Complexity and ultrastructure of infectious extracellular vesicles from cells infected by nonenveloped virus

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 secreted from host cells. Using biochemical and biophysical methods we identify multiple components in these secreted vesicles, including PV virions; positive and negative-sense viral RNA; essential viral replication proteins; ribosomal and regulatory cellular RNAs; and numerous host cell proteins, such as regulators of cellular metabolism and structural remodeling. Using cryo-electron tomography, we visualize the near-native three-dimensional architecture of secreted infectious extracellular vesicles containing both virions and a unique mat-like structure. Based on our biochemical data (western blot, RNA-Seq, and mass spectrometry), these mat-like structures are expected to be comprised of unencapsidated RNA and proteins. Our data show that, prior to cell lysis, non-enveloped viruses are secreted within infectious vesicles that also transport viral and host RNAs and proteins.\n\nImportanceThe family of picornaviridae is comprised of small positive-sense RNA viruses, many of which are significant human pathogens. Picornaviruses exploit secreted extracellular vesicles for cell-to-cell viral transmission without cell lysis, and poliovirus serves as a model system for picornaviruses that are not protected by a surrounding membrane (non-enveloped viruses). The structure and contents of these vesicles secreted by virus-infected cells are described here. In addition to mature virions, these vesicles carry negative-sense, template viral RNA and essential replication proteins, as well as cellular resources from the host. Their complex contents may comprise an enhanced virulence factor for propagation of infection, and understanding their structure and function is helping elucidate the mechanism by which extracellular vesicles contribute to the spread of non-enveloped virus infection.