The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.