Specific Packaging of Nodaviral RNA2 Requires the N-Terminus of the Capsid Protein

Abstract

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

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

“…To distinguish between these two possibilities, we decided to take advantage of a capsid protein mutant (⌬31) that lacks N-terminal residues 2 through 31. This mutant is able to package RNA1 independently of RNA2 in FHV-infected Drosophila cells (17). Thus, we reasoned that this mutant would package RNA1 in the absence of replicating RNA2 unless RNA2 replication was key to packaging FHV RNAs.…”

“…The absence of significant complementarity between RNA1 and RNA2, however, does not support this model. Moreover, it was recently shown that RNA1 can be packaged in the absence of RNA2 by a capsid protein variant that lacks N-terminal residues 2 through 31, further suggesting that a noncovalent complex between RNA1 and RNA2 is not formed prior to packaging (17). An alternate hypothesis is that the packaging of genomic RNAs occurs in a sequential manner, similar to what has been described for bacteriophage 6 (19).…”

Morphological Changes in the T=3 Capsid of Flock House Virus during Cell Entry

“…After incubation for 4 h at 27°C, radiolabeled virus was purified as described above. [ 3 H]uridinelabeled FHV was generated and purified as previously described (30).…”

“…Drosophila cells were transfected with FHV RNA1 and RNA2 using Cytofectene (Bio-Rad) as a transfection reagent. Details of this procedure are described elsewhere (30).…”

“…FHV was purified as previously described (30). Briefly, infected or transfected cells were lysed with NP-40, and cell debris was removed by low-speed centrifugation.…”