RNA transfer from poliovirus 135S particles across membranes is mediated by long umbilical connectors.
During infection, the binding of poliovirus to its cell surface receptor at 37°C triggers an expansion of the virus in which internal polypeptides that bind to membranes are externalized. Subsequently, in a poorly understood process, the viral RNA genome is transferred directly across an endosomal membrane, and into the host cell cytoplasm, to initiate infection. Here, cryoelectron tomography demonstrates the results of 37°C warming of a poliovirus-receptor-liposome model complex that was produced using Ni-nitrilotriacetic acid lipids and His-tagged receptor ectodomains. In total, 651 subtomographic volumes were aligned, classified, and averaged to obtain detailed pictures, showing both the conversion of virus into its expanded form and the passage of RNA into intact liposomes. Unexpectedly, the virus and membrane surfaces were located ∼50 Å apart, with the 5-fold axis tilted away from the perpendicular, and the solvent spaces between them were spanned by either one or two long "umbilical" density features that lie at an angle to the virus and membrane. The thinner connector, which sometimes appears alone, is 28 to 30 Å in diameter and has a footprint on the virus surface located close to either a 5-fold or a 3-fold axis. The broader connector has a footprint near the quasi-3-fold hole that opens upon virus expansion and is hypothesized to include RNA, shielded from enzymatic degradation by polypeptides that include the N-terminal extension of VP1 and capsid protein VP4. The implications of these observations for the mechanism of RNase-protected RNA transfer in picornaviruses are discussed.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
Mike
M
Strauss
Hazel C
HC
Levy
Mihnea
M
Bostina
David J
DJ
Filman
James M
JM
Hogle
D055032:Electron Microscope Tomography; D008081:Liposomes / Q000378:metabolism*; D008954:Models, Biological; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D009942:Organometallic Compounds; D017955:Poliovirus / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D012367:RNA, Viral / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D013696:Temperature; D053585:Virus Attachment; D053586:Virus Internalization
D008081:Liposomes; D009942:Organometallic Compounds; D012367:RNA, Viral; D011991:Receptors, Virus; C066100:poliovirus receptor; C088321:nickel nitrilotriacetic acid; D009571:Nitrilotriacetic Acid