Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER

Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.     

Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40 into cells devoid of caveolae

Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)-deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1-independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.     

Role of endosomes in simian virus 40 entry and infection

After binding to its cell surface receptor ganglioside GM1, simian virus 40 (SV40) is endocytosed by lipid raft-mediated endocytosis and slowly transported to the endoplasmic reticulum, where partial uncoating occurs. We analyzed the intracellular pathway taken by the virus in HeLa and CV-1 cells by using a targeted small interfering RNA (siRNA) silencing screen, electron microscopy, and live-cell imaging as well as by testing a variety of cellular inhibitors and other perturbants. We found that the virus entered early endosomes, late endosomes, and probably endolysosomes before reaching the endoplasmic reticulum and that this pathway was part of the infectious route. The virus was especially sensitive to a variety of perturbations that inhibited endosome acidification and maturation. Contrary to our previous models, which postulated the passage of the virus through caveolin-rich organelles that we called caveosomes, we conclude that SV40 depends on the classical endocytic pathway for infectious entry.     

Interaction of endocytotic vacuoles with the inner nuclear membrane in simian virus 40 entry into CV-1 cell nucleus.

The transfer of endocytosed simian virus 40 (SV40) to the nuclear position was investigated ultrastructurally using cationized ferritin (CF), ferritin labelled concanavalin A (Fer-Con A) and Con A as cell membrane markers. In the cells incubated with these markers and SV40 at 4 degrees C, and then chased for 2 h at 37 degrees C in serum-free medium, ferritin particles representing CF and/or Fer-Con A binding sites were found in vacuoles with SV40. The membrane of some vacuoles seemed to be in contact with the outer nuclear membrane. Several ferritin particles were located in the perinuclear cisterna and within the nucleoplasm, but not within the nuclear pores. In addition, there were vacuoles with ferritin particles and SV40 near the nuclear membrane, which looked like a single diaphragm with heterochromatins inside it. The outer nuclear and vacuole membranes were often obscure in the areas where the vacuole was very close to the diaphragm. In the case of cells incubated with CF, SV40 and Con A at 4 degrees C, chased for 2 h at 37 degrees C, and then reacted with horseradish peroxidase (HRP), HRP activity showing Con A-binding sites was also observed along the nuclear side of the inner nuclear membrane as well as in the perinuclear cisterna along the outer membrane. These results confirm that SV40-induced endocytotic vacuoles fuse with the outer nuclear membrane, and further indicate that some endocytotic vacuoles may well interact directly with the diaphragm, suggesting another path for migration of SV40 into CV-1 cell nuclei besides the path going through the process of fusion of the vacuole membrane with the outer nuclear membrane.     

Plasmalemmal vesicle associated protein (PV1) modulates SV40 virus infectivity in CV-1 cells

Plasmalemmal vesicle associated protein (Plvap/PV1) is a structural protein required for the formation of the stomatal diaphragms of caveolae. Caveolae are plasma membrane invaginations that were implicated in SV40 virus entry in primate cells. Here we show that de novo Plvap/PV1 expression in CV-1 green monkey epithelial cells significantly reduces the ability of SV40 virus to establish productive infection, when cells are incubated with low concentrations of the virus. However, in presence of high viral titers PV1 has no effect on SV40 virus infectivity. Mechanistically, PV1 expression does not reduce the cell surface expression of known SV40 receptors such as GM1 ganglioside and MHC class I proteins. Furthermore, PV1 does not reduce the binding of virus-like particles made by SV40 VP1 protein to the CV-1 cell surface and does not impact their internalization when cells are incubated with either high or low VLP concentrations. These results suggest that PV1 protein is able to block SV40 infectivity at low but not at high viral concentration either by interfering with the infective internalization pathway at the cell surface or at a post internalization step.     

Release of simian virus 40 virions from epithelial cells is polarized and occurs without cell lysis.

We have investigated the process of release of simian virus 40 (SV40) virions from several monkey kidney cell lines. High levels of virus release were observed prior to any significantly cytopathic effects in all cell lines examined, indicating that SV40 utilizes a mechanism for escape from the host cell which does not involve cell lysis. We demonstrate that SV40 release was polarized in two epithelial cell types (Vero C1008 and primary African green monkey kidney cells) grown on permeable supports; release of virus occurs almost exclusively at apical surfaces. In contrast, equivalent amounts of SV40 virions were recovered from apical and basal culture fluids of nonpolarized CV-1 cells. SV40 virions were observed in large numbers on apical surfaces of epithelial cells and in cytoplasmic smooth membrane vesicles. The sodium ionophore monensin, an inhibitor of vesicular transport, was found to inhibit SV40 release without altering viral protein synthesis or infectious virus production.     

