Alternate usage of two dimerization initiation sites in HIV-2 viral RNA in vitro.
An essential step in the replication cycle of all retroviruses is the dimerization of genomic RNA prior to or during encapsidation and budding. In HIV-1, a stem-loop structure in the genomic RNA called the dimerization initiation site, or DIS, has been well characterized. However, the identification of the structure(s) necessary for dimerization of HIV-2 genomic RNA has been less straightforward, as reflected by recent conflicting reports. Here, using a variety of mutant and wild-type RNA constructs and a systematic analysis of experimental conditions, we demonstrate that two dimerization sites in HIV-2 RNA are clearly discernible under different experimental conditions. A short sequence overlapping the primer binding site acts as the default dimerization site for wild-type viral RNA transcripts of several lengths provided that dimerization incubation conditions do not include a high heat step (>50 degrees C), and electrophoresis is carried out under mild conditions that do not deplete the RNA of magnesium. However, some RNA constructs are able to dimerize through stem-loop 1 (SL1), which is the structure homologous to the HIV-1 DIS, under certain experimental conditions. Interestingly, deletion or mutation of the default PBS dimerization site leads to efficient usage of the SL1 dimerization site. This study defines conditions under which each site may be used for dimerization and demonstrates, furthermore, the facility with which the two sites can substitute for each other. This is suggestive of a switching mechanism that may be used in the viral replication cycle.
Lanchy
Jean-Marc
JM
Division of Biological Sciences, The University of Montana, Science Complex Room 202, Missoula, MT 59812-1002, USA.
Lodmell
J Stephen
JS
D001483:Base Sequence; D001665:Binding Sites; D019281:Dimerization; D015497:HIV-1 / Q000235:genetics; D015498:HIV-2 / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016372:RNA, Antisense / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014158:Transcription, Genetic
D016372:RNA, Antisense; D012367:RNA, Viral