Membrane fusion process of Semliki Forest virus. II: Cleavage-dependent reorganization of the spike protein complex controls virus entry

The envelope of the Semliki Forest virus (SFV) contains two transmembrane proteins, E2 and E1, in a heterodimeric complex. The E2 subunit is initially synthesized as a precursor protein p62, which is proteolytically processed to the mature E2 form before virus budding at the plasma membrane. The p62 (E2) protein mediates binding of the heterodimer to the nucleocapsid during virus budding, whereas E1 carries the entry functions of the virus, that is, cell binding and low pH-mediated membrane fusion activity. We have investigated the significance of the cleavage event for the maturation and entry of the virus. To express SFV with an uncleaved p62 phenotype, BHK-21 cells were transfected by electroporation with infectious viral RNA transcribed from a full-length SFV cDNA clone in which the p62 cleavage site had been changed. The uncleaved p62E1 heterodimer was found to be used for the formation of virus particles with an efficiency comparable to the wild type E2E1 form. However, in contrast to the wild type virus, the mutant virus was virtually noninfectious. Noninfectivity resulted from impaired uptake into cells, as well as from the inability of the virus to promote membrane fusion in the mildly acidic conditions of the endosome. This inability could be reversed by mild trypsin treatment, which converted the viral p62E1 form into the mature E2E1 form, or by treating the virus with a pH 4.5 wash, which in contrast to the more mild pH conditions of endosomes, effectively disrupted the p62E1 subunit association. We conclude that the p62 cleavage is not needed for virus budding, but regulates entry functions of the E1 subunit by controlling the heterodimer stability in acidic conditions.     

Inhibition of the membrane fusion machinery prevents exit from the TGN and proteolytic processing by furin

The Semliki Forest virus (SFV) glycoprotein precursor p62 is processed to the E2 and E3 during the transport from the trans-Golgi network (TGN) to the cell surface. We have studied the regulation of the membrane fusion machinery (Rab/N-ethylmaleimide (NEM)-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)-SNAP receptor) in this processing. Activation of the disassembly of this complex with recombinant NSF stimulated the cleavage of p62 in permeabilized cells. Inactivation of NSF with a mutant alpha-SNAP(L294A) or NEM treatment inhibited processing of p62. Rab GDP dissociation inhibitor inhibited the cleavage. Inactivation of NSF blocks the transport of SFV glycoproteins and vesicular stomatitis virus G-glycoprotein from the TGN membranes to the cell surface. The results support the conclusion that inhibition of membrane fusion arrests p62 in the TGN and prevents its processing by furin.     

Opposite polarity of virus budding and of viral envelope glycoprotein distribution in epithelial cells derived from different tissues

We compared the surface envelope glycoprotein distribution and the budding polarity of four RNA viruses in Fischer rat thyroid (FRT) cells and in CaCo-2 cells derived from a human colon carcinoma. Whereas both FRT and CaCo-2 cells sort similarly influenza hemagglutinin and vesicular stomatitis virus (VSV) G protein, respectively, to apical and basolateral membrane domains, they differ in their handling of two togaviruses, Sindbis and Semliki Forest virus (SFV). By conventional EM Sindbis virus and SFV were shown to bud apically in FRT cells and basolaterally in CaCo-2 cells. Consistent with this finding, the distribution of the p62/E2 envelope glycoprotein of SFV, assayed by immunoelectronmicroscopy and by domain-selective surface biotinylation was predominantly apical on FRT cells and basolateral on CaCo-2 cells. We conclude that a given virus and its envelope glycoprotein can be delivered to opposite membrane domains in epithelial cells derived from different tissues. The tissue specificity in the polarity of virus budding and viral envelope glycoprotein distribution indicate that the sorting machinery varies considerably between different epithelial cell types.     

