Cain D, Erlwein O, Grigg A, Russell R A, McClure M O
Department of G.U. Medicine and Communicable Diseases, Jefferiss Research Trust Laboratories, Wright-Fleming Institute, Imperial College School of Medicine at St. Mary's, London W2 1PG, United Kingdom.
J Virol. 2001 Apr;75(8):3731-9. doi: 10.1128/JVI.75.8.3731-3739.2001.
The retroviral RNA genome is dimeric, consisting of two identical strands of RNA linked near their 5' ends by a dimer linkage structure. Previously it was shown that human foamy virus (HFV) RNA transcribed in vitro contained three sites, designated SI, SII, and SIII, which contributed to the dimerization process (O. Erlwein, D. Cain, N. Fischer, A. Rethwilm, and M. O. McClure, Virology 229:251-258, 1997). To characterize these sites further, a series of mutants were designed and tested for their ability to dimerize in vitro. The primer binding site and a G tetrad in SI were dispensable for dimerization. However, a mutant that changed the 3' end of SI migrated slower on nondenaturing gels than wild-type RNA dimers. The sequence composition of the SII palindrome, consisting of 10 nucleotides, proved to be critical for in vitro dimerization, since mutations within this sequence or replacement of the sequence with a different palindrome of equal length impaired in vitro dimerization. The length of the palindrome also seems to play an important role. A moderate extension to 12 nucleotides was tolerated, whereas an extension to 16 nucleotides or more impaired dimerization. When nucleotides flanking the palindrome were mutated in a random fashion, dimerization was unaffected. Changing the SIII sequence also led to decreased dimer formation, confirming its contribution to the dimerization process. Interesting mutants were cloned into the infectious molecular clone of HFV, HSRV-2, and were transfected into BHK-21 cells. Mutations in SII that reduced dimerization in vitro also abolished virus replication. In contrast, constructs containing mutations in SI and SIII replicated to some extent in cell culture after an initial drop in viral replication. Analysis of the SIM1 mutant revealed reversion to the wild type but with the insertion of an additional two nucleotides. Analysis of cell-free virions demonstrated that both replication-competent and replication-defective mutants packaged nucleic acid. Thus, efficient dimerization is a critical step for HFV to generate infectious virus, but HFV RNA dimerization is not a prerequisite for packaging.
逆转录病毒RNA基因组是二聚体，由两条相同的RNA链组成，它们在5'端附近通过二聚体连接结构相连。此前研究表明，体外转录的人类泡沫病毒（HFV）RNA含有三个位点，分别命名为SI、SII和SIII，它们参与二聚化过程（O. Erlwein、D. Cain、N. Fischer、A. Rethwilm和M. O. McClure，《病毒学》229:251 - 258，1997年）。为了进一步表征这些位点，设计了一系列突变体并测试它们在体外二聚化的能力。SI中的引物结合位点和一个G四联体对于二聚化是可有可无的。然而，一个改变了SI 3'端的突变体在非变性凝胶上的迁移速度比野生型RNA二聚体慢。由10个核苷酸组成的SII回文序列的序列组成被证明对体外二聚化至关重要，因为该序列内的突变或用等长的不同回文序列替换该序列会损害体外二聚化。回文序列的长度似乎也起着重要作用。适度延长至12个核苷酸是可以容忍的，而延长至16个核苷酸或更多则会损害二聚化。当回文序列两侧的核苷酸以随机方式突变时，二聚化不受影响。改变SIII序列也会导致二聚体形成减少，证实了它对二聚化过程的贡献。将有趣的突变体克隆到HFV的感染性分子克隆HSRV - 2中，并转染到BHK - 21细胞中。体外二聚化能力降低的SII突变也会消除病毒复制。相比之下，含有SI和SIII突变的构建体在病毒复制最初下降后在细胞培养中仍能部分复制。对SIM1突变体的分析显示其回复为野生型，但额外插入了两个核苷酸。对无细胞病毒粒子的分析表明，具有复制能力和复制缺陷的突变体都能包装核酸。因此，高效二聚化是HFV产生感染性病毒的关键步骤，但HFV RNA二聚化不是包装的先决条件。
