Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus.

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.


Complexity and ultrastructure of infectious extracellular vesicles from cells infected by non-enveloped virus
(a) Proteins from lysates of PV-and mock-infected cells at 3, 4, 5, 6, and 7 hours post infection (hpi) were probed by western blot. The blot was first probed for GAPDH (top panel) to show similar loading in all lanes. The blot was then stripped, and re-probed using polyclonal anti-3D antibodies. All species containing the 3D protein are expected to be labeled by the anti-3D antibody (e.g. Garmarnik & Andino (1998). Genes and Development 12:2293-2304, and Teterina et al., (2001. J. Virology 75:3841-3850); these additional species are shown in parentheses. (b) Proteins from lysates of mock-infected (3 and 7 hpi) and PV-infected cells (3, 4, 5, 6, and 7 hpi) were probed for PV 3A; it is expected that 3AB is also labeled by this antibody, as indicated by parentheses. (c) Proteins from lysates of PV-infected (Inf) and mock-infected (Mock) cells at 7 hpi were probed for PV 2C.
3AB: purified PV 3AB protein Bl: blank lanes MW: molecular weight markers. Vesicles were run on tricine gels, and probed with (a) anti-VP capsid protein antibodies; the membrane on the left is shown for its molecular weight markers (MW) and poliovirus stock solution, (b) anti-C antibodies; Mock 1 (vesicles from mock-infected cells) and Infect 1 (vesicles from PV-infected cells) are from one experiment, and Mock 2 and Infect 2 are from an independent experiment, (c) anti-3D antibodies; Mock prep 1 (vesicles from mock-infected cells) and Infected prep 1 (vesicles from PV-infected cells) are from one experiment, and Mock prep 2 and Infected prep 2 are from an independent experiment, (d) anti-3A antibodies; lanes are samples of vesicles secreted by mock-and PV-infected cells at 7 hpi. All species containing the protein probed for are expected to be labeled by that antibody (see, e.g., Garmarnik & Andino The titer of poliovirus stock solutions was determined by plaque assay before (black) and after (white) freeze-thaw, detergent, and RNAse treatment. The y-axis indicates the viral titer (plaqueforming units) while the x-axis indicates three independent experiments.

Supplementary Figure 5. Virions were rarely seen dispersed within the bundled actin filaments carried by infectious microvesicles.
Successive 1 nanometer thick slices through a reconstructed cryo-electron tomogram (a-d).
Traced models of (a'-d') depict the disappearance and appearance of individual virions (red hexagons) among actin filaments (black arrows). The scale bar is 100 nm.

Supplemental Movie. Cryotomogram of a microvesicle from poliovirus-infected cells
Movie of an electron cryotomogram of a Class III microvesicle isolated from poliovirus-infected cells. Labeled, and later shown in isosurface-rendered models in the tomogram, are virions, vesicle membranes, mat-like structures, and the inner vesicular structure. Additional labels indicate representative annexin-V beads that were used to isolate the sample, and fiducial gold beads that were added to aid alignment of tilt series data. Scale bar, 100 nm.
Supplemental Table S1. Proteins Identified in Microvesicles Isolated from Cells that were:

Table S1. Proteomic profiles of microvesicles from PV-and mock-infected cells at 8 hpi.
We report the full proteomic profile of proteins that were identified in both of two independent LC/MS experiments. Microvesicles from PV-infected cells carry 65 identified host protein matches (columns to the left of the green column, including Accession number, protein name, -10lgP, P, and aggregated area from one of the experiments). Microvesicles from mock-infected cells carry five host protein matches (columns to the right of the green column). All proteins identified in samples from mock-infected cells were also identified in samples from PV-infected cells. All proteomic analyses were conducted at a specified precursor ion (MS1) error tolerance of 10 ppm, a fragment ion (MS/MS) error tolerance of 0.02 Da. and a target-decoy false discovery threshold of 0.1%.