Analysis of RNA Packaging in Wild-Type and Mosaic Protein Capsids of Flock House Virus Using Recombinant Baculovirus Vectors

Tang

et al. 2004

J Virol

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...

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions

Tang

et al. 2004

J Virol

“…The construction of DNA transfer vectors for the generation of recombinant baculoviruses AcR1␦ and AcR2␦ with the BacPAK expression system kit (BD Biosciences) was previously described (13). These vectors contain the cDNA of FHV RNA1 and RNA2, respectively, with the hepatitis delta virus (HDV) ribozyme sequence (22) (2), in which the cDNA of RNA1 is fused to the HDV ribozyme sequence at its 3Ј end, was utilized.…”

Section: Methodsmentioning

confidence: 99%

“…We recently showed that FHV replication can be initiated in Sf21 cells from recombinant baculovirus vectors (13). Specifically, we demonstrated that the coinfection of Sf21 cells with baculoviruses containing the cDNA for RNA1 and RNA2 launches self-directed RNA replication that leads to the formation of progeny FHV particles.…”

mentioning

confidence: 99%

Capsid Protein Synthesis from Replicating RNA Directs Specific Packaging of the Genome of a Multipartite, Positive-Strand RNA Virus

Venter

Krishna

Schneemann

2005

J Virol

Self Cite

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.Flock house virus (FHV), a member of the Nodaviridae family, is a nonenveloped, icosahedral insect virus whose genome consists of two positive-strand RNA molecules (3, 28). The bipartite nature of the FHV genome organizes its nonstructural and structural genes onto RNA1 (3.1 kb) and RNA2 (1.4 kb),respectively. An open reading frame (ORF) that nearly spans the length of RNA1 encodes protein A (112 kDa), the RNAdependent RNA polymerase which directs the replication of RNA1, RNA2, and a 387-nucleotide (nt) subgenomic RNA3 (7). RNA3 corresponds to the 3Ј end of RNA1 and encodes two small proteins, B1 and B2 (10). No function has been ascribed to B1, while B2 functions as an inhibitor of RNA silencing (15). Interestingly, RNA3 is also required as a transactivator of RNA2 replication, but the mechanism by which this is achieved is not completely understood (6). RNA2 encodes the 43-kDa capsid precursor protein ␣, which is the only structural protein required for the assembly of FHV provirions (7, 9). Each provirion consists of 180 subunits of protein ␣, arranged with Tϭ3 quasiequivalent symmetry, and the two genomic RNAs. Provirions are not infectious and undergo an autocatalytic maturation process in which protein ␣ cleaves into proteins ␤ (38 kDa) and ␥ (5 kDa). Both cleavage products remain associated with the matured, infectious virion (9, 26).FHV RNA1 and RNA2 are packaged into a single virion but the mechanism by which FHV copackages its multipartite genome is still unknown (14). One hypothesis is that an interaction between RNA1 and RNA2 occurs prior to packaging, allowing the genome to be encapsidated as a complex. The absence of significant complementarity between RNA1 and RNA2, however, does ...

Methods in Molecular Biology

Production and Application of Insect Virus-Based VLPs

Gopal

Schneemann

2018

Virus-like particles (VLPs) are self-assembling platforms composed of viral structural proteins. They are used for a variety of purposes, ranging from the study of virus assembly to vaccine development. VLPs can be produced in plants, bacteria, yeast, and insect and mammalian cells. The baculovirus expression system is one of the most commonly used systems for production of VLPs in eukaryotic cells. This chapter provides a brief overview of the main strategies used to generate recombinant baculoviruses and the applications of insect virus-derived VLPs in basic and applied research. It then describes detailed protocols for generation of recombinant baculoviruses, screening for their expression of VLPs in insect cells, and VLP purification.