Heterologous RNA encapsidated in Pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions

Abstract

The genome of some icosahedral RNA viruses plays an essential role in capsid assembly and structure. In T=3 particles of the nodavirus Pariacoto virus (PaV), a remarkable 35% of the single-stranded RNA genome is icosahedrally ordered. This ordered RNA can be visualized at high resolution by X-ray crystallography as a dodecahedral cage consisting of 30 24-nucleotide A-form RNA duplex segments that each underlie a twofold icosahedral axis of the virus particle and interact extensively with the basic N-terminal region of 60 subunits of the capsid protein. To examine whether the PaV genome is a specific determinant of the RNA structure, we produced virus-like particles (VLPs) by expressing the wild-type capsid protein open reading frame from a recombinant baculovirus. VLPs produced by this system encapsidated similar total amounts of RNA as authentic virus particles, but only about 6% of this RNA was PaV specific, the rest being of cellular or baculovirus origin. Examination of the VLPs by electron cryomicroscopy and image reconstruction at 15.4-A resolution showed that the encapsidated RNA formed a dodecahedral cage similar to that of wild-type particles. These results demonstrate that the specific nucleotide sequence of the PaV genome is not required to form the dodecahedral cage of ordered RNA.

FIG. 1.

Analysis of VLPs produced from bCAP in Se-1 cells. (A) Electron cryomicrographs of gradient purified PaV and VLPs recorded at underfocus of 2.9 and 2.6 μm, respectively. The scale bar represents 500 Å. (B) Protein composition of wt PaV and VLPs. The proteins in purified particles were resolved by electrophoresis on an SDS-10% polyacrylamide gel and visualized by Coomassie blue staining. The wt PaV capsid proteins are indicated on the left (the cleaved form of capsid protein initiated at Met25 is indicated as 25 beta), and molecular mass markers are shown to the right in kilodaltons.

FIG. 2.

Ultracentrifugation analysis of radiolabeled PaV and VLPs. Particles were labeled with either [³⁵S]Met-Cys (VLPs) or [³H]uridine (PaV) and purified by pelleting through 30% sucrose cushions. The particles were mixed and sedimented through a 15 to 45% sucrose gradient (A) or centrifuged to equilibrium on a CsCl gradient (B). Thirty fractions were collected, and the radioactive counts in each fraction were determined. The top of each gradient is on the left.

FIG. 3.

Analysis of encapsidated RNAs. Duplicate samples of RNA extracted from purified PaV (lanes 2 and 6), VLPs (lanes 3 and 7), or Se-1 cells that were mock infected (lanes 4 and 8) or infected with bCAP (lanes 5 and 9) were resolved by electrophoresis on a denaturing 1% agarose-formaldehyde gel. The amount of RNA loaded was 1 μg in lanes 2 and 3; 2 μg in lanes 4 and 5; 50 ng in lanes 6 and 7; and 500 ng in lanes 8 and 9. The gel was divided in two, and RNA was visualized by staining with Sybr-gold (A) or was transferred to Nytran nylon membranes and hybridized with probes for PaV RNA1 and -2 (B). The sizes of RNA markers (lane 1) and the positions of PaV RNA1 and -2 are shown to the left of panels A and B, respectively.

FIG. 4.

Quantitation of RNA2 sequences encapsidated in VLPs. RNA was extracted from purified PaV and VLPs and was analyzed by RNase protection. (A) RNAs were quantitated by spectrometry and annealed with an excess of a ³²P-labeled RNA2 probe. In the absence of template (lane 7) the probe was completely digested by RNase A/T1, and in the absence of RNase the probe remained intact (lane 8). The protected RNA in 20-, 30-, 40-, and 50-ng samples of wt virion RNA (lanes 1 to 4) was compared to that protected in 100-ng samples of VLP RNA (lanes 5 and 6). A phosphorimage of the dried gel is shown. (B) Quantitation of the protected RNAs from the gel shown in panel A is shown graphically in panel B. Each circle represents a data point from which the standard curve line was calculated. The black diamond represents the value obtained for protected RNA in 100 ng of VLP RNA (the average of the samples in lanes 5 and 6). The dotted line shows the intercept with the standard curve and an estimate for the equivalent amount of RNA2 sequences protected in wt virus RNA. Data pooled from this and several other experiments indicated that VLPs contained 22% (±1.4%) of the RNA2 content of wt PaV particles.

FIG. 5.

Three-dimensional reconstructed images of wt PaV and VLPs. CryoEM maps of PaV and VLP are shown for the total densities (A to C) and difference maps (D to F) generated by subtraction of the capsid protein density as determined by the crystal structure of PaV. The PaV reconstructions are shown at 23-Å resolution (A and D) (27), and the VLP map was determined at 15.4-Å resolution (C and F) and is also shown computationally filtered at 22 Å (B and E). The RNA cage identified in wt PaV is also visible in the VLPs.

FIG. 6.

Superimposition of VLP and wt PaV RNA. The 15.4-Å VLP difference map (semitransparent) is shown superimposed on the model of the RNA cage (blue ribbons) from the PaV crystal structure (27).