A novel yeast-based recombination method to clone and propagate diverse HIV-1 isolates.

Abstract:

: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.

journal_name

Biotechniques

journal_title

BioTechniques

authors

Dudley DM,Gao Y,Nelson KN,Henry KR,Nankya I,Gibson RM,Arts EJ

doi

10.2144/000113119

subject

Has Abstract

pub_date

2009-05-01 00:00:00

pages

458-67

issue

6

eissn

0736-6205

issn

1940-9818

pii

000113119

journal_volume

46

pub_type

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