to BioTechniques free email alert service to receive content updates.
A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates
Dawn M. Dudley*1, 2, Yong Gao*2, Kenneth N. Nelson2, Kenneth R. Henry2, Immaculate Nankya1, Richard M. Gibson2, Eric J. Arts1, 2
1, Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA
2, Division of Infectious Diseases, Department of Medicine, Case Western Reserve University, Cleveland, OH, USA
BioTechniques, Vol. 46, No. 6, May 2009, pp. 458–467
Full Text (PDF)
Supplementary Material

Replication studies on human immunodeficiency virus 1 (HIV-1) rely on a few laboratory strains that are divergent from dominant HIV-1 subtypes in the epidemic. Several phenotypic differences between diverse HIV-1 isolates and subtypes could affect vaccine development and treatment, but this research field lacks robust cloning/virus production systems to study drug sensitivity, replication kinetics, or to develop personalized vaccines. Extreme HIV-1 heterogeneity leaves few restriction enzyme sites for bacterial cloning strategies. In this study, we describe an alternative approach that involves direct introduction of any HIV-1 coding regions (e.g., any gene from a patient sample) into an HIV-1 DNA vector using yeast recombination. This technique uses positive and negative selectable markers in yeast and avoids the need for purification and screening of the DNA substrates and cloning products. Replication-competent virus is then produced from a modified mammalian 293T packaging cell line transfected with this yeast-derived HIV-1 vector. Although HIV-1 served as the prototype, this cloning strategy is now being developed for other diverse virus species such as hepatitis C virus and influenza virus.


The study of primary virus isolates is necessary to understand many aspects of virus-host interactions and specific viral characteristics (1,2,3,4,5), but virus isolation/propagation is extremely challenging for many virus species and strains. An alternative approach involves cloning specific viral genes into expression or viral genomic vectors to assess function. Expression vectors can be used to produce pseudo-typed viruses, but they limit studies to specific steps in the virus replication cycle (6,7,8,9). Cloning into full-length viral genomes followed by a method for direct virus production provides a more flexible virus replication system to study drug sensitivity, phenotypic resistance in patients on drug treatment, fitness, mechanisms of viral replication, and host-virus interactions (10,11,12).

Viruses such as human immunodeficiency virus type 1 (HIV-1) or hepatitis C virus (HCV) are highly heterogeneous and lack convenient, conserved, or unique restriction enzyme sites for cloning (13,14,15). In the past, molecular analyses of HIV-1 replication involved only a handful of subtype B laboratory strains (e.g., NL4–3, HXB2, Ada, BH10) but these are not representative of the majority of circulating HIV-1 isolates in the infected population. Several studies have now examined possible phenotypic differences among the 10 different HIV-1 subtypes and at least 33 circulating recombinant forms, which can differ by as much as 25% at the nucleotide level (16,17,18,19,20). Any differences in viral phenotypes are typically mapped to specific genetic sequences, but this characterization requires the difficult cloning of diverse genetic elements. As a consequence, few groups have performed drug resistance/susceptibility studies or in-depth analyses on replicative fitness using diverse HIV-1 subtypes. Of these, most have relied on restriction enzyme–based cloning into an expression vector for gene complementation/pseudotyping rather than cloning into a full-length viral genome. These studies, even when successful, prove to be time-consuming and often introduce foreign genetic elements.

An alternative method to bacterial cloning involves transfecting a human cell line (e.g., MT4 cells) (i) with a proviral DNA vector with a deletion in a specific gene and (ii) an HIV-1 PCR product derived from a patient isolate in order to replace the deleted gene (10,13,14). Homologous recombination in mammalian cells can then produce a full-length HIV-1 genome and, ultimately, an infectious virus. Although this method is relatively simple, it is also plagued by the poor efficiency of eukaryotic recombination, an inability to recover the cloned genome, a slow outgrowth of recombined virus, and a heavy selection bias for a few randomly generated clones from a swarm of patient HIV-1 sequences.

Recently, we adopted a yeast-based recombination/gap repair technique to shuttle any HIV-1 genetic element into an HIV-1 vector (15). This technique has several advantages over mammalian recombination or classical bacterial cloning in that yeast recombination is highly efficient, precise, and involves negative selectable markers that nearly ensure insertion of the new genetic element (21,22,23,24). One major disadvantage of this first-generation technique is the need for restriction enzyme/ligation-based subcloning to produce infectious full-length proviral DNA vectors. In this study, we describe an improved yeast-based recombination method that allows any HIV-1 gene, coding region, or potentially even full-length genome from any subtype to be cloned into an HIV-1 DNA vector. Using a single cloning step and a highly efficient negative/positive selection method, this system avoids restriction enzyme digestions, gel purifications, in vitro ligations, and screening for correct inserts/vectors. The resulting HIV-1 DNA is then shuttled into a 293T packaging cell line that provides a short complementing HIV-1 genome in trans for subsequent virus propagation.

Materials and methods

Yeast strain and growth conditions

Saccharomyces cerevisiae Hanson (MYA-906), MAT α ade6 can1 his3 leu2 trp1 URA3 was grown at 30°C in appropriate media depending on the cloning step [yeast extract peptone dextrose (YEPD), complete (C) minimal media -LEU-URA3, C-LEU, or C-LEU/5-fluoro-1,2,3,6-tetra-hydro-2,6-dioxo-4-pyrimidine carboxylic acid (5-FOA)]. Transformations were performed using the lithium acetate (LiAc) method described by Coligan et al. (22).

Cell culture

293T packaging cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin). 293T cells stably transfected with a plasmid expressing the 5′ long-terminal repeat (LTR) (293Tcplt) were maintained in DMEM with 10% FBS and 100 mg/mL zeocin (Invitrogen, Carlsbad, CA). U87. CD4.CCR5 and U87.CD4.CXCR4 cells were cultured in 15% FBS-DMEM supplemented with 300 μg/mL G418, 1 μg/mL puromycin and antibiotics. U87 cell lines were obtained from Dr. Hongkui Deng and Dr. Dan Littman through the AIDS Research and Reference Reagent Program (Germantown, MD, USA).

  1    2    3    4    5