Kits comprising plus-sense single stranded rna viral vectors and methods for producing polypeptides using the kits

A mixture comprising 2, 3, or more different vectors of the following vectors a) to c):

• a) at least one first plus-sense single stranded RNA viral vector,
• b) at least one second plus-sense single stranded RNA viral vector,
• C) at least one third and/or additional plus-sense single stranded RNA viral vector,
• wherein
• (A)
• (i) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
• (ii) the coat protein ORF of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
• (iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
• (iv) the first plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and
• (v) the second plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived, and
• (vi) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector comprise an RNA replicon which is able to replicate in plant cells, and
• wherein
• (B)
• (i) the at least one third plus-sense single-stranded viral vector(s) comprise(s) at least one functional heterologous ORF, and
• (ii) the at least one third plus-sense single-stranded viral vector(s) is/are derived from the same plant virus as the first or second plus-sense single-stranded viral vector, and
• (iii) the coat protein ORF of the virus from which the at least one third plus-sense single-stranded viral vector(s) are derived is completely deleted in the at least one third plus-sense single stranded RNA viral vector(s), and
• wherein
• (C)
• (ii) the at least one additional plus-sense single-stranded viral vector(s) comprise(s) at least one functional heterologous ORF, and
• (ii) the at least one additional plus-sense single-stranded viral vector(s) is/are derived from the same plant virus as the first or second plus-sense single-stranded viral vector.

A plant or plant cell

• a) comprising at least one first plus-sense single stranded RNA viral vector and at least one second plus-sense single stranded RNA viral vector, and optionally at least one third and/or additional plus-sense single stranded RNA viral vector(s),
• wherein
• (A)
• (i) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
• (ii) the coat protein ORF of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
• (iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
• (iv) the first plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and
• (v) the second plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived, and
• (vi) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector comprise an RNA replicon which is able to replicate in plant cells, and
• wherein
• (B)
• (i) the at least one third plus-sense single-stranded viral vector(s) comprise(s) at least one functional heterologous ORF, and
• (ii) the at least one third plus-sense single-stranded viral vector(s) is/are derived from the same plant virus as the first or second plus-sense single-stranded viral vector, and
• (iii) the coat protein ORF of the virus from which the at least one third plus-sense single-stranded viral vector(s) are derived is completely deleted in the at least one third plus-sense single stranded RNA viral vector(s), and
• wherein
• (C)
• (i) the at least one additional plus-sense single-stranded viral vector(s) comprise(s) at least one functional heterologous ORF, and
• (ii) the at least one additional plus-sense single-stranded viral vector(s) is/are derived from the same plant virus as the first or second plus-sense single-stranded viral vector.

A method for producing in a plant, or plant tissue, or plant cell a heterooligomeric polypeptide and/or two or more polypeptides, comprising providing to at least one plant cell

• (x) at least one first plus-sense single stranded RNA viral vector, and
• (y) at least one second plus-sense single stranded RNA viral vector, and
• (z) optionally at least one third and/or additional plus-sense single stranded RNA viral vector,
• wherein
• (i) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector are derived from different plant viruses, and
• (ii) the coat protein ORF of the virus from which the first vector is derived is completely deleted in the first plus-sense single stranded RNA viral vector, and
• (iii) the coat protein ORF of the virus from which the second vector is derived is completely deleted in the second plus-sense single stranded RNA viral vector, and
• (iv) the first plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the second plus-sense single-stranded viral vector is derived, and
• (v) the second plus-sense single stranded RNA viral vector comprises a functional coat protein ORF of the virus from which the first plus-sense single-stranded viral vector is derived, and
• (vi) the first plus-sense single-stranded viral vector and the second plus-sense single-stranded viral vector comprise an RNA replicon which is able to replicate in plant cells, and
• wherein
• (B)
• (i) the at least one third plus-sense single-stranded viral vector(s) comprise(s) at least one functional heterologous ORF, and
• (ii) the at least one third plus-sense single-stranded viral vector(s) is/are derived from the same plant virus as the first or second plus-sense single-stranded viral vector, and
• (iii) the coat protein ORF of the virus from which the at least one third plus-sense single-stranded viral vector(s) are derived is completely deleted in the at least one third plus-sense single stranded RNA viral vector(s), and
• wherein
• (C)
• (i) the at least one additional plus-sense single-stranded viral vector(s) comprise(s) at least one functional heterologous ORF, and
• (ii) the at least one additional plus-sense single-stranded viral vector(s) is/are derived from the same plant virus as the first or second plus-sense single-stranded viral vector,
• and further wherein
• a) at least two third plus-sense single stranded RNA viral vectors are provided to at least one plant cell if the first plus-sense single stranded RNA viral vector and the second plus-sense single stranded RNA viral vector do not comprise a functional heterologous ORF, and
• b) wherein at least two of the viral vectors according to (x) to (z) comprise different heterologous ORFs.

A method for producing in a plant, or plant tissue, or plant cell a heterooligomeric polypeptide and/or two or more polypeptides, comprising providing to a plant, or plant tissue, or plant cell at least two plus-sense single stranded RNA viral vectors,

• a) wherein the at least two plus-sense single stranded RNA viral vectors are derived from the same virus, and
• b) wherein at least two plus-sense single stranded RNA viral vectors comprise different functional heterologous ORFs, and
• c) wherein the at least two plus-sense single stranded RNA viral vectors are devoid of the functional coat protein ORF of the virus from which the vectors are derived, and
• d) wherein the at least two plus-sense single stranded RNA viral vectors comprise an RNA replicon which is able to replicate in plant cells.

A mixture of Agrobacterium cells, in particular Agrobacterium tumefaciens cells,

• a) wherein a plurality of Agrobacterium cells comprise at least one plus-sense single stranded RNA viral vector comprising at least one functional heterologous ORF,
• i) wherein the at least one plus-sense single stranded RNA viral vector is devoid of the functional coat protein ORF, and
• ii) wherein the at least one plus-sense single stranded RNA viral vector comprises an RNA replicon which is able to replicate in plant cells, and
• b) wherein at least two Agrobacterium cells comprise different plus-sense single stranded RNA vectors comprising different functional heterologous ORF, and
• c) wherein the different plus-sense single stranded RNA vectors comprise different functional heterologous ORFs are derived from the same virus,
• preferably
• i) wherein the virus from which the plus-sense single stranded RNA viral vectors are derived is a Potexvirus, in particular PVX virus (potato virus X), and/or
• ii) wherein the coat protein ORF of the virus from which the plus-sense single stranded RNA viral vectors are derived, is completely deleted in the plus-sense single stranded RNA viral vectors.

A PVX virus vector, wherein

• a) the functional coat polypeptide ORF of the PVX virus is completely deleted, and
• b) the PVX viral vector comprises an RNA replicon which is able to replicate in plant, cells, and
• c) the PVX virus vector optionally comprises at least one functional heterologous ORF, and
• d) the PVX virus vector optionally comprises a functional coat polypeptide ORF of a different virus.