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Abstract
BackgroundThe majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions.ResultsHere, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments.ConclusionsSince the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments.
Highlights
The majority of viruses enter host cells via endocytosis
The nearly identical rates of fusion and endosome maturation appear to control non-selective ASLVA fusion with early and intermediate compartments in TVA800 cells. These findings show that ASLV-A can enter cells from distinct intracellular compartments and that the sites of entry are determined by the receptor isoforms
Considering that the Rab5 accumulation and acidification of endosomal lumen are tightly correlated (Figure 5B,C) and that the rate of low pH-dependent ASLV-A fusion is not affected by the receptor isoform (Figure 5C), the sites of entry are likely determined by the kinetic competition between endosome maturation and virus fusion
Summary
The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Viral entry routes have been traditionally studied by blocking one or more endocytic pathways and/or preventing endosome maturation (reviewed in [4]) These strategies include pharmacological interventions, silencing the expression of proteins involved in vesicular trafficking or over-expression of their dominant-negative mutants. Agents that raise endosomal pH block entry of viruses whose fusion is triggered by acidic conditions, but can interfere with pH-independent steps of viral entry [5,6,7] due to the coupling between endosome maturation and acidification of their lumen [8,9,10,11] Another widely used approach to elucidate the viral entry pathways is based on virus colocalization with endosomal cargo or markers for specific intracellular compartments in fixed cells.
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