MOLECULAR BIOTECHNOLOGY Volume 28, 2004 Minimized Version of the HIV-1 pNL4-3 Plasmid 87 RESEARCH 87 Molecular Biotechnology © 2004 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2004/28:2/87–95/$25.00 Abstract *Author to whom all correspondence and reprint requests should be addressed: 1 Division of Microbiology, Hospital de Conxo, Complejo Hospitalario Universitario de Santiago (CHUS), Santiago de Compostela, Spain. E-mail: mlabad@teleline.es Construction and Characterization of a Minimized Version of the HIV-1 pNL4-3 Plasmid and Its Application for Pseudotyping HIV-1 Vectors María Luisa Abad,* Tomás Verdura, 1 Alicia Vela, 1 María José Iglesias, 1 Dolores Gutiérrez, Manuel Veiga, 1 Antonio Aguilera, 1 and Benito J. Regueiro 1 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 termi- nal 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 selec- tion 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 transfec- tion, as a more helpful alternative to the traditionally used cotransfection method. Index Entries: HIV-1; retroviral vectors; pNL4-3; pCHUS; pseudotypes. 1. Introduction The molecular study of human immunodefi- ciency virus type 1 (HIV-1) has been facilitated by the use of infectious molecular clones. Typi- cally, these clones are large plasmids containing HIV-1 proviral genomes. The pUC-based pNL4- 3 plasmid (14,833-bp in size) (1) is one of the most widely used vectors for in vitro genetic alter- ation of the HIV-1 proviral sequences. This genetic manipulation is necessary for multiple research applications, such as phenotypic assays used for the assessment of HIV-1 drug susceptibility. Pheno- typic testing directly measures the drug suscepti- bility of patient-derived viruses in culture assays that are based on recombinant virus assays (2). Viruses can be generated through recombination of plasma-derived reverse transcriptase/protease sequences and modified HIV-1 vectors. However, the engineering of HIV-1 proviral constructs is one of the major tasks in a molecular biology labora- tory working in HIV research. Many sophisticated retroviral constructions cannot be easily and rap- idly engineered in a routine way. For example, the necessary endonuclease restriction sites might not be available or could be unsuitable for the desired constructions. Thus, an optimized vector system is highly desirable. For these reasons, we have constructed pCHUS, a new minimal retroviral vector based on the pNL4-3 plasmid, which may be useful to facilitate the in vitro designing of HIV-1-based vectors and to increase their applications.