Analysis of the functional and host range-determining regions of the murine ectropic and amphotropic retrovirus envelope proteins.
A series of Moloney murine leukemia virus (Mo-MuLV) envelope gene constructs were analyzed for biological activity. Three classes of recombinant envelopes were examined: insertions, deletions, and chimeras. Insertion (4 to 5 amino acids) and deletion (31 to 62 amino acids) mutants spanned most of the SU (gp70)-coding region and were all biologically inactive. Radioimmunoprecipitation demonstrated that the mutant envelope proteins were incorrectly processed. The Pr80env envelope precursor proteins failed to obtain the proper posttranslational modifications and were not cleaved into SU (gp70) and TM (p15E), suggesting that disruption of Pr80env structure prevents intracellular transport and processing. To analyze the functional domains of the SU portion of the Env protein, we assembled several chimeric constructs. In these constructs, portions of the ecotropic Mo-MuLV envelope gene were replaced with corresponding sequences from the 4070A amphotropic MuLV envelope. Using a retroviral vector pseudotyping assay, 5 of 12 chimeric envelope proteins were shown to be biologically active. Host range was determined by retroviral vector transduction of the appropriate cell, by viral interference studies, and by the productive infection of Chinese hamster ovary cells expressing the murine ecotropic receptor. These results permit assignment of the amino acids responsible for host range determination. Ecotropic host range is determined by the first 88 amino acids of the Mo-MuLV SU, while the amphotropic host range-determining region spans the first 157 amino acids of the 4070A SU.
Morgan
R A
RA
Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Nussbaum
O
O
Muenchau
D D
DD
Shu
L
L
Couture
L
L
Anderson
W F
WF
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003673:Defective Viruses / Q000235:genetics* / Q000378:metabolism; D015686:Gene Products, env / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015752:Genes, env; D051379:Mice; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017384:Sequence Deletion; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D015686:Gene Products, env; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D001616:beta-Galactosidase