Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.
Princeton Neuroscience Institute, Princeton University, Princeton, New Jersey, USA.
J Virol. 2021 Feb 24;95(6). doi: 10.1128/JVI.02286-20.
Latent and recurrent productive infection of long-living cells, such as neurons, enables alphaherpesviruses to persist in their host populations. Still, the viral factors involved in these events remain largely obscure. Using a complementation assay in compartmented primary peripheral nervous system (PNS) neuronal cultures, we previously reported that productive replication of axonally delivered genomes is facilitated by pseudorabies virus (PRV) tegument proteins. Here, we sought to unravel the role of tegument protein UL13 in this escape from silencing. We first constructed four new PRV mutants in the virulent Becker strain using CRISPR/Cas9-mediated gene replacement: (i) PRV Becker defective for UL13 expression (PRV ΔUL13), (ii) PRV where UL13 is fused to eGFP (PRV UL13-eGFP), and two control viruses (iii and iv) PRV where VP16 is fused with mTurquoise at either the N terminus (PRV mTurq-VP16) or the C terminus (PRV VP16-mTurq). Live-cell imaging of PRV capsids showed efficient retrograde transport after axonal infection with PRV UL13-eGFP, although we did not detect dual-color particles. However, immunofluorescence staining of particles in mid-axons indicated that UL13 might be cotransported with PRV capsids in PNS axons. Superinfecting nerve cell bodies with UV-inactivated PRV ΔUL13 failed to efficiently promote escape from genome silencing compared to UV-PRV wild type and UV-PRV UL13-eGFP superinfection. However, UL13 does not act directly in the escape from genome silencing, as adeno-associated virus (AAV)-mediated UL13 expression in neuronal cell bodies was not sufficient to provoke escape from genome silencing. Based on this, we suggest that UL13 may contribute to initiation of productive infection through phosphorylation of other tegument proteins. Alphaherpesviruses have mastered various strategies to persist in an immunocompetent host, including the induction of latency and reactivation in peripheral nervous system (PNS) ganglia. We recently discovered that the molecular mechanism underlying escape from latency by the alphaherpesvirus pseudorabies virus (PRV) relies on a structural viral tegument protein. This study aimed at unravelling the role of tegument protein UL13 in PRV escape from latency. First, we confirmed the use of CRISPR/Cas9-mediated gene replacement as a versatile tool to modify the PRV genome. Next, we used our new set of viral mutants and AAV vectors to conclude the indirect role of UL13 in PRV escape from latency in primary neurons, along with its spatial localization during retrograde capsid transport in axons. Based on these findings, we speculate that UL13 phosphorylates one or more tegument proteins, thereby priming these putative proteins to induce escape from genome silencing.
潜伏和反复的生产性感染长寿细胞，如神经元，使α疱疹病毒能够在其宿主群体中持续存在。尽管如此，在这些事件中涉及的病毒因素在很大程度上仍然不清楚。我们以前曾在分隔的原代周围神经系统 (PNS) 神经元培养物中使用补体测定法报告说，轴突传递的基因组的生产性复制是由伪狂犬病病毒 (PRV) 被膜蛋白促进的。在这里，我们试图揭开被膜蛋白 UL13 在这种沉默逃逸中的作用。我们首先使用 CRISPR/Cas9 介导的基因替换在毒力 Becker 株中构建了四个新的 PRV 突变体：（i）PRV Becker 缺陷型 UL13 表达（PRV ΔUL13），（ii）UL13 融合到 eGFP 的 PRV（PRV UL13-eGFP），和两个对照病毒（iii 和 iv）VP16 在 N 末端（PRV mTurq-VP16）或 C 末端（PRV VP16-mTurq）融合到 mTurquoise 的 PRV。PRV 衣壳的活细胞成像显示，在用 PRV UL13-eGFP 感染轴突后，有效逆行运输，尽管我们没有检测到双色颗粒。然而，在中间轴突中的颗粒的免疫荧光染色表明 UL13 可能与 PRV 衣壳一起在 PNS 轴突中转运。与 UV-PRV 野生型和 UV-PRV UL13-eGFP 超感染相比，用 UV 失活的 PRV ΔUL13 再次感染神经细胞体未能有效促进从基因组沉默中逃逸。然而，UL13 并非直接参与从基因组沉默中逃逸，因为腺相关病毒 (AAV) 介导的 UL13 在神经元细胞体中的表达不足以引发从基因组沉默中逃逸。基于此，我们建议 UL13 可能通过磷酸化其他被膜蛋白来促进生产性感染的开始。α疱疹病毒已经掌握了各种策略来在免疫功能正常的宿主中持续存在，包括潜伏和外周神经系统 (PNS) 神经节中的再激活。我们最近发现，α疱疹病毒伪狂犬病病毒 (PRV) 从潜伏中逃逸的分子机制依赖于一种结构病毒被膜蛋白。本研究旨在揭示被膜蛋白 UL13 在 PRV 从潜伏中逃逸中的作用。首先，我们证实了使用 CRISPR/Cas9 介导的基因替换作为修改 PRV 基因组的通用工具。接下来，我们使用我们的新病毒突变体集和 AAV 载体得出结论，即在原代神经元中，UL13 在 PRV 从潜伏中逃逸中具有间接作用，以及在轴突中逆行衣壳运输期间的空间定位。基于这些发现，我们推测 UL13 磷酸化一个或多个被膜蛋白，从而使这些假定的蛋白原初化以诱导从基因组沉默中逃逸。
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