The pathway by which Semliki Forest virus (SFV), a membrane-containing animal virus, enters BHK-21 cells was studied morphologically and biochemically . After attaching to the cell surface, the majority of viruses was rapidly trapped into coated pits, internalized by endocytosis in coated vesicles, and sequestered into intracellular vacuoles and lysosomes . Direct penetration of viruses through the plasma membrane was never observed .To assess the possible involvement of lysosomes in the release of the genome into the cytoplasm, the effect of five lysosomotropic agents, known to increase the lysosomal pH, was tested. All of these agents inhibited SFV infectivity and one, chloroquine (the agent studied in most detail), inhibited a very early step in the infection but had no effect on binding, endocytosis, or intracellular distribution of SFV . Thus, the inhibitory effect was concluded to be either on penetration of the nucleocapsid into the cytoplasm or on uncoating of the viral RNA .Possible mechanisms for the penetration of the genome into the cytoplasm were studied in vitro, using phospholipid-cholesterol liposomes and isolated SFV . When the pH was 6 .0 or lower, efficient fusion of the viral membranes and the liposomal membranes occurred, resulting in the transfer of the nucleocapsid into the liposomes . Infection of cells could also be induced by brief low pH treatment of cells with bound SFV under conditions where the normal infection route was blocked .The results suggest that the penetration of the viral genome into the cytosol takes place intracellularly through fusion between the limiting membrane of intracellular vacuoles and the membrane of viruses contained within them . The low pH required for fusion together with the inhibitory effect of lysosomotropic agents implicate lysosomes, or other intracellular vacuoles with sufficiently low pH, as the main sites of penetration . KEY WORDS coated vesicleslysosomes " from the extracellular space to the cytosol . This endocytosis -membrane fusion process requires transport through one or more lysosomotropic agents membrane barriers . For animal viruses the mechanisms involved are poorly understood . Data from To infect a cell, a virus must transfer its genome electron microscopy suggest that penetration oc-404 J . CELL BIOLOGY

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The high molecular mass glycosaminoglycan hyaluronan (HA) can become modified by the covalent attachment of heavy chains (HCs) derived from the serum protein inter-alpha-inhibitor (IalphaI), which is composed of three subunits (HC1, HC2 and bikunin) linked together via a chondroitin sulfate moiety. The formation of HC.HA is likely to play an important role in the stabilization of HA-rich extracellular matrices in the context of inflammatory disease (e.g. arthritis) and ovulation. Here, we have characterized the complexes formed in vitro between purified human IalphaI and recombinant human TSG-6 (an inflammation-associated protein implicated previously in this process) and show that these complexes (i.e. TSG-6 x HC1 and TSG-6 x HC2) act as intermediates in the formation of HC x HA. This is likely to involve two transesterification reactions in which an ester bond linking an HC to chondroitin sulfate in intact IalphaI is transferred first onto TSG-6 and then onto HA. The formation of TSG-6 x HC1 and TSG-6 x C2 complexes was accompanied by the production of bikunin x HC2 and bikunin x HC1 by-products, respectively, which were observed to break down, releasing free bikunin and HCs. Both TSG-6 x HC formation and the subsequent HC transfer are metal ion-dependent processes; these reactions have a requirement for either Mg2+ or Mn2+ and are inhibited by Co2+. TSG-6, which is released upon the transfer of HCs from TSG-6 onto HA, was shown to combine with IalphaI to form new TSG-6 x HC complexes and thus be recycled. The finding that TSG-6 acts as cofactor and catalyst in the production of HC x HA complexes has important implications for our understanding of inflammatory and inflammation-like processes.

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TSG-6, the secreted product of tumor necrosis factorstimulated gene-6, is not constitutively expressed but is up-regulated in various cell-types during inflammatory and inflammation-like processes. The mature protein is comprised largely of contiguous Link and CUB modules, the former binding several matrix components such as hyaluronan (HA) and aggrecan. Here we show that this domain can also associate with the glycosaminoglycan heparin/heparan sulfate. Docking predictions and sitedirected mutagenesis demonstrate that this occurs at a site distinct from the HA binding surface and is likely to involve extensive electrostatic contacts. Despite these glycosaminoglycans binding to non-overlapping sites on the Link module, the interaction of heparin can inhibit subsequent binding to HA, and it is possible that this occurs via an allosteric mechanism. We also show that heparin can modify another property of the Link module, i.e. its potentiation of the anti-plasmin activity of inter-␣-inhibitor (I␣I). Experiments using the purified components of I␣I indicate that TSG-6 only binds to the bikunin chain and that this is at a site on the Link module that overlaps the HA binding surface. The association of heparin with the Link module significantly increases the anti-plasmin activity of the TSG-6⅐I␣I complex. Changes in plasmin activity have been observed previously at sites of TSG-6 expression, and the results presented here suggest that TSG-6 is likely to contribute to matrix remodeling, at least in part, through down-regulation of the protease network, especially in locations containing heparin/heparan sulfate proteoglycans. The differential effects of HA and heparin on TSG-6 function provide a mechanism for its regulation and functional partitioning in particular tissue microenvironments.TSG-6 1 (the secreted product of tumor necrosis factor-stimulated gene-6), a protein composed mainly of contiguous Link and CUB modules, is not constitutively expressed in adult tissues but is up-regulated in many different inflammatory diseases that often involve remodeling of the extracellular matrix (ECM) (1). These include rheumatoid arthritis and osteoarthritis (2, 3), Kawasaki disease (4), systemic lupus erythematosus (5), and asthma. 2 TSG-6 is also expressed in normal physiological processes involving ECM reorganization, notably following blood vessel wall injury (6), during ovulation (6 -10), and in cervical ripening (11). TSG-6 binds to several components of the ECM through its Link module domain; i.e. the glycosaminoglycans (GAGs) hyaluronan (HA) (12-17) and chondroitin-4-sulfate (13), as well as the G1 domain of aggrecan (18) and pentraxin-3 (19, 20). Interactions between TSG-6 and the serine protease inhibitor inter-␣-inhibitor (I␣I) have also been described (21-27). I␣I is a proteoglycan and consists of three polypeptides (heavy chain 1 (HC1), heavy chain 2 (HC2) and bikunin), covalently linked by a chondroitin sulfate moiety, which originates from Ser-10 of bikunin (28). There is evidence that TSG-6 mediates the cross-li...