Comparison of Subacute Sclerosing Panencephalitis and Measles Viruses: an Electron Microscope Study

The ultrastructure of CV-1 cells infected with subacute sclerosing panencephalitis (SSPE) viruses was compared with that of CV-1 cells infected with the wild or Edmonston strain of measles virus. Both SSPE viruses and the measles viruses produced two types of nucleocapsid structures: smooth filaments, 15 to 17 nm in diameter, and granular filaments, 22 to 25 nm. The smooth and granular filaments produced by SSPE and measles virus did not differ in appearance. In CV-1 cells infected with SSPE viruses, smooth filaments formed large intranuclear inclusions and granular filaments occupied a large area of the cytoplasm, but always spared the area under the cell membrane. Particles budding from the surface of these cells contained no nucleocapsids. In CV-1 cells infected with measles virus, only small aggregates of smooth filaments were seen in the nuclei. Granular filaments in the cytoplasm predominantly occupied the area under the cell membrane, and were aligned beneath the cell membrane in a parallel fashion and assembled into budding particles. These differences between SSPE and measles virus may be regarded as quantitative, but they do distinguish SSPE viruses from measles virus. Moreover, the formation of large nuclear inclusions filled with smooth filaments appears to be a characteristic process of SSPE, but not of measles, since this type of inclusion is invariably seen in SSPE brain tissues, brain cultures derived from them, and CV-1 cells infected with SSPE viruses.     

The relationship between ER-multivesicular body membrane contacts and the ESCRT machinery

Activated EGFR (epidermal growth factor receptor) undergoes ESCRT (endosomal sorting complex required for transport)-mediated sorting on to ILVs (intraluminal vesicles) of endosomes before degradation in the lysosome. Sorting of endocytosed EGFR on to ILVs removes the catalytic domain of the EGFR from the cytoplasm, resulting in termination of receptor signalling. EGFR signalling is also subject to down-regulation through receptor dephosphorylation by the ER (endoplasmic reticulum)-localized PTP1B (protein tyrosine phosphatase 1B). PTP1B on the cytoplasmic face of the ER interacts with endocytosed EGFR via direct membrane contacts sites between the ER and endosomes. In the present paper, we review the relationship between ER-endosome membrane contact sites and ILV formation, and their potential role in the regulation of EGFR sorting on to ILVs, through PTP1B-mediated dephosphorylation of both EGFR and components of the ESCRT machinery.     

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.     

Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells.     

Group translocation of the ribose moiety of inosine by vesicles of plasma membrane from T(3 cells transformed by Simian virus 40.

Plasma membrane vesicles are isolated from Simian virus 40-transformed Balb/c mouse 3T3 (SV-3T3) cells. These membrane vesicles contain no significant contamination by mitochondria, endoplasmic reticulum, or lysosomes as determined by marker enzyme analysis. The use of [U-14C] inosine as a transport substrate results in the accumulation of labeled ribose-1P as transport product by the plasma membrane vesicles. This suggests the action of purine nucleoside phosphorylase (the enzyme which mediates the phosphorolysis of inosine to ribose-1-P and hypoxanthine0 before, during, or after the transport step. Neither inosine nor significant amounts of hypoxanthine are found intravesicularly. The Km for inosine, the substrate in this reaction which leads to the accumulation of ribose-1-P by the plasma membrane vesicles, is 35 to 45 muM while the Vmax for ribose-1-P accumulation is 100 to 120 pmol/min/mg of plasma membrane protein...     

Vesicular Transport of Progeny Parvovirus Particles through ER and Golgi Regulates Maturation and Cytolysis

Progeny particles of non-enveloped lytic parvoviruses were previously shown to be actively transported to the cell periphery through vesicles in a gelsolin-dependent manner. This process involves rearrangement and destruction of actin filaments, while microtubules become protected throughout the infection. Here the focus is on the intracellular egress pathway, as well as its impact on the properties and release of progeny virions. By colocalization with cellular marker proteins and specific modulation of the pathways through over-expression of variant effector genes transduced by recombinant adeno-associated virus vectors, we show that progeny PV particles become engulfed into COPII-vesicles in the endoplasmic reticulum (ER) and are transported through the Golgi to the plasma membrane. Besides known factors like sar1, sec24, rab1, the ERM family proteins, radixin and moesin play (an) essential role(s) in the formation/loading and targeting of virus-containing COPII-vesicles. These proteins also contribute to the transport through ER and Golgi of the well described analogue of cellular proteins, the secreted Gaussia luciferase in absence of virus infection. It is therefore likely that radixin and moesin also serve for a more general function in cellular exocytosis. Finally, parvovirus egress via ER and Golgi appears to be necessary for virions to gain full infectivity through post-assembly modifications (e.g. phosphorylation). While not being absolutely required for cytolysis and progeny virus release, vesicular transport of parvoviruses through ER and Golgi significantly accelerates these processes pointing to a regulatory role of this transport pathway.     

