The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by the extension of a tRNA primer bound near the 5' end of the genomic RNA at a position termed the primer-binding site (PBS). The PBS is an 18-nucleotide region of the HIV-1 genome complementary to cellular tRNA(Lys). To further investigate the sequence requirements for the PBS in reverse transcription, deletions in the PBS were created and subcloned into a plasmid containing the infectious HIV-1 proviral genome. The mutations deleted the entire PBS (delta PBS) or the first 9 (delta 1-9), the second 9 (delta 10-18), or 12 (delta 7-18) nucleotides of the PBS. An additional mutation in the PBS was created in which the second nine nucleotides were deleted and nine additional nucleotides were substituted [Lys(1-9)]. The transfection of plasmids containing the wild-type or mutant proviral genomes into tissue culture cells resulted in expression of the HIV-1 gag and env gene products, as determined by immunoprecipitation using sera from AIDS patients. HIV-1 virus was released from the transfected cells, as determined by analysis of the supernatants for reverse transcriptase activity. The infectivity of the viruses derived from the transfection was examined by coculture experiments with SupT1 cells, which support high-level replication of HIV-1. The transfection of plasmids containing HIV-1 proviral genomes with the delta PBS and PBS (delta 1-9) mutations did not produce infectious virus. In contrast, the HIV-1 proviral genomes with the delta 10-18, delta 7-18, and Lys(1-9) mutations in the PBS produced infectious virus upon transfection, although the kinetics of appearance was significantly delayed for the mutant viruses compared with the wild type. To further explore the nature of this defect, the PBS region from integrated proviral genomes was amplified by polymerase chain reaction and individual DNA products were subcloned into M13mp19, followed by a sequence analysis of the PBS region from individual M13 phage clones. In each of the PBS regions examined, the 18-nucleotide PBS complementary to tRNA(Lys) was present. However, nucleotide deletions and insertions were found 3' to the PBS from the samples derived from the transfection of plasmids containing mutant proviral genomes. Upon reinfection, the revertant viruses maintained the deletions 3' to the PBS and had kinetics of replication similar to that of the wild-type virus.(ABSTRACT TRUNCATED AT 400 WORDS)