A Yeast Recombination-Based Cloning System to Produce Chimeric HIV-1 Viruses and Express HIV-1 Genes

Differential phenotypes or properties of HIV-1 gene products in primary virus isolates are difficult to assess due to interference by the high degree of sequence variation across the entire genome. Thus, chimeric viruses provide a powerful tool to study the function of single gene products or genetic elements in the context of a neutral viral genomic backbone. In this chapter, we describe how to produce HIV-1 chimeric viruses utilizing a yeast-based homologous recombination cloning technique to insert env sequences first into a yeast cloning vector and then into the common pNL4-3 virus backbone. This technique is not limited to the env gene, but can be used to build chimeric viruses with any HIV-1 gene or genetic element. This cloning technique involves the use of a shuttle vector that can replicate in yeast and bacterial cells. Along with acting as a shuttle vector for subsequent subcloning into pNL4-3, this construct pRec/env can also be used to express to the env gene product, gp120/gp41, on the surface of mammalian cells. The chimeric viruses produced by this cloning method are capable of undergoing multiple rounds of replication and are therefore very useful to study drug sensitivity, coreceptor usage, and viral fitness as influenced by a single gene or gene fragment of a primary HIV-1 isolate from any group M subtype.