Investigating the Biological Relevance of In Vitro-Identified Putative Packaging Signals at the 5' Terminus of Satellite Tobacco Necrosis Virus 1 Genomic RNA

Abstract

Satellite tobacco necrosis virus 1 (STNV-1) is a model system for in vitro RNA encapsidation studies (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114), leading to the identification of degenerate packaging signals (PSs) proposed to be involved in the recognition of its genome by the capsid protein (CP). The aim of the present work was to investigate whether these putative PSs can confer selective packaging of STNV-1 RNA in vivo and to assess the prospects of using decoy RNAs in antiviral therapy. We have developed an in planta packaging assay based on the transient expression of STNV-1 CP and have assessed the ability of the resulting virus-like particles (VLPs) to encapsidate mutant STNV-1 RNAs expected to have different encapsidation potential based on in vitro studies. The results revealed that >90% of the encapsidated RNAs are host derived, although there is some selectivity of packaging for STNV-1 RNA and certain host RNAs. Comparison of the packaging efficiencies of mutant STNV-1 RNAs showed that they are encapsidated mainly according to their abundance within the cells, rather than the presence or absence of the putative PSs previously identified from in vitro studies. In contrast, subsequent infection experiments demonstrated that host RNAs represent only <1% of virion content. Although selective encapsidation of certain host RNAs was noted, no direct correlation could be made between this preference and the presence of potential PSs in the host RNA sequences. Overall, the data illustrate that the differences in RNA packaging efficiency identified through in vitro studies are insufficient to explain the specific packaging of STNV-1 RNA.IMPORTANCE Viruses preferentially encapsidate their own genomic RNA, sometimes as a result of the presence of clearly defined packaging signals (PSs) in their genome sequence. Recently, a novel form of short degenerate PSs has been proposed (N. Patel, E. C. Dykeman, R. H. A. Coutts, G. P. Lomonossoff, et al., Proc Natl Acad Sci U S A 112:2227-2232, 2015, https://doi.org/10.1073/pnas.1420812112; N. Patel, E. Wroblewski, G. Leonov, S. E. V. Phillips, et al., Proc Natl Acad Sci U S A 114:12255-12260, 2017, https://doi.org/10.1073/pnas.1706951114) using satellite tobacco necrosis virus 1 (STNV-1) as a model system for in vitro studies. It has been suggested that competing with these putative PSs may constitute a novel therapeutic approach against pathogenic single-stranded RNA viruses. Our work demonstrates that the previously identified PSs have no discernible significance for the selective packaging of STNV-1 in vivo in the presence and absence of competition or replication: viral sequences are encapsidated mostly on the basis of their abundance within the cell, while encapsidation of host RNAs also occurs. Nevertheless, the putative PSs identified in STNV-1 RNA may still have applications in bionanotechnology, such as the in vitro selective packaging of RNA molecules.

Keywords: Nicotiana benthamiana; STNV-1; TNV-A; agroinfiltration; encapsidation; infection; packaging signals.