Effects of alterations of primer-binding site sequences on human immunodeficiency virus type 1 replication

The human immunodeficiency virus type 1 genomic RNA primer-binding site (PBS) sequence comprises 18 nucleotides which are complementary to those at the 3 ' end of the replication initiation primer tRNAALYS. To investigate the role of the PBS in viral replication, we either deleted the original wild-type PBS (complementary to tRNA3YS) or replaced it with DNA sequences complementary to either tRNAlys or tRNAPhe. Transfection of COS cells with such molecular constructs yielded similar levels of viral progeny that were indistinguishable with regard to viral proteins and tRNA content. Virus particles derived from PBS-deleted molecular clones were noninfectious for MT-4, Jurkat, and CEM-T4 cells. However, infectious viruses were derived from constructs in which the PBS had been altered to sequences complementary to either tRNA'LYs or tRNAPhe, although mutated forms showed significant lags in replication efficiency in comparison with wild types. Molecular analysis of reverse-transcribed DNA in cells infected by the mutated viruses indicated that both tRNA1LY and tRNAPhe could function as primers for reverse transcription during the early stages of infection. Sequencing of full-length proviral DNA, obtained 6 days after infection, revealed the mutated PBS, indicating that a complete cycle of reverse transcription had occurred. During subsequent rounds of infection, reversion of the mutated PBS to wild-type sequences was observed, accompanied by increased production of viral gene products. Reversion to wild-type PBS sequences was confirmed both by specific PCR analysis, using distinc