Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha

Abstract

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.

Nucleotide-Resolution Profiling of RNA Recombination in the Encapsidated Genome of a Eukaryotic RNA Virus by Next-Generation Sequencing

“…Data acquisition FHV virions and VLPs were generated in cell culture and purified with multiple sucrose gradients as is well established for structural and biochemical studies. 30 We thus obtained highly purified viral RNA for RNAseq analysis by virtue of its encapsidation inside FHV virions and VLPs. We did not employ any additional RNA-selection steps, such as PCR or poly-A capture, which would yield a non-random population of RNA molecules.…”

Role of Mitochondrial Membrane Spherules in Flock House Virus Replication

“…Total RNA generated in polymerase assays was treated with micrococcal nuclease (New England Bio-Labs), RNase H (New England BioLabs), or RNase A (Roche Diagnostics) according to the manufacturers' instructions. In addition, total RNA products were resolved alongside ssRNA1 and ssRNA2 generated by in vitro transcription as previously described (24) in the presence of 0.1 mM [␣-33 P]CTP.…”

Mapping RNA-capsid interactions and RNA secondary structure within authentic virus particles using next-generation sequencing

“…Furthermore, U2515 and U2576 were located in the subgenomic promoter region (47, 58) which are also adjacent to a RNA 1 internal cis-acting replication element (intRE, nts 2322-2501) (47). Similarly, on RNA 2 ( Figure 8b ), we noticed PAR-CL site U534 is adjacent to a RNA 2 cis-acting regulatory site (59), and U1155 which is within a site required for specific packaging of both RNAs (18). A previously predicted stem loop site (nts 168-249) on RNA 2 serves as a RNA 2 packaging signal (24).…”

“… (a) On RNA 1, four highly conserved defective interfering RNA regions (DI regions 1-4)(34) were shown in pink; four RNA 1 replication regulatory elements were shown in orange (5’Cis (46), 5’intRE(47), 3’intRE(47), and (67)); two RNA 3 regulatory elements and a putative RNA 3 subgenomic promoter region were shown in green (RNA3cis(dis) (47), RNA3cis(prox)(47)); the candidate PAR-CL sites (listed in Figure 4e) were shown as red bars on bottom. (b) On RNA 2, three conserved defection interfering RNA regions were shown in pink (DI regions 1-3 (34)); a mid-genome cis-acting replicational element(59) and a 3’ cis-acting regulatory element(67) were shown in brown; a RNA 2 packaging signal (24) and a capsid site essential for RNA 1&2 specificity (18) are shown in green.…”