title: High throughput functional analysis of HIV-1 env genes without cloning.
abstract: Functional human immunodeficiency virus type 1 (HIV-1) env genes have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The cytomegalovirus (CMV) promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3 Delta env backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions. This allows for quick analysis of multiple env genes from HIV-1 infected individuals.
authors.affiliation: Duke Human Vaccine Institute and Center for HIV-AIDS Vaccine Immunology, Department of Medicine, Duke University Medical Center, 112 RPIII, Research Drive, Box 3347, Durham, NC 27710, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Kirchherr
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Lu
authors.forename: Webster
authors.initials: W
authors.lastname: Kasongo
authors.forename: Victor
authors.initials: V
authors.lastname: Chalwe
authors.forename: Lawrence
authors.initials: L
authors.lastname: Mwananyanda
authors.forename: Rosemary M
authors.initials: RM
authors.lastname: Musonda
authors.forename: Shi-Mao
authors.initials: SM
authors.lastname: Xia
authors.forename: Richard M
authors.initials: RM
authors.lastname: Scearce
authors.forename: Hua-Xin
authors.initials: HX
authors.lastname: Liao
authors.forename: David C
authors.initials: DC
authors.lastname: Montefiori
authors.forename: Barton F
authors.initials: BF
authors.lastname: Haynes
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
mesh_terms: D000163:Acquired Immunodeficiency Syndrome; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D015686:Gene Products, env / Q000302:isolation & purification*; D015752:Genes, env; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D015024:Zambia
chemical_list: D015686:Gene Products, env