Year-end Sale % OFF 
Abstract
The genome of all retroviruses consists in two homologous RNA molecules associated near their 5' end in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for several functions of the retroviral cycle such as encapsidation, reverse transcription, and translation. In human immunodeficiency virus type 1 (HIV-1), a region downstream of the splice donor site was initially postulated to mediate dimerization. However, we recently showed that the dimerization initiation site is located upstream of the splice donor site and suggested that dimerization may initiate through a loop-loop interaction. Here, we show that single base mutations in the palindromic loop of the dimerization initiation site completely abolish dimerization, while introduction of compensatory mutations restores the process. Furthermore, two single nucleotide mutants that are unable to form homodimers efficiently codimerize, while the wild type RNA and the compensatory mutant, which both form homodimers, are unable to codimerize. These results unambiguously prove the interaction between the palindromic loops of each monomer. By contrast, none of the deletions that we introduced downstream of the splice donor site abolishes dimerization. However, deletions of two purine tracts located in the vicinity of the initiation codon of the gag gene significantly decrease the thermal stability of the HIV-1 RNA dimer.
Highlights
The genomeof all retroviruses consistsin two homol- Dimerization of retroviral RNAs can he induced in uitro by ogous RNA molecules associated near their5’ end in a the nucleocapsid protein (7) and by cations (15, 16).In human region called the dimer linkage structure
Since HIV-1 genomic RNA contains both the DIS and the 3'-DLS, RNA 1-615 is a better model of the genomic RNA than between dimerization and encapsidation (6-11), we constructed mutants containing either adeletion of the complete stem-loop (Fig. 2, mutant DLSA326-339) or a substitution of the GGAGG335 loop sequence (Fig. 2, mutant DLSS331-334)
Mutations inthe 3"DLS-We showed that the sequences Sequences GGG355 and GAGAG362 are located at the botlocated downstream of the S.D. site increases the thermal sta- tom and at the apex of a small stem-loopimmediately following bility of the HIV-1 RNAdimer (Table I) (20)
Summary
Plasmid pJCB was obtained by inverse PCR on the plasmidpHIVCG-4 (15). The resulting pJCB plasmid contains a new PvuII restriction site a t the 3' end of the HW-I sequence. For the RNA dimer stability experiments, the samples were incubated for 30 minat 30 "C, the temperature was gradually increased by 7 "C steps. After a 5-min incubation at the appropriate temperature, an aliquot was loaded on gel after addition of glycerol (20%final concentration). In order to ensure that all T, data published in the present paper are directly comparable,the denaturation experiments were performedat the same time with three mutants and the wild type [1-615] RNA, as internal control, and all samples were loaded on the same agarose gel
Sign-in/Register to view highlights & summary 
Published Version
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
