John Heuser, Robyn Roth, Lyusienna Loultcheva & Jennifer ... Heuser Lectures/PDF_Slide_Thu… ·...
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Application of "deep-etch" EM to the study of Vaccinia virus morphogenesis and dissemination John Heuser, Robyn Roth, Lyussiena Loultcheva and Jennifer Scott Department of Cell Biology and Physiology, Washington University, St. Louis, MO and Patricia Szajner * , Andrea Weisberg * , and Bernard Moss * Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD The surprise starting-point of this study: immature vaccinia viruses are exposed so beautifully by "deep-etch"EM! (This involves quick freezing infected cells while still alive, "deep etching" them by vacuum-sublimation of the ice, and platinum replication for transmission electron microscopy). Those viruses just "grazed" (by fracturing right over the tops of them) reveal a honeycomb lattice never seen before (....or not properly seen before). Those actually freeze-fractured reveal beneath the honeycomb surface-lattice a membrane envelope composed of a single lipid bilayer. . Those freeze-fractured more deeply (and actually avulsed from the cell) leave behind the other half of the lipid bilayer, (presumably, still stabilized by a honeycomb lattice on their backside, facing down into the underlying ice) Overexpressing the viral coat protein (D13) leads not to spheres, but the accumulation of huge sheets of honeycomb lattice that are flat . (I'll gladly buy you a drink if you can tell me why they are flat, not spherical.) Anti-D13 antibody of course labels the sheets, but only on their freeze- fractured edges, indicating that the epitope we used must be buried in the honeycomb-lattice. Same antibody-labeling of normal immature viruses also labels the fractured edges of the honeycomb surface. (Note how "scalped" viruses would give the impression of D13 being inside the virus, in traditional immunogold-EM of cryosections, but it's actually not!) Same antibody-labeling of normal immature viruses also labels the fractured edges of the honeycomb surface. (Note how "scalped" viruses would give the impression of D13 being inside the virus, in traditional immunogold-EM of cryosections, but it's actually not!) The fun is not over! Once closed and complete, the D13 honeycomb gets removed and a different crystalline lattice forms on the "mature" virus. This "mature" form gets engulfed by a host- cell membrane to create the final "enveloped" virus. Freeze-fracture reveals the wrapping event in exquisite detail. (The origin of the wrapping membrane is still at question: Golgi (TGN) or endosomal?) Freeze-drying of infected cells displays emergence of the enveloped virus at the cell surface, driven by microtubule- motor interactions (inside,not shown). (Here actin polymerization is beginning to generate a 'comet-tail" under the virus; but don't get too excited, just go hear Michael Way's talk.) The mysterious conversion from cell- attached viruses that are nonfusigenic (above) to fusigenic (below) involves a dramatic reorganization of their envelope. (Remind me to tell you what activates this conversion...heh, heh.) When activated, the envelope ruptures, exposing the naked mature virus directly to the cell surface. (This is pretty dangerous for the cell!) The result can be direct fusion of the virus's single bilayer with the cell surface, here demonstrated by the beginning of dispersion of the viral coat into the plasmalemma. Within tens of seconds, the viral coat begins to break up into the individual elements that originally comprised it: what EM'ists have since the 1960's called "mulberries". (An unfusedvirus is shown in the upper left, for size- comparison.) The three-dimensional, topological views provided by "deep-etch" electron microscopy (DEEM) have begun to resolve several controversies that have plagued poxvirus research. First, because DEEM starts with 'quick-freezing' of living cells, it prevents fixative-induced membrane distortions that have confused earlier analyses of virogenesis. Importantly, it shows that the primary intracellular vaccinia capsule is a single membrane bilayer, coated over its entire external surface with a polygonal lattice of protein. This is confirmed by freeze-fracturing of IV's, which invariably yields only one fracture plane through their single membrane (and also yields many crossfractures of its external honeycomb lattice, which demonstrate why it looked like a series of 'spikes' of p65 in earlier thin-sections). The tight organization of this honeycomb network explains how the single IV membrane can remain stable in the cytoplasm during its formation from crescent-shaped precursors. DEEM also demonstrates that the honeycomb network on the IV is replaced by a very different, compact paracrystalline coat on the on the exterior of the IMV, coincident with the condensation of its core. DEEM further reveals that this surface-maturation is accompanied by a collapse of host- cell cytoplasm onto the surface of the IMV (which otherwise tends to withdraw from the IV's surface-honeycomb during deep-etching). With this collapse comes activation of a sort of autophagocytosis of the IMV to form the IEV. Further steps in viral maturation and dissemination are best seen by switching from "deep-etching" to complete freeze- drying of cells, an procedural extension that permits straightforward visualization of the external surfaces of quick-frozen cells. This graphically displays discharged CEVs still attached to infected cells, and also displays the inevitable breakdown of the host- membrane that surrounds these CEVs, resulting in the re-exposure and freeing of IMVs from within CEVs. Finally, DEEM clearly reveals the fusion of such released IMVs with the plasma membranes of uninfected cells, as it clearly displays the delivery and dispersion of protein-components of the IMV coat directly into these cells' surface membranes. Application of "deep-etch" EM to the study of Vaccinia virus morphogenesis and dissemination John Heuser, Robyn Roth, Lyusienna Loultcheva & Jennifer Scott and Patricia Szajner * , Andrea Weisberg * , and Bernard Moss * Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD
Transcript of John Heuser, Robyn Roth, Lyusienna Loultcheva & Jennifer ... Heuser Lectures/PDF_Slide_Thu… ·...
