Coexpression of human immunodeficiency virus envelope proteins and tat from a single simian virus 40 late replacement vector.
A Sal I-Xho I fragment containing the genes encoding tat, art, and the envelope proteins from the BH10 clone of human immunodeficiency virus (HIV) was inserted into a simian virus 40 (SV40)-based eukaryotic expression vector. The vector is a shuttle vector that replicates to high copy numbers in both Escherichia coli and eukaryotic cells permissive for SV40 replication. Transfection of the HIV DNA-containing vector (pSVSX1) into the CV-1 monkey cell line gave high levels of expression of the envelope glycoproteins gp160 and gp120 in 20-30% of the transfected cells. By several criteria, the proteins were indistinguishable from those produced during infection. The proteins were localized to the cytoplasm and plasma membrane, and some of the gp120 was shed into the culture medium. Approximately 0.5 microgram of envelope protein could be extracted from 10(6) cells. This is at least 100 times higher than the levels found in HIV-infected H9 cells. In addition, a trans-activation assay performed with pSVSX1 and a plasmid containing the gene for chloramphenicol acetyltransferase under the control of the HIV long terminal repeat demonstrated that a functional tat gene product also was expressed. Thus, this transient vector system provides an abundant source of native envelope protein for purification and characterization and also will be useful for studies dealing with the regulation of HIV gene expression.
Rekosh
D
D
Department of Biochemistry, State University of New York, Buffalo 14214.
Nygren
A
A
Flodby
P
P
Hammarskjöld
M L
ML
Wigzell
H
H
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D006678:HIV / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics*; D014162:Transfection; D014759:Viral Envelope Proteins / Q000235:genetics*
D014759:Viral Envelope Proteins