Abstract
ABSTRACTInfectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell.IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection.
Highlights
Infectious rotavirus particles are triple-layered, icosahedral assemblies
We showed in previous work that during infection of BSC-1 cells, recoated TLPs (rcTLPs) become insensitive to elution by EDTA within a few minutes of attachment [8]
We found that for the rhesus rotavirus (RRV) rcTLPs and BSC-1 cells in our experiments, about 5% of the particles that attach to the cells synthesize mRNA and that the number of transcribing particles per cell corresponds to the multiplicity of infection (MOI)
Summary
Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. RNA synthesis by even a single, uncoated particle can initiate infection in a cell. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. RNA synthesis by even a single virus particle can initiate a productive infection. Imaging of single virus particles as they enter cells, made possible by advances in fluorescence microscopy [4], offers a way to connect in situ observation of the entry process with virion structure and biochemistry. The result of successful penetration is release of the double-layered particle (DLP) into the cytosol, with loss of the two outer-layer proteins, VP4 and VP7 [10, 11]. VP4, activated by cleavage to VP8* and VP5* [12, 13], is the molecular agent of penetration [14, 15], with folding back of the VP5* trimer likely to be the essential membrane-disruptive step [16, 17]
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