Complementary RNA Viral Vectors for Plant Protein Co-Expression
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Solution Overview
Problem
The challenge in producing complex proteins using viral vectors in plants lies in the spatial segregation of different viral vectors during infection, making it difficult to express multiple proteins simultaneously in the same cell, particularly when using vectors derived from the same genome, which also poses biosafety concerns due to potential environmental contamination.
Innovation Solution
A kit comprising two plus-sense single-stranded RNA viral vectors derived from different plant viruses, where the coat protein ORF of one vector is deleted and replaced with the functional coat protein ORF of the other, allowing systemic co-infection and co-expression while ensuring containment by preventing solo systemic infection of healthy plants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If two viral vectors derived from the same genome are co-inoculated in one plant, then high yields of multiple proteins can be produced, but the vectors segregate spatially and cannot co-express in the same cell
Solution Approach 1:
The invention divides the viral vector system into two complementary segments: Vector 1 contains gene A but lacks coat protein, while Vector 2 contains gene B but lacks replicase. Neither vector alone can complete the replication cycle, but together they enable simultaneous expression of multiple proteins in the same cell without spatial segregation
Solution Approach 2:
The invention introduces a complementary viral vector system where each vector provides functions that the other lacks. Vector 1 provides the coat protein function that Vector 2 needs, while Vector 2 provides the replicase function that Vector 1 needs, enabling both vectors to co-exist and co-express in the same plant cells
2Productivity
If complete viral genomes with capability to infect plants systemically are used, then high protein production is achieved, but environmental contamination and biosafety risks increase
Solution Approach 1:
The invention extracts and removes the harmful systemic infection capability from the viral vectors by deleting essential genes (coat protein in Vector 1, replicase in Vector 2). This eliminates the ability of individual vectors to cause environmental contamination while preserving the ability to produce recombinant proteins when both vectors are present together
Solution Approach 2:
The invention converts the potential harm of systemic infection into a benefit by using the requirement for both vectors to enable controlled protein production. The very genetic deletions that prevent systemic infection also ensure that only the intended recombinant proteins are produced when both vectors are co-introduced, eliminating unwanted viral propagation
3Adaptability or versatility
If multiple non-competitive viral vectors are used to produce complex proteins, then protein diversity increases, but the burden on the infected plant increases
Solution Approach 1:
The invention merges the essential viral functions (replicase, coat protein, movement proteins) into a shared complementary system between two vectors. This allows multiple complex proteins to be produced using only two viral vector constructs rather than requiring separate complete viral genomes for each protein, reducing the overall burden on the infected plant
Data Source
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AI summary
The present invention relates to kits comprising plus-sense single stranded RNA viral vectors, as well as mixtures of these vectors and uses thereof, and methods for producing in a plant, or plant tissue, or plant cell simultaneously two or more polypeptides using the kits and vectors.