Adaptive mutation in nuclear export protein allows stable transgene expression in a chimaeric influenza A virus vector.
The development of influenza virus vectors with long insertions of foreign sequences remains difficult due to the small size and instable nature of the virus. Here, we used the influenza virus inherent property of self-optimization to generate a vector stably expressing long transgenes from the NS1 protein ORF. This was achieved by continuous selection of bright fluorescent plaques of a GFP-expressing vector during multiple passages in mouse B16f1 cells. The newly generated vector acquired stability in IFN-competent cell lines and in vivo in murine lungs. Although improved vector fitness was associated with the appearance of four coding mutations in the polymerase (PB2), haemagglutinin and non-structural (NS) segments, the stability of the transgene expression was dependent primarily on the single mutation Q20R in the nuclear export protein (NEP). Importantly, a longer insert, such as a cassette of 1299 nt encoding two Mycobacterium tuberculosis Esat6 and Ag85A proteins, could substitute for the GFP transgene. Thus, the inherent property of the influenza virus to adapt can also be used to adjust a vector backbone to give stable expression of long transgenes.
Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.
Irina
I
Kuznetsova
Research Institute of Influenza, Russian Academy of Medical Sciences, Moscow, Russia.
Anna-Polina
AP
Shurygina
Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.
Brigitte
B
Wolf
Avir Green Hills Biotechnology AG, Vienna, Austria.|Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.
Markus
M
Wolschek
Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.
Florian
F
Enzmann
Research Institute for Biological Safety Problems, Gvardeyskyi, Kazakhstan.
Abylay
A
Sansyzbay
Research Institute for Biological Safety Problems, Gvardeyskyi, Kazakhstan.
Berik
B
Khairullin
Research Institute for Biological Safety Problems, Gvardeyskyi, Kazakhstan.
Nurlan
N
Sandybayev
Research Institute of Influenza, Russian Academy of Medical Sciences, Moscow, Russia.
Marina
M
Stukova
Research Institute of Influenza, Russian Academy of Medical Sciences, Moscow, Russia.
Oleg
O
Kiselev
Research Institute of Influenza, Russian Academy of Medical Sciences, Moscow, Russia.
Andrej
A
Egorov
Comprehensive Cancer Center, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.|Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090, Vienna, Austria.
Michael
M
Bergmann
D021581:Active Transport, Cell Nucleus; D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009980:Influenza A virus / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019562:Viral Load; D017361:Viral Nonstructural Proteins / Q000235:genetics* / Q000378:metabolism*; D014779:Virus Replication
C085820:INS1 protein, influenza virus; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D049452:Green Fluorescent Proteins