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 T‫3؍‬ 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-Å 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.Analysis of the three-dimensional structure of icosahedral viruses has provided insights into the function and biology of virus particles. The techniques used rely on the symmetry of the particles, and therefore less is known about the structure of the encapsidated genome. The nucleic acid is seen only where it is arranged with symmetry that correlates with that of the icosahedrally ordered protein shell. Nevertheless, a portion of the genome has been visualized in several icosahedral viruses with positive-sense RNA genomes (reviewed in reference 22). Although these viruses contain single-stranded genomes, the RNA visualized by X-ray crystallography in both Tϭ1 and Tϭ3 virus particles predominantly resembles A-form duplex RNA, suggesting that the conformation of the RNA encapsidated in these particles is influenced by factors other than the genome sequence. In the 3-Å X-ray crystallographic structure of Pariacoto virus (PaV), a Tϭ3 nodavirus, an unprecedented 1,500 nucleotides (nt) of the 4,322-nt genome were visible (27), providing an opportunity to examine the factors involved in ordering of the RNA.The structure and assembly of viruses from the Alphanodavirus genus of the Nodaviridae family have been studied extensively (reviewed in references 10 and 25). Nodaviruses are positive-sense RNA viruses that have 30-nm nonenveloped particles with Tϭ3 icosahedral symmetry. Each particle is assembled from 180 copies of a capsid protein precursor (alpha), which is autocatalytically cleaved following assembly into the two mature capsid proteins beta and gamma (7). A copy of each of the two genome segments, RNA1 (3 kb) and RNA2 (1.3 kb), are coencapsidated in each virus particle (16,21). Authentic nodavirus particles do not encap...

Infectious myonecrosis virus has a totivirus-like, 120-subunit capsid, but with fiber complexes at the fivefold axes

“…This pattern is remarkably similar to that of the single-stranded RNA genome of nodaviruses (28,29), except that in IMNV the pattern extends over multiple layers. In nodaviruses the pattern is proposed to reflect RNA-capsid interactions that promote both short-and long-range base pairing within the genome, resulting in a complex RNA path dominated by formation of duplex stems that interact with the capsid along the edges of the dodecahedron.…”

Construction and Crystal Structure of Recombinant STNV Capsids

“…If, for example, all ordered RNA sites were occupied with 3-bp stems topped by short tetra-loops, this would constitute 600 nt, or about half the length of the STNV genome. The structure reported here does not, of course, contain the native genome, but the synthetic gene is similar in secondary structure, 17 and it has been shown that Pariacoto Virus (PaV) 27 and Flock House Virus (FHV) 28 impose an architecture on encapsidated heterologous RNA similar to that observed for their genomic RNAs in the native viruses. RNA fragments (red sticks) found in the current X-ray map, superimposed on the neutron scattering density.…”

The T=1 Capsid Protein of Penicillium chrysogenum Virus Is Formed by a Repeated Helix-Rich Core Indicative of Gene Duplication

“…The size of the visualized genome is variable, and the ordered ssRNA is located at the icosahedral 3-fold or 2-fold axis of the virion. In Pariacoto virus (PaV), the ssRNA forms a dodecahedral cage in close contact with the protein shell that constitutes ϳ35% of the encapsidated RNA (31,68). Cryo-EM studies with rotaviruses also showed dsRNA organized into a dodecahedral shell beneath the protein capsid (37,52).…”