Cell, Vol. 23, 423-431, February 1981, Copyright 0 1981 by MIT Fluorescence Photobleaching Recovery Measurements Reveal Differences in Envelopment of Sindbis and Vesicular Stomatitis Viruses David C. Johnson and Milton J. Schlesinger Department of Microbiology and Immunology Elliot L. Elson Department of Biological Chemistry Washington University School of Medicine St. Louis, Missouri 63110 Summary Fluorescence photobleaching recovery (FPR) mea- surements of virus glycoproteins on the surfaces of cells infected with vesicular stomatitis virus (VSV) and Sindbis virus showed that the VSV glycoprotein (G) remained mobile throughout the infectious cy- cle, whereas Sindbis virus glycoproteins (El, E2) were partially mobile early after infection and im- mobile at later times when greater amounts of these proteins were on the cell surface. A highly mobile fraction of Sindbis virus glycoproteins was detected throughout the replication cycle of a temperature- sensitive mutant unable to form virus particles. Thus immobilization of El and E2 was the result of increasing surface glycoprotein concentrations and virus budding. Together with other data, which in- cluded the detection of El and E2 in particles as soon as these proteins were transported to the cell surface, the FPR results suggest that Sindbis virus assembly initiates on intracellular vesicles, where glycoproteins aggregate and bind nucleocapsids. In contrast, our FPR data on VSV support a model previously suggested by others, in which a small fraction of cell-surface G is immobilized into bud- ding sites formed by interactions with virus matrix and nucleoproteins. FPR measurements also pro- vide direct evidence for strong interactions between El and E2, as well as between El and PE2, the precursor form of E2. Introduction Complex biomolecular structures frequently are ca- pable of self-assembly. This implies that coded within the components of such structures are determinants of the pathways and mechanisms for their assembly. The morphogenesis of viruses such as the small bac- teriophages and plant viruses, containing only protein and nucleic acid, are prime examples of this principle. For animal viruses that contain a lipid envelope, self- assembly is more complicated, for it requires a com- bining of both soluble and membrane-bound compo- nents, which are synthesized and transported to the sites of virus assembly by different pathways in the animal cell. Replication of small, enveloped animal viruses depends upon several host-cell systems, which have complex structural and regulatory inter- actions with each other. It is therefore interesting to compare the mechanisms of assembly of different kinds of enveloped viruses to discover the extent to which differences in virus structure are correlated with and determine differences in morphogenic pathways. This information is useful to characterize both the viruses themselves and the operation of host-cell syn- thetic and transport systems. We have been studying the final events in the as- sembly pathways of two enveloped RNA animal vi- ruses, Sindbis virus and vesicular stomatitis virus (VSV). Our approach centers on measurements of the rates of lateral diffusion of virus glycoproteins on the surfaces of infected cells, by the method of fluores- cence photobleaching recovery (FPR). This method has been used to measure the lateral mobilities of a number of cell-surface proteins and lipid probes in several different kinds of cells (Axelrod et al., 1976; Schlessinger et al., 1976; Cherry, 1979; Schlessinger and Elson, 1981). The mobility of a molecule can be decreased by interaction with other cell-surface or cytoplasmic components (Schlessinger et al., 1977; Elson and Reidler. 1979). Hence we use our mea- surements as an indication of the interactions under- gone by glycoproteins on the cell surface as they are assembled into nascent budding viruses. This meth- odology has already been applied to a study of the interaction between the matrix protein (M) and the glycoprotein (G) of VSV (Reidler et al., 1981). Sindbis virus and VSV differ substantially in struc- ture. VSV is a bullet-shaped particle (65 x 180 nm) in which the M protein intervenes between a helical nucleocapsid and a lipid envelope, which contains a single species of glycoprotein (G). Sindbis virus is a somewhat smaller (diameter = 60 nm) icosahedral particle containing a nucleocapsid of cubic symmetry surrounded by a lipid envelope containing two species of glycoproteins, El and E2. It has no separate matrix protein. Current views of the assembly of these viruses have developed from biochemical and electron micro- scopic studies. Extensive biochemical data have been reported on the biosynthesis of the glycoproteins of these two viruses and the pathways by which they move to the cell surface (reviewed in Schlesinger and Kaariainen, 1980; Lodish et al., 1981). The final stages of virus envelopment have been elegantly stud- ied by electron microscopic techniques (Acheson and Tamm, 1967; Grimley et al., 1968; Coward et al., 1971; Zajac and Hummeler, 1971 ; Birdwell et al., 1973; Brown and Smith, 1975; Brown and Reidel, 1977). Based on these data, models have been pro- posed for the molecular events that occur as virus components interact (Brown et al., 1972; Cartwright et al., 1972; Lenard and Compans, 1974). Garoff and Simons (1974) have suggested that, for Semliki forest virus-a strain closely related to Sindbis virus-the virus nucleocapsid acts as a nucleation site that pro- motes an accumulation and immobilization of glyco-