Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors
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Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors. / Bahrami, Shervin; Jespersen, Thomas; Pedersen, Finn Skou; Duch, Mogens R.; Jespersen, Thomas.
In: Gene, Vol. 315, 02.10.2003, p. 51-61.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Mutational library analysis of selected amino acids in the receptor binding domain of envelope of Akv murine leukemia virus by conditionally replication competent bicistronic vectors
AU - Bahrami, Shervin
AU - Jespersen, Thomas
AU - Pedersen, Finn Skou
AU - Duch, Mogens R.
AU - Jespersen, Thomas
PY - 2003/10/2
Y1 - 2003/10/2
N2 - The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV) and the neomycin phosphotransferase II (Neo) selection marker from the same transcript. Envelope expression was achieved by inserting an internal ribosome entry site (IRES) between the neo and the env genes. We found the structure of the linker between the IRES element and env to be critical for sufficient envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells indicated that the wild type aspartic acid or another hydrophilic residue at position 86 is an important determinant for envelope function.
AB - The envelope protein of retroviruses is responsible for viral entry into host cells. Here, we describe a mutational library approach to dissect functional domains of the envelope protein involving a retroviral vector, which expresses both the envelope protein of Akv murine leukemia virus (MLV) and the neomycin phosphotransferase II (Neo) selection marker from the same transcript. Envelope expression was achieved by inserting an internal ribosome entry site (IRES) between the neo and the env genes. We found the structure of the linker between the IRES element and env to be critical for sufficient envelope expression. This vector functions as a replication competent mini-virus in a culture of NIH 3T3 derived semi-packaging cells that express the viral Gag and Pol proteins. Titers comparable to those of wild type virus were achieved by this system. To test this vector system, we created a random mutational library of Arg 85 and Asp 86 in the first variable region of Akv envelope protein. Homologous amino acids to Asp 86 in Moloney and Friend murine leukemia viruses are thought to be directly involved in receptor binding. Subsequent selection of mutants capable of infecting murine NIH 3T3 cells indicated that the wild type aspartic acid or another hydrophilic residue at position 86 is an important determinant for envelope function.
KW - Amino Acid Sequence
KW - Amino Acids
KW - Animals
KW - Arginine
KW - Aspartic Acid
KW - Base Sequence
KW - Binding Sites
KW - Cell Line
KW - Codon
KW - Gene Library
KW - Gene Products, gag
KW - Gene Products, pol
KW - Genes, Viral
KW - Genetic Vectors
KW - Humans
KW - Leukemia Virus, Murine
KW - Mice
KW - Mutation
KW - NIH 3T3 Cells
KW - Protein Structure, Tertiary
KW - Receptors, Virus
KW - Transfection
KW - Viral Envelope Proteins
KW - Viral Structural Proteins
KW - Virus Replication
M3 - Journal article
C2 - 14557064
VL - 315
SP - 51
EP - 61
JO - Gene
JF - Gene
SN - 0378-1119
ER -
ID: 33017769