Tucker Jessica M, Larango Morgan E, Wachsmuth Lucas P, Kannan Natarajan, Garfinkel David J
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.
PLoS Genet. 2015 Oct 9;11(10):e1005571. doi: 10.1371/journal.pgen.1005571. eCollection 2015 Oct.
A novel form of copy number control (CNC) helps maintain a low number of Ty1 retrovirus-like transposons in the Saccharomyces genome. Ty1 produces an alternative transcript that encodes p22, a trans-dominant negative inhibitor of Ty1 retrotransposition whose sequence is identical to the C-terminal half of Gag. The level of p22 increases with copy number and inhibits normal Ty1 virus-like particle (VLP) assembly and maturation through interactions with full length Gag. A forward genetic screen for CNC-resistant (CNCR) mutations in Ty1 identified missense mutations in GAG that restore retrotransposition in the presence of p22. Some of these mutations map within a predicted UBN2 domain found throughout the Ty1/copia family of long terminal repeat retrotransposons, and others cluster within a central region of Gag that is referred to as the CNCR domain. We generated multiple alignments of yeast Ty1-like Gag proteins and found that some Gag proteins, including those of the related Ty2 elements, contain non-Ty1 residues at multiple CNCR sites. Interestingly, the Ty2-917 element is resistant to p22 and does not undergo a Ty1-like form of CNC. Substitutions conferring CNCR map within predicted helices in Ty1 Gag that overlap with conserved sequence in Ty1/copia, suggesting that p22 disturbs a central function of the capsid during VLP assembly. When hydrophobic residues within predicted helices in Gag are mutated, Gag level remains unaffected in most cases yet VLP assembly and maturation is abnormal. Gag CNCR mutations do not alter binding to p22 as determined by co-immunoprecipitation analyses, but instead, exclude p22 from Ty1 VLPs. These findings suggest that the CNCR alleles enhance retrotransposition in the presence of p22 by allowing productive Gag-Gag interactions during VLP assembly. Our work also expands the strategies used by retroviruses for developing resistance to Gag-like restriction factors to now include retrotransposons.
一种新型的拷贝数控制（CNC）方式有助于在酿酒酵母基因组中维持低数量的Ty1逆转录病毒样转座子。Ty1产生一种编码p22的替代转录本，p22是Ty1逆转录转座的反式显性负抑制剂，其序列与Gag的C端一半相同。p22的水平随拷贝数增加，并通过与全长Gag相互作用抑制正常的Ty1病毒样颗粒（VLP）组装和成熟。对Ty1中耐CNC（CNCR）突变进行的正向遗传筛选鉴定出GAG中的错义突变，这些突变在p22存在的情况下恢复逆转录转座。其中一些突变位于整个Ty1/长末端重复逆转录转座子家族中预测的UBN2结构域内，其他突变聚集在Gag的一个中心区域，该区域被称为CNCR结构域。我们生成了酵母Ty1样Gag蛋白的多序列比对，发现一些Gag蛋白，包括相关Ty2元件的Gag蛋白，在多个CNCR位点含有非Ty1残基。有趣的是，Ty2-917元件对p22有抗性，并且不经历Ty1样的CNC形式。赋予CNCR的替代突变位于Ty1 Gag中预测的螺旋内，这些螺旋与Ty1/copia中的保守序列重叠，表明p22在VLP组装过程中干扰衣壳的核心功能。当Gag中预测螺旋内的疏水残基发生突变时，在大多数情况下Gag水平不受影响，但VLP组装和成熟异常。通过共免疫沉淀分析确定，Gag CNCR突变不会改变与p22的结合，而是将p22排除在Ty1 VLP之外。这些发现表明，CNCR等位基因通过在VLP组装过程中允许有效的Gag-Gag相互作用，在p22存在的情况下增强逆转录转座。我们的工作还扩展了逆转录病毒用于产生对Gag样限制因子抗性的策略，使其现在包括逆转录转座子。
