The proline-rich region of the ecotropic Moloney murine leukaemia virus envelope protein tolerates the insertion of the green fluorescent protein and allows the generation of replication-competent virus.
Sequences encoding the green fluorescent protein (GFP) were inserted into the envelope protein (Env) of ecotropic Moloney murine leukaemia virus, MoMLV. Insertion of these sequences into the proline-rich region (PRR) of Env resulted in a chimeric GFP-Env protein that allowed retrovirus vector transduction of murine cells with titres similar to wild-type Env. However, N-terminal extension with GFP did not result in a functional Env protein. GFP sequences were then inserted into the Env PRR of E-MO virus, a MoMLV that carries epidermal growth factor sequences at the N terminus of its Env protein. The resulting virus, GFP-EMO1, replicates to the same titres as the parental virus. In a chronically infected cell culture, GFP-EMO1 was genetically stable. However, additional insertions of sequences that led to recombination or that may have been incompatible with virus replication were deleted and decreased virus titre. In summary, Env PRR can be used to tag individual virus particles with GFP, which leaves other regions available for modification in studies aimed at altering virus tropism.
Erlwein
Otto
O
Institute for Biomedical Research, Georg-Speyer Haus, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt/Main, Germany.
Buchholz
Christian J
CJ
Paul Ehrlich Institute, Medical Biotechnology, Paul-Ehrlich-Str. 51-59, D-63225 Langen, Germany.
Schnierle
Barbara S
BS
Institute for Biomedical Research, Georg-Speyer Haus, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt/Main, Germany.
D016475:3T3 Cells; D000818:Animals; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008979:Moloney murine leukemia virus / Q000235:genetics / Q000502:physiology*; D011392:Proline / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D049452:Green Fluorescent Proteins; D011392:Proline