Imaging Single Retrovirus Entry through Alternative Receptor Isoforms and Intermediates of Virus-Endosome Fusion

PLoS Pathogens

Naveen K. Jha¹  Olga Latinovic¹  Gennadiy Novitskiy¹  Erik Martin¹  Gregory B. Melikyan¹  Mariana Marin¹  Kosuke Miyauchi¹  John A. T. Young²  John Naughton² 

[1] Institute of Human Virology and Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America;Nomis Center for Immunobiology and Microbial Pathogenesis, The Salk Institute for Biological Studies, La Jolla, California, United States of America

关键词: Cell fusion;    Membrane fusion;    Endosomes;    Viral entry;    Virions;    Transmembrane receptors;    Endocytosis;    Lipids;   

DOI  :  10.1371/journal.ppat.1001260

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

【 授权许可】

CC BY