Chimeric Envs were constructed using a domain exchange strategy that has been described previously [24, 25] . The primer sequences and their HXB2 locations are shown below. A PCR screen was performed to identify transformants in which the fragments ligated together in the correct orientation using forward primer EnvA and the reverse primer that was used to amplify the exchanged domain. Colonies that were positive by PCR screen were inoculated into LB-Ampicillin broth for overnight cultures, and the plasmid was prepared using the QIAprep Spin Miniprep Kit. Env chimeras were then screened for biological function as described above. For chimeras that produced functional Env pseudotypes, the plasmids were re-transfected into 293T cells on a larger scale to produce a working pseudotype virus stock. Transfection supernatants were collected at 72 hours post-transfection, clarified by low speed centrifugation, aliquoted into 0.5 ml or less portions, and stored at 280uC. The titer of each pseudovirus stock was determined by infecting JC53-BL13 cells with 5-fold serial dilutions of virus as described previously. All Env chimeras were confirmed by nucleotide sequencing of the entire env gene. The 0-month Env backbones (minus the target domains) with pcDNA3.1 vector sequences were PCR amplified using primers that anneal to conserved regions adjacent to the target domain primers. These primers amplify away from the target domains. A 59 phosphate group (Phos) was added to these primer sets during synthesis to facilitate ligation to the target domain amplicons. The primer sequences and their HXB2 locations were as follows: