Koehler Alexander, Kolesnikova Larissa, Welzel Ulla, Schudt Gordian, Herwig Astrid, Becker Stephan
Institut für Virologie, Philipps-Universität Marburg, Marburg, Germany.
Institut für Virologie, Philipps-Universität Marburg, Marburg, Germany German Center for Infection Research (DZIF), Institut für Virologie, Philipps-Universität Marburg, Marburg, Germany
J Virol. 2015 Nov 18;90(3):1444-54. doi: 10.1128/JVI.02670-15. Print 2016 Feb 1.
Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling.
The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.
马尔堡病毒（MARV）可在人类和非人类灵长类动物中引发严重出血热，但在啮齿动物中仅引起短暂的非致命性疾病。然而，MARV在啮齿动物中的连续传代可增强感染，导致致命性疾病。适应豚鼠的MARV在病毒基质蛋白VP40的第184位含有一个突变（VP40D184N）。D184N突变对在新宿主中复制效力的贡献尚不清楚。在本研究中，我们证明，在豚鼠细胞中，含有VP40中D184N突变的重组MARV[rMARVVP40(D184N)]比野生型重组MARV（rMARVWT）生长至更高滴度。此外，rMARVVP40(D184N)在豚鼠细胞中表现出更高的感染性。VP40功能的比较分析表明，D184N突变既不影响VP40的干扰素（IFN）拮抗功能，也不影响其膜结合能力。然而，在豚鼠细胞中，D184N突变改善了VP40诱导的病毒样颗粒（VLP）的产生以及其他病毒蛋白向出芽位点的募集，这导致与VP40诱导的感染性VLP（iVLP）相比，VP40D184N诱导的iVLP具有更高的感染性。此外，VP40在抑制病毒RNA合成中的功能受到D184N突变的影响，特别是在豚鼠细胞中，从而允许更高的转录和复制速率。我们的结果表明，rMARVVP40(D184N)在豚鼠细胞中病毒适应性的提高是由于VP40D184N更好的病毒组装功能及其对病毒转录和复制的较低抑制作用，而不是对VP40介导的IFN信号抑制的调节。
病毒在新宿主中传代所实现的毒力增加伴随着病毒基因组中的突变。分析这些突变如何影响病毒蛋白的功能以及病毒在新宿主细胞内生长的能力有助于理解增加毒力的分子机制。使用反向遗传学方法，我们证明，在适应豚鼠的MARV中检测到的MARV VP40中的单个突变赋予了rMARVVP40(D184N)在豚鼠细胞中的复制优势。我们的研究表明，rMARV VP40D184N的这种复制优势基于VP40在iVLP组装以及转录和复制调节方面功能的改善，而不是基于VP40对抗宿主先天免疫的能力。