Hyaluronate-binding protein of simian virus 40-transformed 3T3 cells: membrane distribution and reconstitution into lipid vesicles

Hyaluronate-binding protein (HABP) has been extracted in detergent from the membranes of simian virus 40-transformed 3T3 (SV-3T3) cells (Underhill et al, J Biol Chem 258:8086-8091, 1983). When SV-3T3 cells were treated with trypsin prior to isolation and dissolution of the membranes, no hyaluronate-binding activity could be detected. This indicates that all of the detectable HABP of SV-3T3 cells is located on the external surface of the plasma membrane rather than on internal membranes, which would be inaccessible to the trypsin. The detergent-extracted HABP from SV-3T3 membranes was reconstituted into the membrane of lipid vesicles, which were formed by addition of exogenous phosphatidylcholine and cholic acid to the extracts followed by removal of detergent by dialysis against 0.02 M Tris pH 8.0 in the presence of protease inhibitors. Reconstitution was assessed by sedimentation in a discontinuous sucrose gradient and by gel filtration on Sepharose 4B in the presence and absence of detergent. The characteristics of binding of hyaluronate to the reconstituted HABP were then compared with those studied previously for the original membrane-bound HABP and the detergent-extracted HABP (Underhill et al, J Biol Chem 258:8086-8091, 1983). It was observed previously that binding of hyaluronate to HABP in the cell membranes was of higher affinity and specificity than to HABP in the detergent extracts of these membranes. It was found here that reconstitution of the extracted HABP into the membranes of lipid vesicles led to restoration of affinity of binding to the level observed in the original cell membranes. However, whereas chondroitin sulfate does not compete significantly for binding of hyaluronate to cell membrane-bound HABP, partial competition was observed for the reconstituted HABP as well as for detergent-extracted HABP. Thus, it is concluded that the high affinity of binding of hyaluronate to the plasma membrane of SV-3T3 cells is in part dependent on insertion of the HABP in the membrane, but that other interactions, not duplicated in our reconstitution experiments, must be necessary for the specificity of the HABP.     

Synthesis of GnRH analogues and their application in targeted gene delivery systems

A set of GnRH analogues containing the nuclear localization signal (NLS) of the SV-40 virus was synthesized using solid phase peptide synthesis and chemical ligation techniques. Selective chemical ligation was achieved through hydrazone formation upon the interaction of NLS hydrazide and GnRH analogue modified with pyruvic acid. The efficiency of the synthesized compounds was demonstrated in experiments on transfection of various human cancer cell lines with reporter luciferase and β-galactosidase genes, as well as suicide thymidine kinase gene of HSV-1. Selectivity of the peptide-DNA complex effect on cancer cells is achieved as a result of its penetration through the cell membrane via GnRH receptor-mediated endocytosis pathway.     

Direct evidence that the N-terminal heptad repeat of Sendai virus fusion protein participates in membrane fusion.

Recent studies have demonstrated the importance of heptad repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal heptad repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and heptad repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the heptad repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal heptad repeat in assisting the fusion peptide in mediating membrane fusion.     

Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles

Viral infections frequently cause endoplasmic reticulum (ER) stress in host cells leading to stimulation of the ER-associated degradation (ERAD) pathway, which subsequently targets unassembled glycoproteins for ubiquitylation and proteasomal degradation. However, the role of the ERAD pathway in the viral life cycle is poorly defined. In this paper, we demonstrate that hepatitis C virus (HCV) infection activates the ERAD pathway, which in turn controls the fate of viral glycoproteins and modulates virus production. ERAD proteins, such as EDEM1 and EDEM3, were found to increase ubiquitylation of HCV envelope proteins via direct physical interaction. Knocking down of EDEM1 and EDEM3 increased the half-life of HCV E2, as well as virus production, whereas exogenous expression of these proteins reduced the production of infectious virus particles. Further investigation revealed that only EDEM1 and EDEM3 bind with SEL1L, an ER membrane adaptor protein involved in translocation of ERAD substrates from the ER to the cytoplasm. When HCV-infected cells were treated with kifunensine, a potent inhibitor of the ERAD pathway, the half-life of HCV E2 increased and so did virus production. Kifunensine inhibited the binding of EDEM1 and EDEM3 with SEL1L, thus blocking the ubiquitylation of HCV E2 protein. Chemical inhibition of the ERAD pathway neither affected production of the Japanese encephalitis virus (JEV) nor stability of the JEV envelope protein. A co-immunoprecipitation assay showed that EDEM orthologs do not bind with JEV envelope protein. These findings highlight the crucial role of the ERAD pathway in the life cycle of specific viruses.     

