Encapsidation of Host RNAs by Cucumber Necrosis Virus Coat Protein during both Agroinfiltration and Infection.
Next-generation sequence analysis of virus-like particles (VLPs) produced during agroinfiltration of cucumber necrosis virus (CNV) coat protein (CP) and of authentic CNV virions was conducted to assess if host RNAs can be encapsidated by CNV CP. VLPs containing host RNAs were found to be produced during agroinfiltration, accumulating to approximately 1/60 the level that CNV virions accumulated during infection. VLPs contained a variety of host RNA species, including the major rRNAs as well as cytoplasmic, chloroplast, and mitochondrial mRNAs. The most predominant host RNA species encapsidated in VLPs were chloroplast encoded, consistent with the efficient targeting of CNV CP to chloroplasts during agroinfiltration. Interestingly, droplet digital PCR analysis showed that the CNV CP mRNA expressed during agroinfiltration was the most efficiently encapsidated mRNA, suggesting that the CNV CP open reading frame may contain a high-affinity site or sites for CP binding and thus contribute to the specificity of CNV RNA encapsidation. Approximately 0.09% to 0.7% of the RNA derived from authentic CNV virions contained host RNA, with chloroplast RNA again being the most prominent species. This is consistent with our previous finding that a small proportion of CNV CP enters chloroplasts during the infection process and highlights the possibility that chloroplast targeting is a significant aspect of CNV infection. Remarkably, 6 to 8 of the top 10 most efficiently encapsidated nucleus-encoded RNAs in CNV virions correspond to retrotransposon or retrotransposon-like RNA sequences. Thus, CNV could potentially serve as a vehicle for horizontal transmission of retrotransposons to new hosts and thereby significantly influence genome evolution.


OBJECTIVE
Viruses predominantly encapsidate their own virus-related RNA species due to the possession of specific sequences and/or structures on viral RNA which serve as high-affinity binding sites for the coat protein. In this study, we show, using next-generation sequence analysis, that CNV also encapsidates host RNA species, which account for ∼0.1% of the RNA packaged in CNV particles. The encapsidated host RNAs predominantly include chloroplast RNAs, reinforcing previous observations that CNV CP enters chloroplasts during infection. Remarkably, the most abundantly encapsidated cytoplasmic mRNAs consisted of retrotransposon-like RNA sequences, similar to findings recently reported for flock house virus (A. Routh, T. Domitrovic, and J. E. Johnson, Proc Natl Acad Sci U S A 109:1907-1912, 2012). Encapsidation of retrotransposon sequences may contribute to their horizontal transmission should CNV virions carrying retrotransposons infect a new host. Such an event could lead to large-scale genomic changes in a naive plant host, thus facilitating host evolutionary novelty.
University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada.
Kankana
K
Ghoshal
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada.
Jane
J
Theilmann
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada.
Ron
R
Reade
Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada.
Ajay
A
Maghodia
University of British Columbia, Faculty of Land and Food Systems, Vancouver, British Columbia, Canada Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, Summerland, British Columbia, Canada dann.rochon@agr.gc.ca.
D'Ann
D
Rochon
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D022181:COP-Coated Vesicles / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D018553:Cucumis sativus / Q000821:virology*; D004591:Electrophoresis, Polyacrylamide Gel; D019143:Evolution, Molecular; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D018749:RNA, Plant / Q000378:metabolism*; D018626:Retroelements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D017864:Tombusvirus / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000648:ultrastructure
D018749:RNA, Plant; D018626:Retroelements