Cholesterol is required in the exit pathway of Semliki Forest virus

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a membrane fusion reaction triggered by low pH. For fusion to occur cholesterol is required in the target membrane, as demonstrated both in in vitro fusion assays and in vivo for virus infection of a host cell. In this paper we examine the role of cholesterol in postfusion events in the SFV life cycle. Cholesterol-depleted insect cells were transfected with SFV RNA or infected at very high multiplicities to circumvent the fusion block caused by the absence of cholesterol. Under these conditions, the viral spike proteins were synthesized and transported to the site of p62 cleavage with normal kinetics. Surprisingly, the subsequent exit of virus particles was dramatically slowed compared to cholesterol-containing cells. The inhibition of virus production could be reversed by the addition of cholesterol to depleted cells. In contrast to results with SFV, no cholesterol requirement for virus exit was observed for the production of either the unrelated vesicular stomatitis virus or a cholesterol-independent SFV fusion mutant. Thus, cholesterol was only critical in the exit pathway of viruses that also require cholesterol for fusion. These results demonstrate a specific and unexpected lipid requirement in virus exit, and suggest that in addition to its role in fusion, cholesterol is involved in the assembly or budding of SFV.     

Insertion of the two cleavage sites of the respiratory syncytial virus fusion protein in Sendai virus fusion protein leads to enhanced cell-cell fusion and a decreased dependency on the HN attachment protein for activity

Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (F(RSV)) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (F(SeV)), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both F(RSV) cleavage sites in F(SeV) resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric F(SeV) mutants containing both F(RSV) cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.     

Assembly of the semliki forest virus membrane glycoproteins in the membrane of the endoplasmic reticulum in vitro

A cell-free system has been constructed to study the mechanism by which a single messenger RNA directs the synthesis of proteins destined for two different cellular locations. The Semliki Forest virus (SFV) 26 S mRNA codes for the viral capsid protein (C protein) and the membrane proteins p62 and E1. The three virus proteins are read in this order from the messenger RNA using one initiation site. The C protein is left on the cytoplasmic side and the p62 and the El proteins are inserted into the endoplasmic reticulum membrane. Translation of 26 S mRNA in a HeLa cell-free system in the presence of microsomes from dog pancreas reproduced the segregation, and proteolytic processing and glycosylation observed in infected cells. The signal for membrane binding was in the amino-terminal end of p62. The results indicate that the membrane proteins become inserted in the nascent state. The cleavage between p62 and El was coupled to membrane insertion. If the membranes were added after a period corresponding to the synthesis of about 100 amino acids of the p62 protein, segregation, glycosylation and cleavage between p62 and E1 failed to occur.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.     

The Role of the Membrane-spanning Domain Sequence in Glycoprotein-mediated Membrane Fusion

The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.     

Interactions between the transmembrane segments of the alphavirus E1 and E2 proteins play a role in virus budding and fusion

The alphavirus envelope is built by heterodimers of the membrane proteins E1 and E2. The complex is formed as a p62E1 precursor in the endoplasmic reticulum. During transit to the plasma membrane (PM), it is cleaved into mature E1-E2 heterodimers, which are oligomerized into trimeric complexes, so-called spikes that bind both to each other and, at the PM, also to nucleocapsid (NC) structures under the membrane. These interactions drive the budding of new virus particles from the cell surface. The virus enters new cells by a low-pH-induced membrane fusion event where both inter- and intraheterodimer interactions are reorganized to establish a fusion-active membrane protein complex. There are no intact heterodimers left after fusion activation; instead, an E1 homotrimer remains in the cellular (or viral) membrane. We analyzed whether these transitions depend on interactions in the transmembrane (TM) region of the heterodimer. We observed a pattern of conserved glycines in the TM region of E1 and made two mutants where either the glycines only (SFV/E1(4L)) or the whole segment around the glycines (SFV/E1(11L)) was replaced by leucines. We found that both mutations decreased the stability of the heterodimer and increased the formation of the E1 homotrimer at a suboptimal fusion pH, while the fusion activity was decreased. This suggested that TM interactions play a role in virus assembly and entry and that anomalous or uncoordinated protein reorganizations take place in the mutants. In addition, the SFV/E1(11L) mutant was completely deficient in budding, which may reflect an inability to form multivalent NC interactions at the PM.     

