The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses

Abstract

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

Fig. 1.

Scanning electron microscopy of G25D AcMNPV polyhedra (A) and infectious WNPV polyhedra (B). (B, Inset) WNPV polyhedra in which imprints of virus particles are apparent. This is not observed for the G25D polyhedra, which do not contain virions and have a smooth surface.

Fig. 2.

Structure of the baculovirus polyhedrin protein. Cartoon representations of the WNPV polyhedrin molecule (A and B) and its trimeric assembly (C and D) in orthogonal views. In A and B, the polypeptide chain is colored in a blue-to-red gradient from the N-terminus to the C-terminus (CT). The polyhedrin has a jelly-roll fold with two sheets, BIDG and FCHE. Helix H1/2 and the CT loop extend away from the main body of the trimer in orthogonal directions. Three regions are disordered, and dotted lines represent the missing loops. First, only weak electron density was visible for the H1/2 helix so that only the main chain was traced with a short break between residues 37 and 39. Second, the short loop comprising residues 142–145 had no interpretable electron density and was not modeled. Third, the region comprising residues 171–202 was disordered apart from a short helix (186–194). (C and D) Trimer is represented in orthogonal views, with scale bars representing 75 Å. The trimer has the shape of a chalice with a tripod base (H1/2) and prominent protrusions (CT).

Fig. 3.

Tetrahedral clusters in polyhedra. (A) Cartoon representation of a tetrahedral cluster with different colors for each of the four WNPV polyhedrin trimers. (B) Close-up view of the contact area between trimers within the tetrahedral cluster highlighting salt bridges between residues Glu₂₁₄ and Arg₁₃₀ and a disulfide bond between two Cys₁₃₁ residues (Inset). The side chain of the cysteine is in two alternate conformations, and conformation A in one molecule forms a disulfide bond with the symmetry-related cysteine in conformation B. (C) Close-up view of the interactions between the four trimers mediated by domain swapping of the N-terminal regions of three trimers (red, yellow, and green) with the central jelly-roll domains of the fourth trimer (blue).

Fig. 4.

Architecture of baculovirus polyhedra. (A) Cartoon representation of two tetrahedral clusters (yellow and blue) showing their tight packing in polyhedra according to the I23 crystallographic translations. Symmetry elements are indicated as gray triangles and ellipses. The C-terminal (CT) loops project outward from the unit cell (red ribbon), and disulfide bonds stabilize the clusters (side chain represented as magenta spheres). (B) Surface representation of two unit cells with the CT loops highlighted in red. These cells pack as indicated by the arrow and interlock with extensive interactions mediated by the CT loop. This forms an extremely dense matrix with only a 30-Å wide cavity delimited by helices H1/2 and no continuous solvent channels. (C) Surface (Bottom) and ribbon (Top) representations of the anchoring interactions mediated by CT loops.

Fig. 5.

Comparison of baculovirus and cypovirus polyhedra. The distinct folds and trimeric organizations of nucleopolyhedrovirus and cypovirus polyhedrin proteins are shown in cartoon (A and D) and surface (B and E) representations. The cartoon representations of the tetrahedral clusters (C and F) highlight the different localizations and functions of the N-terminal helical regions of the two classes of polyhedrins. The central helices H1/2 stabilize the tetrahedral cluster in baculovirus polyhedra, whereas helices H1 extend away from the cluster in cypovirus polyhedra. Molecules are colored in a blue-to-red gradient from the N-terminus to the C-terminus (CT).

Fig. 6.

Molecular arms in baculovirus and cypovirus polyhedra. Surface representation of the interaction between a central trimer and a neighboring tetrahedral cluster in baculovirus (A) and cypovirus (B) polyhedra. The tetrahedral cluster is represented in gray except for the molecular arms clamping the central trimer, which are highlighted in green and indicated with black arrows. As described in the text, these arms are the C-terminus of baculovirus polyhedrin and the N-terminal helix of the cypovirus polyhedrin.