ABSTRACT

Interactions between the cytoplasmic domains of viral transmembrane proteins and host machinery often determine the outcome of viral infection. The M2 protein of influenza A has been identified as a key player in autophagy-mediated viral replication. Here, we describe the engineering and validation of an antibody specific for the cytoplasmic domain of the M2 protein. Through phage and yeast display selection techniques, we obtained an antibody that recognizes: 1) the M2 cytoplasmic domain purified from bacterial inclusion bodies and refolded, 2) full-length M2 recombinant protein expressed in mammalian cells, and 3) native M2 protein in influenza A infected cells. This antibody can serve as a molecular tool to enhance our knowledge of protein–protein interactions between influenza A virus and the host cell machinery. We anticipate the methods described herein will further the development of antibodies specific to the cytoplasmic domains of transmembrane proteins.

摘要

病毒跨膜蛋白的细胞质结构域与宿主机器之间的相互作用通常决定病毒感染的结果。甲型流感的 M2 蛋白已被确定为自噬介导的病毒复制的关键参与者。在这里，我们描述了对 M2 蛋白的细胞质域特异的抗体的工程和验证。通过噬菌体和酵母展示选择技术，我们获得了一种抗体，可识别：1) 从细菌包涵体中纯化并重新折叠的 M2 细胞质结构域，2) 在哺乳动物细胞中表达的全长 M2 重组蛋白，以及 3) 流感中的天然 M2 蛋白受感染的细胞。这种抗体可以作为一种分子工具来增强我们对甲型流感病毒与宿主细胞机制之间蛋白质-蛋白质相互作用的了解。