Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Abstract

Next-generation sequencing is a valuable tool in our growing understanding of the genetic diversity of viral populations. Using this technology, we have investigated the RNA content of a purified nonenveloped single-stranded RNA virus, flock house virus (FHV). We have also investigated the RNA content of virus-like particles (VLPs) of FHV and the related Nudaurelia capensis omega virus. VLPs predominantly package ribosomal RNA and transcripts of their baculoviral expression vectors. In addition, we find that 5.3% of the packaged RNAs are transposable elements derived from the Sf21 genome. This observation may be important when considering the therapeutic use of VLPs. We find that authentic FHV virions also package a variety of host RNAs, accounting for 1% of the packaged nucleic acid. Significant quantities of host messenger RNAs, ribosomal RNA, noncoding RNAs, and transposable elements are readily detected. The packaging of these host RNAs elicits the possibility of horizontal gene transfer between eukaryotic hosts that share a viral pathogen. We conclude that the genetic content of nonenveloped RNA viruses is variable, not just by genome mutation, but also in the diversity of RNA transcripts that are packaged.

Fig. 1.

Extraction of RNA from purified authentic FHV virions and VLPs of FHV and NωV. Particles of (A) FHV VLPs, (B) NωV VLPs, and (C) authentic FHV virions were visualized by negative-stain electron microscopy. Scale bars correspond to 50 nm. FHV VLPs form particles 30 nm in diameter that are indistinguishable from authentic FHV virions. NωV VLPs form 40 nm particles. RNA extracted from (D) FHV VLPs, (E) NωV VLPs, and (F) authentic FHV virions was visualized on a 0.8% nondenaturing agarose gel stained with ethidium bromide. FHV virions package two viral RNAs (RNA 1 = 3.1 kb, RNA 2 = 1.4 kb) as indicated. Molecular weights of VLP RNAs are indicated next to the gels.

Fig. 2.

Summaries of the alignments of reads from each dataset, grouped into functional RNA classes. (A) FHVVLP-RNAseq, (B) NωVVLP-RNAseq, and (C) FHVvirion-RNAseq. Color-coded: peach, viral RNA; green, mRNA; blue, rRNA; gold, transposon RNA; purple, ncRNA; and yellow, microsatellite markers from ref. . In A and B, assembled contigs that did not have significant homology to other known sequences are indicated as unknown. Dm, Drosophila melanogaster; FHV, flock house virus; Sf, Spodoptera frugiperda; AcMNPV, Autographa californica multiple nuclear polyhedrosis virus.

Fig. 3.

RT-PCR analysis confirms that full-length RNA transcripts are packaged inside authentic FHV virions. RNAs from each functional class were amplified and analyzed by gel electrophoresis: (A) ′1731′ retrotransposon ORF 1; (B) 18S rRNA; (C) 7SL ncRNA; and (D) enolase mRNA. Lane 1, molecular weight standard 1 Kb plus DNA ladder (Invitrogen); lane 2, amplified from DL-1 DNA (without reverse transcription); lane 3, amplified from DL-1 RNA; lane 4, amplified from RNA encapsidated by FHV virions; lane 5, same as lane 4 but without a reverse transcription step. The expected product lengths are indicated on the left of each gel.