Role of Distal Zinc Finger of Nucleocapsid Protein in Genomic RNA Dimerization of Human Immunodeficiency Virus Type 1; No Role for the Palindrome Crowning the R-U5 Hairpin

Abstract Genomic RNA isolated from HIV-1 variously mutated in nucleocapsid protein (NC) was characterized by nondenaturing gel electrophoresis. Mutations in the C-terminal, the N-terminal, and the linker regions had no effect on genomic RNA dimerization [they are R7R10K11S, P31L, R32G, S3(32–34), and K59L], while a C36S/C39S mutation in the distal zinc knuckle (Cys-His box or zinc finger) inhibited genome dimerization as much as disrupting the kissing-loop domain. The four mutations which inhibited tRNA Lys3 genomic placement (i.e., the in vivo placement of tRNA Lys3 on the primer binding site) had no effect on genome dimerization. Among five mutations which inhibited genome packaging, four had no effect on genome dimerization. Thus the N-terminal and linker regions of NC control genome packaging/tRNA Lys3 placement (two processes which do not require mature NC) but have little influence on genome dimerization and 2-base extension of tRNA Lys3 (two processes which are likely to require mature NC). It has been suggested, based on electron microscopy, that the AAGCUU82 palindrome crowning the R-U5 hairpin stimulates genomic RNA dimerization. To test this hypothesis, we deleted AGCU81 from wild-type viruses and from viruses bearing a disrupted kissing-loop hairpin or kissing-loop domain; in another mutant, we duplicated AGCU81. The loss of AGCU81 reduced dimerization by 2.5 ± 4%; its duplication increased it by 3 ± 6%. Dissociation temperature was left unchanged. We reach two conclusions. First, the palindrome crowning the R-U5 hairpin has no impact on HIV-1 genome dimerization. Second, genomic RNA dimerization is differentially influenced by NC sequence: it is Zn finger dependent but independent of the basic nature of the N-terminal and linker subdomains. We propose that the NC regions implicated in 2-base extension of tRNA Lys3 are required for a second (maturation) step of tRNA placement. Genome dimerization and mature tRNA placement would then become two RNA–RNA interactions sharing similar NC sequence requirements.