Sendai virus internal fusion peptide: structural and functional characterization and a plausible mode of viral entry inhibition

Viral glycoproteins catalyze the fusion between viral and cellular membranes. The fusion protein (F(1)) of Sendai virus has two fusion peptides. One is located at its N-terminus and the other, highly homologous to the HIV-1 and RSV fusion peptides, in the interior of the F(1) protein. A synthetic peptide corresponding to the internal fusogenic domain, namely, SV-201, was found to inhibit virus-cell fusion without interfering with the binding of the virus to the target cells, thus highlighting the importance of this region in Sendai virus-induced membrane fusion. However, its detailed mechanism of inhibition remains unknown. Here, we synthesized a shorter version of SV-201, namely, SV-208, an elongated one, SV-197, and two mutants of SV-201, and compared them functionally and structurally with SV-201. In contrast to SV-201, SV-208 and the two mutants do not inhibit virus-cell fusion. The differences in the oligomerization state of these peptides in aqueous solution and within the membrane, and in their ability to bind to Sendai virions, enabled us to postulate a possible mechanism of viral entry inhibition: SV-201 binds to its target in Sendai virions before the F(1) internal fusion peptide binds to the membrane, therefore blocking the F(1) conformational change required for fusion. In addition, we further characterized the fusogenic activity of the internal fusion peptide, compared to the N-terminal one, and determined its structure in the membrane-bound state by means of fluorescence, CD, and ATR-FTIR spectroscopy. Remarkably, we found that SV-201 and its elongated form, SV-197, are highly potent in inducing fusion of the highly stable large unilamellar vesicles composed of egg phosphatidylcholine, a property found only in an extended version of the HIV-1 fusion peptide. The inhibitory activity of SV-201 and its fusogenic ability are discussed in terms of the "umbrella" model of Sendai virus-induced membrane fusion.     

Routing of the G Protein During Maturation of Festuca Leaf Streak Rhabdovirus in its Plant Host

By immunogold labelling the location of Festuca leaf streak virus glycoprotein (FLSV-G) was investigated in developing phloem and mature leaf parenchyma of Festuca gigantea infected with Festuca leaf streak virus (FLSV: Rhabdotiridae). In developing phloem cells, FLSV-G was detected in endoplasmic reticulum (ER). at perinuclear membranes, and in assembled virions, but neither in Golgi stacks and Golgi vesicles nor at the plasma membrane of infected cells. These results indicate that FLSV-G stays in the ER after transmembrane synthesis, and is not routed through the secretory pathway in F. gigantea. The membranous inclusions, present in infected mature leaf parenchyma cells were found to contain FLSV-G. It is suggested that the, virus-induced membranous inclusions have developed from FLSV-G-containing ER. The residence of FLSV-G in ER (present study) is in contrast to results with vesicular stomatitis virus (VSV; vertebrate rhabdovirus). Here the G protein is known to be routed to the plasma membrane through the secretory pathway.     

Endocytosis of liposomes and intracellular fate of encapsulated molecules: Encounter with a low pH compartment after internalization in coated vesicles

We have compared the intracellular fate of several fluorescent probes and colloidal gold entrapped in negatively charged liposomes. Weakly acidic molecules (carboxyfluorescein) appear in the cytoplasm of CV-1 cells in 30 min; agents that raise lysosomal pH block this process. Highly charged molecules (calcein) and large molecules (FITC-dextran: 18 kd) remain confined to extra-or intracellular vesicles. Thin section electron micrographs show gold-containing liposomes bound to coated pits, in intracellular coated and uncoated vesicles, and in secondary lysosomes, including dense bodies. Free gold was not observed in the cytoplasm. We conclude that negatively charged liposomes are endocytosed and processed intracellularly by the coated vesicle pathway, and acidification of the endocytic vesicle, rather than liposome fusion, permits escape of certain molecules to the cytoplasm.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : I. Biologic, Virologic, and Chemical Properties

Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.