Application of "deep-etch" EM to the study of Vaccinia virus morphogenesis and dissemination
John Heuser, Robyn Roth, Lyussiena Loultcheva and Jennifer Scott Department of Cell Biology and Physiology, Washington University, St. Louis, MO
and Patricia Szajner*, Andrea Weisberg*, and Bernard Moss*
Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD
The surprise starting-point of this study: immature vaccinia viruses are exposed so beautifully by "deep-etch"EM! (This involves quick freezing infected cells while still alive, "deep etching" them by vacuum-sublimation of the ice, and platinum replication for transmission electron microscopy).
Those viruses just "grazed" (by fracturing right over the tops of them) reveal a honeycomb lattice never seen before (....or not properly seen before).
Those actually freeze-fractured reveal beneath the honeycomb surface-lattice a membrane envelope composed of a single lipid bilayer.
Those actually freeze-fractured reveal beneath the honeycomb surface-lattice a membrane envelope composed of a single lipid bilayer.
Those freeze-fractured more deeply (and actually avulsed from the cell) leave behind the other half of the lipid bilayer, (presumably, still stabilized by a honeycomb lattice on their backside, facing down into the underlying ice)
Overexpressing the viral coat protein (D13) leads not to spheres, but the accumulation of huge sheets of honeycomb lattice that are flat. (I'll gladly buy you a drink if you can tell me why they are flat, not spherical.)
Anti-D13 antibody of course labels the sheets, but only on their freeze-fractured edges, indicating that the epitope we used must be buried in the honeycomb-lattice.
Same antibody-labeling of normal immature viruses also labels the fractured edges of the honeycomb surface. (Note how "scalped" viruses would give the impression of D13 being inside the virus, in traditional immunogold-EM of cryosections, but it's actually not!)
Same antibody-labeling of normal immature viruses also labels the fractured edges of the honeycomb surface. (Note how "scalped" viruses would give the impression of D13 being inside the virus, in traditional immunogold-EM of cryosections, but it's actually not!)
The fun is not over! Once closed and complete, the D13 honeycomb gets removed and a different crystalline lattice forms on the "mature" virus. This "mature" form gets engulfed by a host-cell membrane to create the final "enveloped" virus.
Freeze-fracture reveals the wrapping event in exquisite detail. (The origin of the wrapping membrane is still at question: Golgi (TGN) or endosomal?)
Freeze-drying of infected cells displays emergence of the enveloped virus at the cell surface, driven by microtubule-motor interactions (inside,not shown). (Here actin polymerization is beginning to generate a 'comet-tail" under the virus; but don't get too excited, just go hear Michael Way's talk.)
The mysterious conversion from cell-attached viruses that are nonfusigenic (above) to fusigenic (below) involves a dramatic reorganization of their envelope. (Remind me to tell you what activates this conversion...heh, heh.)
When activated, the envelope ruptures, exposing the naked mature virus directly to the cell surface. (This is pretty dangerous for the cell!)
The result can be direct fusion of the virus's single bilayer with the cell surface, here demonstrated by the beginning of dispersion of the viral coat into the plasmalemma.
Within tens of seconds, the viral coat begins to break up into the individual elements that originally comprised it: what EM'ists have since the 1960's called "mulberries". (An unfused virus is shown in the upper left, for size-comparison.)
The three-dimensional, topological views provided by "deep-etch" electron microscopy (DEEM) have begun to resolve several controversies that have plagued poxvirus research. First, because DEEM starts with 'quick-freezing' of living cells, it prevents fixative-induced membrane distortions that have confused earlier analyses of virogenesis. Importantly, it shows that the primary intracellular vaccinia capsule is a single membrane bilayer, coated over its entire external surface with a polygonal lattice of protein. This is confirmed by freeze-fracturing of IV's, which invariably yields only one fracture plane through their single membrane (and also yields many crossfractures of its external honeycomb lattice, which demonstrate why it looked like a series of 'spikes' of p65 in earlier thin-sections). The tight organization of this honeycomb network explains how the single IV membrane can remain stable in the cytoplasm during its formation from crescent-shaped precursors. DEEM also demonstrates that the honeycomb network on the IV is replaced by a very different, compact paracrystalline coat on the on the exterior of the IMV, coincident with the condensation of its core. DEEM further reveals that this surface-maturation is accompanied by a collapse of host-cell cytoplasm onto the surface of the IMV (which otherwise tends to withdraw from the IV's surface-honeycomb during deep-etching). With this collapse comes activation of a sort of autophagocytosis of the IMV to form the IEV. Further steps in viral maturation and dissemination are best seen by switching from "deep-etching" to complete freeze-drying of cells, an procedural extension that permits straightforward visualization of the external surfaces of quick-frozen cells. This graphically displays discharged CEVs still attached to infected cells, and also displays the inevitable breakdown of the host-membrane that surrounds these CEVs, resulting in the re-exposure and freeing of IMVs from within CEVs. Finally, DEEM clearly reveals the fusion of such released IMVs with the plasma membranes of uninfected cells, as it clearly displays the delivery and dispersion of protein-components of the IMV coat directly into these cells' surface membranes.
Application of "deep-etch" EM to the study ofVaccinia virus morphogenesis and dissemination
John Heuser, Robyn Roth, Lyusienna Loultcheva & Jennifer Scottand
Patricia Szajner*, Andrea Weisberg*, and Bernard Moss*
Laboratory of Viral Diseases, NIAID, NIH, Bethesda, MD
