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High throughput functional analysis of HIV-1 env genes without cloning.

Publication ,  Journal Article
Kirchherr, JL; Lu, X; Kasongo, W; Chalwe, V; Mwananyanda, L; Musonda, RM; Xia, S-M; Scearce, RM; Liao, H-X; Montefiori, DC; Haynes, BF; Gao, F
Published in: J Virol Methods
July 2007

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.

Duke Scholars

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Published In

J Virol Methods

DOI

ISSN

0166-0934

Publication Date

July 2007

Volume

143

Issue

1

Start / End Page

104 / 111

Location

Netherlands

Related Subject Headings

  • Zambia
  • Virology
  • Promoter Regions, Genetic
  • Polymerase Chain Reaction
  • Humans
  • HIV-1
  • Genes, env
  • Gene Products, env
  • Cytomegalovirus
  • Cell Line
 

Citation

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Chicago
ICMJE
MLA
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Kirchherr, J. L., Lu, X., Kasongo, W., Chalwe, V., Mwananyanda, L., Musonda, R. M., … Gao, F. (2007). High throughput functional analysis of HIV-1 env genes without cloning. J Virol Methods, 143(1), 104–111. https://doi.org/10.1016/j.jviromet.2007.02.015
Kirchherr, Jennifer L., Xiaozhi Lu, Webster Kasongo, Victor Chalwe, Lawrence Mwananyanda, Rosemary M. Musonda, Shi-Mao Xia, et al. “High throughput functional analysis of HIV-1 env genes without cloning.J Virol Methods 143, no. 1 (July 2007): 104–11. https://doi.org/10.1016/j.jviromet.2007.02.015.
Kirchherr JL, Lu X, Kasongo W, Chalwe V, Mwananyanda L, Musonda RM, et al. High throughput functional analysis of HIV-1 env genes without cloning. J Virol Methods. 2007 Jul;143(1):104–11.
Kirchherr, Jennifer L., et al. “High throughput functional analysis of HIV-1 env genes without cloning.J Virol Methods, vol. 143, no. 1, July 2007, pp. 104–11. Pubmed, doi:10.1016/j.jviromet.2007.02.015.
Kirchherr JL, Lu X, Kasongo W, Chalwe V, Mwananyanda L, Musonda RM, Xia S-M, Scearce RM, Liao H-X, Montefiori DC, Haynes BF, Gao F. High throughput functional analysis of HIV-1 env genes without cloning. J Virol Methods. 2007 Jul;143(1):104–111.
Journal cover image

Published In

J Virol Methods

DOI

ISSN

0166-0934

Publication Date

July 2007

Volume

143

Issue

1

Start / End Page

104 / 111

Location

Netherlands

Related Subject Headings

  • Zambia
  • Virology
  • Promoter Regions, Genetic
  • Polymerase Chain Reaction
  • Humans
  • HIV-1
  • Genes, env
  • Gene Products, env
  • Cytomegalovirus
  • Cell Line