Book of Abstracts: Albany 2005
Stability of Intermediate State in Dimerization of Avian Sarcoma and Leukosis Viruses Genomic RNA
Dimerization of two genomic RNA copies is essential for the assembly of retrovirus particles. The process has been studied in detail and a two-step mechanism has been proposed for the human immunodeficiency virus type 1 (HIV-1). It includes the formation of intermediate state -- kissing hairpin structure (KH) and its subsequent transformation to stable linear dimer (LD). A similar model can be assumed for avian sarcoma and leukosis viruses (ASLV) despite of the lack of homology between dimerization initiation site (DIS) of ASLV RNA and that of HIV-1.
In this work, the structural features of the ASLV DIS were studied with the examples of the avian leukosis virus HPRS-103 and the avian sarcoma virus CT-10 RNA. Three types of parameters effecting stability of KH structure were investigated: i) the length of the DIS hairpin stem; ii) the temperature of incubation; and ii) magnesium concentration.
Dimers of both types were revealed by gel-shift assay for RNA fragments of the two viruses. The rate of transition from kissing hairpin structure to linear dimer increases for shorter stems of DIS hairpins and higher temperature facilitates the spontaneous transition. Although the two viruses have identical sequences (5@'-A-CUGCAG-3@') of RNA hairpin loop, the dimerization of CT-10 RNA fragments required higher RNA concentration comparing to HPRS-103 RNA fragments. The difference can be explained by deletion of an adenine from the hairpin stem of CT-10.
Mg2+ -- dependence stability of loop-loop interaction was revealed using two experimental techniques: ultraviolet-melting of KH dimer and the change in fluorescence emission of 2-aminopurine incorporated in RNA hairpin loop. This studies showed that KH dimer formed by DIS RNA hairpins does exist in solution in the absence of Mg2+ ions but this complex is rather week (Tm = 25° C at 100mM NaCl). The dependence of kissing hairpin structure stability on magnesium concentration was defined.
E. E. Minyat*
Engelhardt Institute of Molecular Biology