Abstract
Enteroviruses support cell-to-cell viral transmission prior to their canonical lytic spread of virus. Poliovirus (PV), a prototype for human pathogenic positive-sense RNA enteroviruses, and picornaviruses in general, transport multiple virions en bloc via infectious extracellular vesicles, 100~1000 nm in diameter, secreted from host cells. Using biochemical and biophysical methods we identify multiple components in secreted microvesicles, including mature PV virions; positive-sense genomic and negative-sense replicative, template viral RNA; essential viral replication proteins; and cellular proteins. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious microvesicles containing both virions and a unique morphological component that we describe as a mat-like structure. While the composition of these mat-like structures is not yet known, based on our biochemical data they are expected to be comprised of unencapsidated RNA and proteins. In addition to infectious microvesicles, CD9-positive exosomes released from PV-infected cells are also infectious and transport virions. Thus, our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral unencapsidated RNAs, viral and host proteins. Understanding the structure and function of these infectious particles helps elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.
Highlights
HeLa cells were cultured in low-glucose DMEM medium supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-streptomycin-glutamine
After three washes with PBS + (PBS supplemented with 0.01 mg/ml MgCl2 and 0.01 mg/ml CaCl2), cells were infected either with PBS + or PBS + and PV stock at a multiplicity of infection (MOI) of 30 virions per cell, titrated by classic plaque assay
For the infectious microvesicle-induced infection assay shown in Fig. 3, infection was first synchronized by addition of infectious microvesicles on ice for 30 min to promote adherence, washed with PBS+, as in[52,53] prior to growth at 37 °C in supplDMEM
Summary
Because PV replication and release of infectious extracellular vesicles decreased when cells were cultured in non-bovine serum media[51], we first infected HeLa cells in FBS-containing media for 4 h, during which the RNA replication rate reaches its maximum. To test for cell viability at the time of vesicle collection (Fig. 1b), HeLa cells were infected with PV at an MOI of 30 for 8 h, stained in situ with a 0.2% Trypan Blue solution in PBS.
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