U5-PBS sequence and infectivity levels of HIV-1 NL4-3 viral mutants at start of PBMC infection. Panel A. HIV-1 U5 and PBS sequence shown (from 5' to 3'). Viral primer binding site (PBS) sequence was altered to be complementary to the 3' terminal 18 nucleotides of tRNAIle, tRNAMet and tRNAPro. The PBS sequence is shadowed. Panel B. Comparison of infectivity of NL4-3 PBS mutants. HIV-1 NL4-3 proviral clones were transfected into 293T cells, incubated for 48 hours, and supernatants were measured for infectious units. For a given sample, the number of infectious units per microliter is equal to the number of blue cells in a well divided by the dilution factor for that well and represents the average of at least two wells. Wild type infectivity levels were set at 100% and mutant virus infectivity was reported as a percentage of wild type. All viruses with altered PBS sequences showed reduced levels of infectivity as compared to wild type. Results presented are representative of three experiments.