Maturation of HIV envelope glycoprotein precursors by cellular endoproteases

The entry of enveloped viruses into its host cells is a crucial step for the propagation of viral infection. The envelope glycoprotein complex controls viral tropism and promotes the membrane fusion process. The surface glycoproteins of enveloped viruses are synthesized as inactive precursors and sorted through the constitutive secretory pathway of the infected cells. To be infectious, most of the viruses require viral envelope glycoprotein maturation by host cell endoproteases. In spite of the strong variability of primary sequences observed within different viral envelope glycoproteins, the endoproteolytical cleavage occurs mainly in a highly conserved domain at the carboxy terminus of the basic consensus sequence (Arg-X-Lys/Arg-Arg downward arrow). The same consensus sequence is recognized by the kexin/subtilisin-like serine proteinases (so called convertases) in many cellular substrates such as prohormones, proprotein of receptors, plasma proteins, growth factors and bacterial toxins. Therefore, several groups of investigators have evaluated the implication of convertases in viral envelope glycoprotein cleavage. Using the vaccinia virus overexpression system, furin was first shown to mediate the proteolytic maturation of both human immunodeficiency virus (HIV-1) and influenza virus envelope glycoproteins. In vitro studies demonstrated that purified convertases directly and specifically cleave viral envelope glycoproteins. Although these studies suggested the participation of several enzymes belonging to the convertases family, recent data suggest that other protease families may also participate in the HIV envelope glycoprotein processing. Their role in the physiological maturation process is still hypothetical and the molecular mechanism of the cleavage is not well documented. Crystallization of the hemagglutinin precursor (HA0) of influenza virus allowed further understanding of the molecular interaction between viral precursors and the cellular endoproteases. Furthermore, relationships between differential pathogenicity of influenza strains and their susceptibility to cleavage are molecularly funded. Here we review the most recent data and recent insights demonstrating the crucial role played by this activation step in virus infectivity. We discuss the cellular endoproteases that are implicated in HIV gp160 endoproteolytical maturation into gp120 and gp41.     

Trypsin action on the growth of Sendai virus in tissue culture cells. IV. Evidence for activation of sendai virus by cleavage of a glycoprotein.

Results obtained by using a reconstitution technique on the Sendai virus envelope confirm that cleavage of one of the envelope glycoproteins (GP2) is prerequisite for activation of hemolytic and cell fusion activities of Sendai virus. The cleavage of GP2 occurs even when free envelope subunits are directly treated with trypsin in the presence of detergent. Trypsin treatment, either of the reconstituted particle or of the free envelope subunits but not of the intact virion, also causes a cleavage of the largest envelope glycoprotein (GP1), suggesting that a site on GP1 sensitive to trypsin becomes exposed during solubilization and reconstitution. The latter cleavage, however, is not associated with any changes in biological activities.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Alphavirus Restriction by IFITM Proteins

Interferon inducible transmembrane proteins (IFITMs) are broad‐spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo‐, flavi‐, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid‐induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH‐induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV.      

Characterization of the R572T point mutant of a putative cleavage site in human foamy virus Env

A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transient env expression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutant env into a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated beta-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.     

Biochemical Reconstitution of Hemorrhagic-Fever Arenavirus Envelope Glycoprotein-Mediated Membrane Fusion

The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.     

Furin processing and proteolytic activation of Semliki Forest virus

The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed "p62," which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.     

