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Virology. 2014 Nov;468-470:454-461. doi: 10.1016/j.virol.2014.08.018. Epub 2014 Sep 20.

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.

Author information

1
Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: mdeymie@emory.edu.
2
Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: dclaibo@emory.edu.
3
Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: zende@emory.edu.
4
Emory Vaccine Center, Yerkes National Primate Research Center, 954 Gatewood Road NE, Atlanta, GA 30329, USA. Electronic address: hannah.ratner@emory.edu.
5
Zambia-Emory HIV Research Project (ZEHRP), B22/737 Mwembelelo, Emmasdale Post Net 412, P/BagE891, Lusaka, Zambia. Electronic address: wkilembe@rzhrg-mail.org.
6
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.
7
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.

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.

KEYWORDS:

Amplification; Cloning; Fitness; HIV-1; Infectious molecular clones; Quasispecies; RNA virus; Transmission

PMID:
25243334
PMCID:
PMC4252503
DOI:
10.1016/j.virol.2014.08.018
[Indexed for MEDLINE]
Free PMC Article

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