Rojek Jillian M, Sanchez Ana B, Nguyen Ngoc Thao, de la Torre Juan-Carlos, Kunz Stefan
Viral Immunobiology Laboratory, Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California 92037, USA.
J Virol. 2008 Aug;82(15):7677-87. doi: 10.1128/JVI.00560-08. Epub 2008 May 28.
The Old World arenavirus Lassa virus (LASV) is the causative agent of severe viral hemorrhagic fever (VHF) in humans and is the most prevalent human pathogen among arenaviruses. The present study investigated the largely unknown mechanisms of cell entry of LASV, a process know to be mediated solely by the virus envelope glycoprotein (GP). To circumvent biosafety restrictions associated with the use of live LASV, we used reverse genetics to generate a recombinant variant of the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) expressing the LASV GP (rLCMV-LASVGP). The rescued rLCMV-LASVGP grew to titers comparable to that of LCMV and showed the receptor binding characteristics of LASV. We used rLCMV-LASVGP to characterize the cellular mechanisms of LASV entry in the context of a productive arenavirus infection. The kinetics of pH-dependent membrane fusion of rLCMV-LASVGP resembled those of the human-pathogenic New World arenavirus Junin virus (JUNV) and other enveloped viruses that use clathrin-mediated endocytosis for entry. However, rLCMV-LASVGP entered cells predominantly via a clathrin-, caveolin-, and dynamin-independent endocytotic pathway similar to the one recently described for LCMV. Productive infection of rLCMV-LASVGP was only mildly affected by a dominant negative mutant of Rab5 and was independent of Rab7, suggesting an unusual mechanism of delivery to endosomes. In addition, rLCMV-LASVGP infection was independent of actin but required intact microtubules. Our data indicate that LASV enters cells via a pathway distinct from the one used by human-pathogenic New World arenaviruses.
旧大陆沙粒病毒拉沙病毒（LASV）是人类严重病毒性出血热（VHF）的病原体，也是沙粒病毒中最常见的人类病原体。本研究调查了LASV进入细胞的机制，这一过程已知仅由病毒包膜糖蛋白（GP）介导，但在很大程度上尚不明确。为规避与使用活LASV相关的生物安全限制，我们利用反向遗传学技术构建了一种表达LASV GP的原型沙粒病毒淋巴细胞性脉络丛脑膜炎病毒（LCMV）的重组变体（rLCMV-LASVGP）。拯救出的rLCMV-LASVGP生长至与LCMV相当的滴度，并表现出LASV的受体结合特性。我们利用rLCMV-LASVGP在有活性的沙粒病毒感染背景下，对LASV进入细胞的细胞机制进行了表征。rLCMV-LASVGP的pH依赖性膜融合动力学类似于人类致病性新大陆沙粒病毒胡宁病毒（JUNV）以及其他利用网格蛋白介导的内吞作用进入细胞的包膜病毒。然而，rLCMV-LASVGP主要通过一种与网格蛋白、小窝蛋白和发动蛋白无关的内吞途径进入细胞，这一途径类似于最近报道的LCMV的途径。rLCMV-LASVGP的有效感染仅受到Rab5显性负性突变体的轻微影响，且不依赖于Rab7，这表明其进入内体的机制不同寻常。此外，rLCMV-LASVGP感染不依赖于肌动蛋白，但需要完整的微管。我们的数据表明，LASV通过一种与人类致病性新大陆沙粒病毒不同的途径进入细胞。
