Abstract
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.
Highlights
Viral fusion proteins mediate the release of nucleocapsid into the cytoplasm by merging the viral and cellular membranes
We imaged the entry of ‘‘labeled’’ ASLV into cells using fluorescence microscopy and found important differences between ASLV entry, depending upon whether the virus was attached to a transmembrane receptor or to a lipid-anchored receptor
A transmembrane receptor supported faster virus uptake followed by the formation of larger and more stable fusion pores, which was somewhat delayed relative to ASLV entry into acidic endosomes
Summary
Viral fusion proteins mediate the release of nucleocapsid into the cytoplasm by merging the viral and cellular membranes. Viruses that rely on low pH to activate their fusion proteins are normally confined to entry through endocytic pathways, whereas those that mediate fusion at neutral pH, following the interactions with cellular receptors, can fuse directly to the plasma membrane or to endocytic compartments [4,5,6,7,8,9,10]. Envelope glycoproteins of viruses entering cells through pHdependent endocytic pathways are capable of mediating cell-cell fusion when expressed in the plasma membrane and exposed to acidic pH. Recent studies revealed that productive virus entry occurs through interplay between the envelope glycoproteins and cellular factors, many of which are endosome-resident [14,15,16,17,18,19]. Fusion should be investigated at the physiological virus entry site(s)
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