Viral tracers are important tools for mapping brain connectomes. The feature of predominant anterograde transneuronal transmission offers herpes simplex virus-1 (HSV-1) strain H129 (HSV1-H129) as a promising candidate to be developed as anterograde viral tracers. In our earlier studies, we developed H129-derived anterograde polysynaptic tracers and TK deficient (H129-dTK) monosynaptic tracers. However, their broad application is limited by some intrinsic drawbacks of the H129-dTK tracers, such as low labeling intensity due to TK deficiency and potential retrograde labeling caused by axon terminal invasion. The glycoprotein K (gK) of HSV-1 plays important roles in virus entry, egress, and virus-induced cell fusion. Its deficiency severely disables virus egress and spread, while only slightly limits viral genome replication and expression of viral proteins. Therefore, we created a novel H129-derived anterograde monosynaptic tracer (H129-dgK) by targeting gK, which overcomes the limitations of H129-dTK. Using our established platform and pipeline for developing viral tracers, we generated a novel tracer by deleting the gK gene from the H129-G4. The gK-deleted virus (H129-dgK-G4) was reconstituted and propagated in the Vero cell expressing wildtype H129 gK (gKwt) or the mutant gK (gKmut, A40V, C82S, M223I, L224V, V309M), respectively. Then the obtained viral tracers of gKmut pseudotyped and gKwt coated H129-dgK-G4 were tested in vitro and in vivo to characterize their tracing properties. H129-dgK-G4 expresses high levels of fluorescent proteins, eliminating the requirement of immunostaining for imaging detection. Compared to the TK deficient monosynaptic tracer H129-dTK-G4, H129-dgK-G4 labeled neurons with 1.76-fold stronger fluorescence intensity, and visualized 2.00-fold more postsynaptic neurons in the downstream brain regions. gKmut pseudotyping leads to a 77% decrease in retrograde labeling by reducing axon terminal invasion, and thus dramatically improves the anterograde-specific tracing of H129-dgK-G4. In addition, assisted by the AAV helper trans-complementarily expressing gKwt, H129-dgK-G4 allows for mapping monosynaptic connections and quantifying the circuit connectivity difference in the Alzheimer’s disease and control mouse brains. gKmut pseudotyped H129-dgK-G4, a novel anterograde monosynaptic tracer, overcomes the limitations of H129-dTK tracers, and demonstrates desirable features of strong labeling intensity, high tracing efficiency, and improved anterograde specificity.

中文翻译：

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一种新型的基于 H129 的顺行单突触示踪剂具有标记强度强、示踪效率高和减少逆行标记的特点

病毒示踪剂是绘制大脑连接组图的重要工具。主要的顺行跨神经元传播的特征为单纯疱疹病毒 1 (HSV-1) 株 H129 (HSV1-H129) 提供了作为顺行病毒示踪剂开发的有希望的候选者。在我们早期的研究中，我们开发了 H129 衍生的顺行多突触示踪剂和 TK 缺陷 (H129-dTK) 单突触示踪剂。然而，它们的广泛应用受到 H129-dTK 示踪剂的一些内在缺陷的限制，例如由于 TK 缺乏导致的低标记强度和轴突末端入侵引起的潜在逆行标记。HSV-1 的糖蛋白 K (gK) 在病毒进入、排出和病毒诱导的细胞融合中起重要作用。它的缺陷严重阻碍了病毒的出口和传播，而仅略微限制病毒基因组复制和病毒蛋白的表达。因此，我们通过靶向 gK 创建了一种新的 H129 衍生的顺行单突触示踪剂 (H129-dgK)，它克服了 H129-dTK 的局限性。使用我们建立的用于开发病毒示踪剂的平台和管道，我们通过从 H129-G4 中删除 gK 基因生成了一种新型示踪剂。gK 缺失病毒 (H129-dgK-G4) 在表达野生型 H129 gK (gKwt) 或突变型 gK (gKmut、A40V、C82S、M223I、L224V、V309M) 的 Vero 细胞中重组和繁殖。然后在体外和体内测试获得的 gKmut 假型和 gKwt 包被的 H129-dgK-G4 病毒示踪剂以表征其示踪特性。H129-dgK-G4 表达高水平的荧光蛋白，无需免疫染色进行成像检测。与 TK 缺陷型单突触示踪剂 H129-dTK-G4 相比，H129-dgK-G4 标记的神经元具有 1.76 倍强的荧光强度，并且在下游大脑区域中显示了 2.00 倍的突触后神经元。gKmut 假型通过减少轴突末端入侵导致逆行标记减少 77%，从而显着改善 H129-dgK-G4 的顺行特异性追踪。此外，在 AAV 辅助反式互补表达 gKwt 的帮助下，H129-dgK-G4 允许绘制单突触连接并量化阿尔茨海默病和对照小鼠大脑中的电路连接差异。gKmut 假型 H129-dgK-G4 是一种新型顺行单突触示踪剂，克服了 H129-dTK 示踪剂的局限性，具有标记强度强、示踪效率高、