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

Genomic RNA isolated from HIV-1 variously mutated in nucleocapsid protein ( NC) was characterized by nondenaturing gel electrophoresis. Mutations in th e C-terminal, the N-terminal, and the linker regions had no effect on genom ic 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-loo p 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 n o effect on genome dimerization. Among five mutations which inhibited genom e packaging, four had no effect on genome dimerization. Thus the N-terminal and linker regions of NC control genome packaging/tRNA(Lys3) placement (tw o processes which do not require mature NC) but have little influence on ge nome dimerization and 2-base extension of tRNA(Lys3) (two processes which a re likely to require mature NC). It has been suggested, based on electron m icroscopy, that the AAGCUU82 palindrome crowning the R-U5 hairpin stimulate s genomic RNA dimerization. To test this hypothesis, we deleted AGCU81 from wild-type viruses and from viruses bearing a disrupted kissing-loop hairpi n 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 con clusions. First, the palindrome crowning the R-U5 hairpin has no impact on HIV-1 genome dimerization. Second, genomic RNA dimerization is differential ly 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 t he NC regions implicated in 2-base extension of tRNA(Lys3) are required for a second (maturation) step of tRNA placement. Genome dimerization and matu re tRNA placement would then become two RNA-RNA interactions sharing simila r NC sequence requirements. (C) 2001 Academic Press.