title: Particle infectivity of HIV-1 full-length genome infectious molecular clones in a subtype C heterosexual transmission pair following high fidelity amplification and unbiased cloning.
abstract: The high genetic diversity of HIV-1 impedes high throughput, large-scale sequencing and full-length genome cloning by common restriction enzyme based methods. Applying novel methods that employ a high-fidelity polymerase for amplification and an unbiased fusion-based cloning strategy, we have generated several HIV-1 full-length genome infectious molecular clones from an epidemiologically linked transmission pair. These clones represent the transmitted/founder virus and phylogenetically diverse non-transmitted variants from the chronically infected individual׳s diverse quasispecies near the time of transmission. We demonstrate that, using this approach, PCR-induced mutations in full-length clones derived from their cognate single genome amplicons are rare. Furthermore, all eight non-transmitted genomes tested produced functional virus with a range of infectivities, belying the previous assumption that a majority of circulating viruses in chronic HIV-1 infection are defective. Thus, these methods provide important tools to update protocols in molecular biology that can be universally applied to the study of human viral pathogens.
authors.affiliation: Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: mdeymie@emory.edu.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Deymier
authors.affiliation: Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: dclaibo@emory.edu.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Claiborne
authors.affiliation: Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: zende@emory.edu.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Ende
authors.affiliation: Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: hannah.ratner@emory.edu.
authors.forename: Hannah K
authors.initials: HK
authors.lastname: Ratner
authors.affiliation: Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka, Zambia. Electronic address: wkilembe@rzhrg-mail.org.
authors.forename: William
authors.initials: W
authors.lastname: Kilembe
authors.affiliation: Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka, Zambia; Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA. Electronic address: sallen5@emory.edu.
authors.forename: Susan
authors.initials: S
authors.lastname: Allen
authors.affiliation: Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA; Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA. Electronic address: eric.hunter2@emory.edu.
authors.forename: Eric
authors.initials: E
authors.lastname: Hunter
mesh_terms: D003001:Cloning, Molecular; D014644:Genetic Variation; D016679:Genome, Viral; D015658:HIV Infections / Q000821:virology; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D010802:Phylogeny; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism
chemical_list: D012367:RNA, Viral
keywords: Amplification; Cloning; Fitness; HIV-1; Infectious molecular clones; Quasispecies; RNA virus; Transmission