Processing of the p62 envelope precursor protein of Semliki Forest virus

The spike protein of Semliki Forest virus is composed of three subunits, E1, E2, and E3, which mediate the fusion of the virus membrane with that of the host cell. E2 and E3 are synthesized as a precursor, p62, which is cleaved post-translationally after an Arg-His-Arg-Arg sequence. In vitro mutagenesis of a cDNA clone of the spike proteins was used to specifically alter amino acids in this cleavage site. Cleavage of p62 was completely blocked by mutation of the proximal Arg residue to Phe, without affecting transport or surface expression of the spike protein. The cleavage mutation resulted in the loss of spike protein fusion activity within the physiological pH range. Fusion activity was restored by cleavage with exogenous chymotrypsin and showed the same low pH dependence as that of wild type. The cleavage sensitivity of newly synthesized p62 was investigated by pulse-chase analysis and chymotrypsin treatment in detergent solution. p62 was sensitive to cleavage immediately following its synthesis. Protein trapped in the rough endoplasmic reticulum or Golgi apparatus by carbonyl cyanide m-chlorophenylhydrazone, monensin, or Brefeldin A treatment was also fully sensitive to cleavage. These results suggest that p62 does not require an organelle-mediated conformational change for processing. Thus, in vivo, the site of p62 processing is probably controlled by the location or activity of the cleavage enzyme, rather than the sensitivity of the p62 substrate.     

Membrane fusion of Semliki Forest virus requires sphingolipids in the target membrane.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pH-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV--liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.     

Proteolytic cleavage of the E2 glycoprotein of murine coronavirus: activation of cell-fusing activity of virions by trypsin and separation of two different 90K cleavage fragments.

In the murine coronavirus mouse hepatitis virus, a single glycoprotein, E2, is required both for attachment to cells and for cell fusion. Cell fusion induced by infection with mouse hepatitis virus strain A59 was inhibited by the addition of monospecific anti-E2 antibody after virus adsorption and penetration. Adsorption of concentrated coronavirions to uninfected cells did not cause cell fusion in the presence of cycloheximide. Thus, cell fusion was induced by E2 on the plasma membrane of infected 17 Cl 1 cells but not by E2 on virions grown in these cells. Trypsin treatment of virions purified from 17 Cl 1 cells quantitatively cleaved 180K E2 to 90K E2 and activated cell-fusing activity of the virions. This proteolytic cleavage yielded two different 90K species which were separable by sodium dodecyl sulfate-hydroxyapatite chromatography. One of the trypsin cleavage products, 90A, was acylated and may be associated with the lipid bilayer. The other, 90B, was not acylated and yielded different peptides than did 90A upon limited digestion with thermolysin or staphylococcal V8 protease. Thus, the cell-fusing activity of a coronavirus required proteolytic cleavage of the E2 glycoprotein, either by the addition of a protease to virions or by cellular proteases acting on E2, which was transported to the plasma membrane during virus maturation. There is a striking functional similarity between the E2 glycoprotein of coronavirus, which is a positive-strand RNA virus, and the hemagglutinin glycoprotein of negative-strand orthomyxoviruses, in that a single glycoprotein has both attachment and protease-activated cell-fusing activities.     

Semliki forest virus core protein fragmentation: Its possible role in nucleocapsid disassembly

Semliki Forest virus (SFV) envelope proteins function as proton pores under mildly acidic conditions and translocate protons across the viral membrane [Schlegel, A., Omar, A., Jentsch, P., Morell, A. and Kemp, F. C. (1991) Biosci. Rep. 11, 243–255]. As a consequence, during uptake of SFV by cells via receptor-mediated endocytosis the nucleocapsid is supposed to be exposed to protons. In this paper the effects of mildly acidic pH on SFV nucleocapsids were examined. A partial proteolytic fragmentation of core proteins was observed when nucleocapsids were exposed to mildly acidic pH. A similar proteolytic event was detected when intact SFV virions were exposed to identical conditions. Protease protection assays with exogenous bromelain provided evidence that the capsid protein degradation was due to an endogenous proteolytic activity and not to a proteolytic contamination. Detergent solubilization of virus particles containing degraded nucleocapsids followed by sucrose gradient centrifugation led to a separation of capsid protein fragments and remaining nucleocapsids. These data are discussed in terms of a putative biological significance, namely that the core protein fragmentation may play a role in nucleocapsid disassembly.