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 ngetikin 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 echofix spring reverb 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.