Laboratory of Protein Structure, International Institute of Molecular and Cell Biologygrid.419362.b, Warsaw, Poland.
Core Facility, International Institute of Molecular and Cell Biologygrid.419362.b, Warsaw, Poland.
J Virol. 2021 Aug 25;95(18):e0084821. doi: 10.1128/JVI.00848-21.
Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: and . The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.
逆转录酶（RTs）利用其 DNA 聚合酶和 RNase H 活性将单链 RNA 转化为双链 DNA（dsDNA），这是逆转录病毒复制的关键过程。泡沫病毒（FVs）具有独特的 RT，它与蛋白酶（PR）结构域融合。该酶的底物结合机制尚不清楚。在这里，我们报告了单体全长狨猴泡沫病毒（MFV）PR-RT 与 RNA/DNA 杂交底物复合物的晶体结构。我们还描述了 MFV PR-RT 与 RNase H 缺失的 dsDNA 底物复合物的结构，其中酶形成不对称同二聚体。全长 MFV PR-RT-dsDNA 复合物的冷冻电镜重建证实了二聚体结构。这些发现代表了泡沫病毒 RT 与核酸结合的第一个结构描述，并证明了它能够根据结合的核酸类型改变其寡聚状态。逆转录酶（RTs）是将单链 RNA 转化为 dsDNA 的有趣酶。它们的活性对于逆转录病毒至关重要，逆转录病毒分为两个亚科，它们的生命周期有很大的不同： 和 。后者家族更为古老，包含五个属。泡沫病毒 RT 的一个独特特征是它们含有 N 端蛋白酶（PR）结构域，而不是在正逆转录病毒酶中存在。迄今为止，还没有报道全长泡沫病毒 PR-RT 与核酸底物相互作用的结构信息。在这里，我们展示了狨猴泡沫病毒（MFV）PR-RT 的晶体和冷冻电镜结构。这些结构揭示了 RNA/DNA 和 dsDNA 底物的结合模式。此外，出乎意料的是，结构和生化数据表明，泡沫病毒 PR-RT 可以采用单体构象，我们在存在 RNA/DNA 杂交物的情况下观察到这种构象，也可以采用不对称二聚体排列，我们在存在 dsDNA 的情况下观察到这种排列。
