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269 Construction and Biological Characterization of an Infectious HIV-1 Subtype C Molecular Clone
M. A. Papathanasopoulos*1, L. Morris1, S. Abdool-Karim2, C. Williamson3, P. K. Ehrenberg4, and F. McCutchan4
1Natl. Inst. for Virology, Johannesburg, South Africa; 2Med. Res. Council, Durban, South Africa; 3Univ. of Cape Town, South Africa; and 4Henry M. Jackson Fndn., Rockville, MD, USA
Background: South Africa will be participating in Phase I HIV-1 subtype C vaccine trials over the next few years. Current understanding of the pathogenesis and molecular biology of HIV-1 is mainly based on analysis of subtype B. Accordingly, molecular reagents for non-subtype B are limited. Infectious HIV-1 molecular clones are needed for studies requiring functional gene products with the defined and uniform genetic and immunological properties of the selected South African vaccine isolates.
Methods: Proviral DNA from the vaccine strain Du151 was isolated from infected PBMCs grown in coculture. The LTR region was PCR amplified and sequenced. Subsequently, primers with relevant restriction sites were designed and used to amplify the 2 LTR fragments. These fragments were used to construct the subtype C LTR reconstitution vector. A 9.2-kb proviral amplicon directionally cloned into this vector provided candidate molecular clones of the full HIV-1 genome for infectivity screening. Several clones that were transfected into 293T cells produced p24 antigen. Positive supernatants containing this initial inoculum were used as viral stocks to infect PHA/IL-2-stimulated seronegative donor PBMCs. A molecular clone capable of spreading infection in PBMCs was selected for further characterization.
Results: We have developed an LTR replacement vector optimized for HIV-1 subtype C isolates. PCR amplification of virtually full-length provirus followed by molecular cloning into this vector precisely regenerated the genomic organization of the entire Du151 provirus. This resulted in generation of infectious molecular clones from the Du151 primary isolate that display biological properties reflecting the cognate parental strain.
Conclusions: Infectious molecular clones of HIV-1 subtype C will facilitate the investigation of subtype-specific differences in virological and immunological properties, and can be used in experimental design of future vaccine candidates against subtype C, as well as reagents to evaluate immunogenicity.
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