Production of human immunodeficiency virus type 1 (HIV-1) pseudoviruses using linear HIV-1 envelope expression cassettes.
HIV-1 pseudoviruses constitute an important tool in HIV-1 vaccine and entry inhibitor research. Single-cycle pseudoviruses carrying functional envelopes are generated by co-transfecting HEK293T cells with pNL4-3.LucR(-)E(-) and Env expression plasmids. However, cloning of Env genes is time consuming and single Env clones are not representative of the diversity of HIV-1 in a patient's blood sample. A new method to construct Env expression cassettes is proposed which can be used for the rapid generation of heterogeneous HIV-1 pseudoviruses without a cloning step. The linear Env expression cassettes are constructed by ligating PCR amplified Env genes between a 5' CMV promoter and 3' SV40 polyadenylation element. The resulting cassettes generate pseudoviruses carrying heterogeneous Env variants of a primary HIV-1 isolate derived from viral RNA or proviral DNA. The influence of cis-acting sequences upstream of the Env gene on infectivity was compared between pseudoviruses generated from plasmids and linear expression cassettes. The results suggest that the presence of these upstream sequences tends to result in higher infectivity of pseudoviruses when present in heterogeneous Env expression cassettes, but they do not enhance infectivity of pseudoviruses generated with homogeneous Env expression constructs. Using linear expression cassettes allows for the rapid production of heterogeneous patient-derived functional Env genes.
Beels
Dominique
D
Laboratory of Immunology, Department of Microbiology, Institute of Tropical Medicine, Nationalestraat 155, 2000 Antwerp, Belgium. dbeels@itg.be
Heyndrickx
Leo
L
Vereecken
Katleen
K
Vermoesen
Tine
T
Michiels
Lieve
L
Vanham
Guido
G
Kestens
Luc
L
D002460:Cell Line; D003001:Cloning, Molecular; D015686:Gene Products, env / Q000378:metabolism; D015694:Gene Products, rev / Q000378:metabolism; D015752:Genes, env; D015757:Genes, rev; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D006801:Humans; D010957:Plasmids; D016133:Polymerase Chain Reaction
D015686:Gene Products, env; D015694:Gene Products, rev