-imposable for the L446A mutant polymerase, whereas the 0-minute time point (grey line) could be differentiated from the later time points for the wild-type polymerase. (d) The time course of the acquisition of turbidity by wild-type and L446A mutant polymerase at a single wavelength, 800 nm.
Figure 3
Figure 3. Computational modeling of polymerase conformations and of docking of two-polymerase fibers with each other
(a-e) Normal mode analysis yielded several different modes of conformational change for the poliovirus polymerase, based on the known full-length structure of a polymerase monomer . The two states in contrasting colors in panels a-e are those within each of the five different normal modes that yielded the highest RMSD when compared with each other (Suppl. Table 2). (f) Two different three-dimensional structures of the poliovirus polymerase determined by X-ray crystallography are superimposed for comparison. The white structure includes all but 100 N-terminal residues of the 3D polymerase, and leaves Interface I intact. The blue structure shows the full-length monomeric poliovirus 3D polymerase . (g) One hundred different two-polymerase Interface I fibers were formed, including the two most different structures from each of the five normal modes. (h) One of the 100 different two-polymerase fibers, with conformation B2 in the ‘thumb contact’ position in blue and conformation D1 in the ‘palm contact’ position in pink. (i) Symmetries capable of self-propagation in a parsimonious manner that were found in the 200,000 structures identified by surface convolution. The direction of the arrow signifies the palm-to-thumb alignment along Interface I. The number of potential solutions that were identified from the 200,000 candidates in each symmetry group is indicated in parentheses.
Figure 4
Figure 4. Effect of selected mutations on lattice formation and turbidity acquisition by poliovirus 3D polymerase
Electron microscopy of indicated polymerases incubated for 24 hours in the absence (left) or presence (middle) of 4 μM U24 RNA are shown, as is the absorbance at 800 nm as a function of time after dilution to 6 μM for each polymerase indicated (right). (a) wild-type, (b) Y32A, (c) S438A, (d) K255A, (e) K431A, and (f) K314A polymerases. White magnification bar, 100 nm.
Figure 5
Figure 5. Characteristics of predicted polymerase complexes with Interface II interactions in complex F2
(a) Ribbon diagram of four polymerase molecules, interacting along two interfaces to form the building block for a two-dimensional lattice. Previously characterized Interface I links the pink and blue polymerases vertically, as in Figure 3h. Computationally predicted Interface II links the blue and pink polymerases from left to right as in the “Anti-Parallel, Single Set of New Contacts” example in Figure 3i. Contact residues on the S438-containing surface are shown in gold, and contact residues on the S87-containing surface are shown in silver. (b) Close-up of proposed Interface II shows all the contacts in gold on the blue, Ser 438-containing, interface and in silver on the pink, Ser87-containing interface. All residues that contain an atom with a van der Waal’s overlap of more than −0.4 of an atom are shown. (c,d) The individual amino acids and their interactions can be seen more clearly when separated into two diagrams.
Figure 6
Figure 6. Growth phenotypes of viruses that contain mutations that affect the stability of Interface II of the predicted F2 lattice
(a) Plaque phenotypes of viruses at 32.5°C and 39.5°C on HeLa cells. (b) Time courses of viral yields at various times after infection of HeLa cells with wild-type and mutant viruses as indicated, at multiplicities of infection of ten plaque-forming units per cell. The values for the final yields are given next to the relevant curve.
Figure 7
Figure 7. Effect of mixing S438A mutant polymerase with wild-type polymerase on lattice formation
Electron microscopy of structures formed by incubating 2 μM wild-type polymerase for 24 hours, followed by negative staining. (b) Structures formed by incubating 2 μM wild-type polymerase in the presence of 2 μM S438A polymerase for 24 hours. (c) Scaled diagrammatic model of polymerase lattice being formed on a membranous vescicle 200 nm in diameter, and being disrupted by the presence of mutant polymerase. Polymerases from the F2 lattice were placed using Chimera software . Magnification Bar 500 Å.
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