Institute of Virology, University of Zurich, Zurich, Switzerland.
Functional Genomic Center Zurich, Zurich, Switzerland.
J Virol. 2024 Nov 19;98(11):e0128224. doi: 10.1128/jvi.01282-24. Epub 2024 Oct 9.
Adeno-associated virus type 2 (AAV2) is a small, non-pathogenic, helper virus-dependent parvovirus with a single-stranded (ss) DNA genome of approximately 4.7 kb. AAV2 DNA replication requires the presence of a helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1) and is generally assumed to occur as a strand-displacement rolling hairpin (RHR) mechanism initiated at the AAV2 3' inverted terminal repeat (ITR). We have recently shown that AAV2 replication supported by HSV-1 leads to the formation of double-stranded head-to-tail concatemers, which provides evidence for a rolling circle replication (RCR) mechanism. We have revisited AAV2 DNA replication and specifically compared the formation of AAV2 replication intermediates in the presence of either HSV-1 or AdV5 as the helper virus. The results confirmed that the AAV2 DNA replication mechanism is helper virus-dependent and follows a strand-displacement RHR mechanism when AdV5 is the helper virus and primarily an RCR mechanism when HSV-1 is the helper virus. We also demonstrate that recombination plays a negligible role in AAV2 genome replication. Interestingly, the formation of high-molecular-weight AAV2 DNA concatemers in the presence of HSV-1 as the helper virus was dependent on an intact HSV-1 DNA polymerase.
AAV is a small helper virus-dependent, non-pathogenic parvovirus. The AAV genome replication mechanism was extensively studied in the presence of AdV as the helper virus and described to proceed using RHR. Surprisingly, HSV-1 co-infection facilitates RCR of the AAV2 DNA. We directly compared AdV5 and HSV-1 supported AAV2 DNA replication and showed that AAV2 can adapt its replication mechanism to the helper virus. A detailed understanding of the AAV replication mechanism expands our knowledge of virus biology and can contribute to increase gene therapy vector production.
腺相关病毒 2 型（AAV2）是一种小的、非致病性的、依赖辅助病毒的细小病毒，具有约 4.7kb 的单链（ss）DNA 基因组。AAV2 DNA 复制需要辅助病毒（如腺病毒 5 型（AdV5）或单纯疱疹病毒 1 型（HSV-1））的存在，通常被认为是从 AAV2 3'反向末端重复序列（ITR）开始的链置换滚动发夹（RHR）机制。我们最近表明，由 HSV-1 支持的 AAV2 复制导致双链头尾连接的形成，这为滚动循环复制（RCR）机制提供了证据。我们重新研究了 AAV2 DNA 复制，并特别比较了在 HSV-1 或 AdV5 作为辅助病毒存在的情况下 AAV2 复制中间体的形成。结果证实，AAV2 DNA 复制机制依赖于辅助病毒，当 AdV5 作为辅助病毒时，遵循链置换 RHR 机制，当 HSV-1 作为辅助病毒时，主要是 RCR 机制。我们还证明重组在 AAV2 基因组复制中作用微不足道。有趣的是，在 HSV-1 作为辅助病毒存在的情况下，高相对分子质量 AAV2 DNA 连接体的形成依赖于完整的 HSV-1 DNA 聚合酶。
AAV 是一种小的、依赖辅助病毒的、非致病性的细小病毒。在 AdV 作为辅助病毒存在的情况下，对 AAV 基因组复制机制进行了广泛研究，并描述为使用 RHR 进行。令人惊讶的是，HSV-1 共感染促进了 AAV2 DNA 的 RCR。我们直接比较了 AdV5 和 HSV-1 支持的 AAV2 DNA 复制，并表明 AAV2 可以使其复制机制适应辅助病毒。对 AAV 复制机制的详细了解扩展了我们对病毒生物学的认识，并有助于提高基因治疗载体的生产。
