Type I interferons (IFNs) are potent mediators of the innate immune response to viral infection. IFNs released from infected cells bind to a receptor (IFNAR) on neighboring cells, triggering signaling cascades that limit further infection. Subtle variations in amino acids can alter IFNAR binding and signaling outcomes. We used a new gene crossbreeding method to generate hybrid, type I human IFNs with enhanced antiviral activity against four dissimilar, highly pathogenic viruses. Approximately 1400 novel IFN genes were expressed in plants, and the resultant IFN proteins were screened for antiviral activity. Comparing the gene sequences of a final set of 12 potent IFNs to those of parent genes revealed strong selection pressures at numerous amino acids. Using three-dimensional models based on a recently solved experimental structure of IFN bound to IFNAR, we show that many but not all of the amino acids that were highly selected for are predicted to improve receptor binding.
Oilseed rape mosaic virus (ORMV) is a tobamovirus taxonomically distinct from the type member of the genus, Tobacco mosaic virus (TMV). Both viruses display a specific host range, although they share certain hosts, such as Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum, on which they induce different symptoms. Using a gain-of-symptom approach, we generated chimeric viruses, starting from a TMV infectious clone, over which different regions of ORMV were exchanged with their corresponding regions in the TMV genome. This approach allowed the association of pathogenicity determinants to certain genes within the ORMV genome. A general trend was observed associating the viral origin of the RNA-dependent RNA-polymerase (RdRp) gene and the gain of symptoms. In A. thaliana and N. benthamiana, chimeric viruses were unable to reproduce the symptoms induced by the parental viruses, leading to disease states which could be described as intermediate, and variable in some cases. In contrast, a hypersensitive reaction caused by both of these viruses on N-gene-bearing tobaccos could be found in resistance reactions to all chimeric viruses, suggesting that the avirulence determinant maps similarly in both viruses. A systemic necrotic spotting typical of non-N-gene tobaccos infected with ORMV was associated with the polymerase domain of RdRp. To our knowledge, this is the first time that this controversial portion of the tobamovirus genome has been identified directly as a pathogenicity determinant. None of the reactions of the chimeric viruses could be correlated with increases or decreases in virus titres in the infections.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.