Construction and characterization of a minimized version of the HIV-1 pNL4-3 plasmid and its application for pseudotyping HIV-1 vectors.
The pUC-based pNL4-3 plasmid is the most widely used vector for in vitro manipulations of the HIV-1 proviral sequences. We have developed a minimal plasmid (pCHUS) based on pNL4-3, which may be useful to facilitate the design of HIV-based constructions. The strategy that has allowed us to construct pCHUS includes the following steps: (1) pNL4-3 digestion by using restriction sites contained within the long terminal repeats (LTRs), (2) recircularization of the fragment containing the pUC18 sequence, (3) amplification of the LTR region restored in the previous step, (4) double digestion of the products obtained in steps 2 and 3, (5) ligation of the fragment containing ColE1+Amp(R) with the LTR fragment, (6) linearization of the intermediate plasmid obtained, and (7) insertion of the fragment containing the proviral genome into the linearized vector. The pCHUS plasmid includes essential information for its replication and antibiotic selection in bacteria, but it lacks all the unnecessary sequences. Our results suggest that pCHUS may be more advantageous than pNL4-3 for in vitro manipulation of the HIV-1 proviral genome. In addition, we describe a potential application of this new vector for pseudotyping HIV-1 particles, using a single plasmid transfection, as a more helpful alternative to the traditionally used cotransfection method.
Abad
María Luisa
ML
Division of Microbiology, Hospital de Conxo, Complejo Hospitalario Universitario de Santiago (CHUS), Santiago de Compostela, Spain. mlabad@teleline.es
Verdura
Tomás
T
Vela
Alicia
A
Iglesias
María José
MJ
Gutiérrez
Dolores
D
Veiga
Manuel
M
Aguilera
Antonio
A
Regueiro
Benito J
BJ
D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication
D049452:Green Fluorescent Proteins