African Swine Fever Virus Uses Macropinocytosis to Enter Host Cells

African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na+/H+ exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved.

非洲猪瘟（African swine fever, ASF）由一种大型高致病性DNA病毒——非洲猪瘟病毒（African swine fever virus, ASFV）引发，该病毒会造成严重的经济损失与疫情扩散威胁。目前尚无针对ASF的特异性防护手段或疫苗，尽管该病在撒哈拉以南非洲持续流行、2007年高加索地区暴发的疫情，以及向周边国家扩散的潜在风险，均构成极高的危害。尽管病毒入侵是开发防护手段的关键靶点，但目前学界对ASFV入侵机制的认知仍十分有限。早期研究曾提出病毒通过受体介导的内吞作用进入细胞，但ASFV所采用的具体入侵机制仍不明确。本研究使用了适配Vero细胞（Vero cells）增殖的ASFV强毒力分离株Ba71（Ba71V）以及强毒株E70，证实ASFV的入侵与内化过程具备巨胞饮（macropinocytosis）的绝大多数特征。通过光学显微镜与电子显微镜联合观察，我们发现病毒可引发细胞质膜扰动、起泡及膜褶皱形成。研究同时发现，病毒粒子的内化过程依赖于肌动蛋白重组、Na+/H+交换蛋白的活性，以及巨胞饮式内吞作用的典型信号传导事件。病毒进入细胞可直接刺激葡聚糖摄取、肌动蛋白极化，以及表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）、磷脂酰肌醇3-激酶-蛋白激酶B（Phosphatidylinositol 3-kinase-Akt, PI3K-Akt）、p21活化激酶1（p21-activated kinase 1, Pak1）和Ras相关C3肉毒杆菌毒素底物1（Ras-related C3 botulinum toxin substrate 1, Rac1）的激活。抑制这些巨胞饮关键调控因子，或使用药物EIPA进行处理，均可显著降低ASFV的入侵效率与感染能力。综上，本研究首次明确了ASFV入侵的完整通路，涵盖病毒摄取所需的关键细胞因子以及所涉及的细胞信号传导过程。