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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
immunology and microbiology
High throughput functional analysis of HIV-1 env genes without cloning
Journal of Virological Methods, Volume 143, No. 1, Year 2007
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Description
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 pSG3Δenv 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. © 2007 Elsevier B.V. All rights reserved.
Authors & Co-Authors
Kirchherr, Jennifer L.
United States, Durham
Duke University Medical Center
Lu, Xiaozhi
United States, Durham
Duke University Medical Center
Kasongo, Webster
Zambia, Ndola
Tropical Diseases Research Centre Ndola
Chalwe, Victor
Zambia, Ndola
Tropical Diseases Research Centre Ndola
Mwananyanda, Lawrence M.
Zambia, Ndola
Tropical Diseases Research Centre Ndola
Musonda, Rosemary Mubanga
Zambia, Ndola
Tropical Diseases Research Centre Ndola
Xia, Shi Mao
United States, Durham
Duke University Medical Center
Scearce, Richard M.
United States, Durham
Duke University Medical Center
Liao, Huaxin
United States, Durham
Duke University Medical Center
Montefiori, David Charles
United States, Durham
Duke University Medical Center
Haynes, Barton F.
United States, Durham
Duke University Medical Center
Gao, Feng
United States, Durham
Duke University Medical Center
Statistics
Citations: 49
Authors: 12
Affiliations: 2
Identifiers
Doi:
10.1016/j.jviromet.2007.02.015
ISSN:
01660934
Research Areas
Genetics And Genomics
Infectious Diseases