Laughrea M, Jetté L
McGill AIDS Centre, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
Biochemistry. 1997 Aug 5;36(31):9501-8. doi: 10.1021/bi970862l.
The genome of all retroviruses consists of two identical RNAs noncovalently linked near their 5' end. Adjacent genomic RNAs from human immunodeficiency virus type 1 (HIV-1) can form loose or tight dimers depending on whether their respective kissing-loop hairpins (nts 248-270 in HIV-1Lai) bond via their autocomplementary sequences (ACS) or via the ACS and stem sequences [Laughrea, M., & Jetté, L. (1996a) Biochemistry 35, 1589-1598]. Loose dimers from HIV-1Mal, but not HIV-1Lai, are stabilized by a sequence (3'DLS) located downstream of the 5' splice junction [Laughrea, M., & Jetté, L. (1996b) Biochemistry 35; 9366-9374]. To understand the ACS-3'DLS interplay in the formation and stability of loose and tight HIV-1 RNA dimers, we replaced the ACS of HIV-1Lai (GCGCGC262) by GUGCAC, GUGCGC (two alternative HIV-1 ACS), or GAGCUC (a non-HIV ACS). For each mutant, RNAs truncated immediately upstream or downstream of the 3'DLS were prepared; their ability to dimerize and their thermal stabilities were compared. The results suggest that the ACS determines whether the 3'DLS participates in RNA dimerization: (1) GAGCUC262 led to poorly stable loose dimers due to the inability of the 3'DLS to stabilize them (the 3'DLS stabilized the GUGCAC and GUGCGC RNAs); (2) GAGCUC262 led to poor formation of tight dimers, due to an inhibitory effect of the 3'DLS (the 3'DLS had little effect on the tight dimerization of the GUGCAC, GUGCGC and GCGCGC RNAs). The results indicate that communication exists between HIV-1 RNA sequences respectively located upstream and downstream of the 5' splice junction; they are consistent with the idea that the 3'DLS plays two ACS-dependent roles in the dimerization process: loose dimer stabilization in HIV-1 RNAs bearing an HIV ACS (unless the ACS already conferred a thermostability equal or superior to that offered by the 3'DLS), and inhibition of tight dimer formation in an HIV-1 RNA bearing a non-HIV ACS.
所有逆转录病毒的基因组均由两条相同的RNA组成，它们在5'端附近通过非共价键相连。来自1型人类免疫缺陷病毒（HIV-1）的相邻基因组RNA可形成松散或紧密的二聚体，这取决于它们各自的吻式环发夹结构（HIV-1Lai中的核苷酸248 - 270）是通过其自身互补序列（ACS）结合，还是通过ACS和茎序列结合[Laughrea, M., & Jetté, L. (1996a) Biochemistry 35, 1589 - 1598]。HIV-1Mal而非HIV-1Lai的松散二聚体可通过位于5'剪接连接下游的一个序列（3'DLS）得以稳定[Laughrea, M., & Jetté, L. (1996b) Biochemistry 35; 9366 - 9374]。为了了解ACS与3'DLS在HIV-1 RNA松散和紧密二聚体形成及稳定性方面的相互作用，我们将HIV-1Lai的ACS（GCGCGC262）替换为GUGCAC、GUGCGC（两种HIV-1的替代ACS）或GAGCUC（一种非HIV的ACS）。对于每个突变体，制备了在3'DLS上游或下游紧邻处截断的RNA；比较了它们二聚化的能力及其热稳定性。结果表明，ACS决定了3'DLS是否参与RNA二聚化：（1）GAGCUC262导致形成的松散二聚体稳定性较差，因为3'DLS无法使其稳定（3'DLS可稳定GUGCAC和GUGCGC RNA）；（2）GAGCUC262导致紧密二聚体形成不佳，这是由于3'DLS的抑制作用（3'DLS对GUGCAC、GUGCGC和GCGCGC RNA的紧密二聚化影响较小）。结果表明，位于5'剪接连接上游和下游的HIV-1 RNA序列之间存在通信；这与3'DLS在二聚化过程中发挥两种依赖于ACS的作用这一观点一致：在带有HIV ACS的HIV-1 RNA中稳定松散二聚体（除非该ACS已经赋予了与3'DLS相当或更高的热稳定性），以及在带有非HIV ACS的HIV-1 RNA中抑制紧密二聚体形成。
