Genetic diversity in natural populations of mammalian reoviruses: tryptic peptide analysis of outer capsid polypeptides of murine, bovine, and human type 1 and 3 reovirus strains.

We have studied the structural relationships between the outer capsid polypeptides of eight murine, bovine, and human isolates of type 1 and 3 mammalian reoviruses. Our results show that the outer capsid polypeptides of reoviruses isolated from different mammalian species, in different years and different geographical areas, have both conserved and unique methionine-containing tryptic peptides. We found that tryptic peptides from mu 1C polypeptides of two human, one murine, and two bovine type 3 isolates and one human and two bovine type 1 reoviruses are highly conserved. Our data show that only one tryptic peptide pattern of the mu 1C polypeptide (encoded by the M2 gene) was present in reoviruses isolated from the three different mammalian species. The mu 1C polypeptide of the type 3 Dearing strain contained one tryptic peptide not found in any other reovirus isolate examined. In marked contrast to the mu 1C polypeptides, the sigma 3 polypeptides (encoded by the S4 gene) of three type 1 and three type 3 isolates were divided into two patterns based on significant differences in their tryptic peptides. In addition, at least seven tryptic peptides were conserved among the sigma 3 polypeptides of all virus strains examined. The sigma 3 polypeptide of the type 3 Dearing strain was distinguishable from the sigma 3 polypeptides of all other strains examined. The one mu 1C and two sigma 3 tryptic peptide patterns were found to occur interchangeably in isolates of type 1 or type 3. About 1/3 of the tyrosine-containing tryptic peptides of sigma 1 polypeptides of four type 3 isolates examined were conserved. Comparison of peptide differences in sigma 1 polypeptides of these isolates showed that each had one or more unique tryptic peptides, suggesting that the S1 genes coding for these polypeptides had undergone genetic drift or, alternatively, that there are at least two tryptic peptide patterns present among the sigma 1 polypeptides of these isolates. Our results suggest that genetic drift and reassortment are the most likely explanation for the extensive genetic diversity found in natural populations of mammalian reoviruses.     

Protective antigens of the vaccinia virus

The comparison of the polypeptide composition of 3 vaccinia virus strains, L-IVP, B-51 and CM-63, has revealed that strains L-IVP and B-51 are similar in their polypeptide composition, while in strain CM-63 capsid polypeptide with a molecular weight of 34000 daltons is absent or has altered electrophoretic mobility. As the result of the isolation of vaccinia envelopes (from strain L-IVP) and the electrophoretic separation of their polypeptides in plates with polyacrylamide gel 10 polypeptides have been obtained in 7 fractions, each containing 1 or 2 polypeptides. The immunization of rabbits with individual fractions has demonstrated that the formation of virus-neutralizing antibodies is induced mainly by 4-5 polypeptides in 3 fractions, having the highest molecular weight (54000-31000 daltons) and constituting about 19% of all proteins in the whole virion. The low-molecular envelopes polypeptides have been found to play no essential role in inducing the formation of virus-neutralizing antibodies. The highest antibody titers (1: 15625) have been detected in antisera to the preparations of whole vaccinia virus envelopes.     

Reovirus polypeptide sigma 3 and N-terminal myristoylation of polypeptide mu 1 are required for site-specific cleavage to mu 1C in transfected cells.

N-myristoylated viral polypeptide mu 1 was produced in COS cells transfected with a transient expression vector containing a DNA copy of the reovirus M2 gene. The mu 1 product was specifically cleaved to polypeptide mu 1C in cells that were cotransfected with the reovirus S4 gene and that expressed polypeptide sigma 3. Studies with site-specific mutants of the M2 gene demonstrated that conversion of mu 1 to mu 1C was dependent on myristoylation and the presence of the proteolytic cleavage sequence asparagine 42-proline 43 in mu 1, as well as on the presence of polypeptide sigma 3. The mu 1C product and polypeptide sigma 3 formed complexes that were immunoprecipitated by sigma 3-directed antibody, and a myristoylation-negative M2 double mutant, G2A-N42T, yielded mu 1 that did not undergo cleavage to mu 1C or bind sigma 3. However, the N42T single mutant did form immunoprecipitable complexes with sigma 3, indicating that binding can occur in the absence of cleavage. Polypeptide sigma 3 alternatively can bind double-stranded RNA and in COS cells stimulates translation of reporter chloramphenicol acetyltransferase mRNA translation, presumably by blocking double-stranded RNA-mediated activation of the eukaryotic initiation factor 2 alpha subunit kinase which inhibits the initiation of protein synthesis. Consistent with these observations and with the formation of mu 1C-sigma 3 complexes, coexpression of M2 with S4 DNA prevented the translational stimulatory effect of polypeptide sigma 3.     

In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3

Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.     

Synthesis of Vaccinia Viral Proteins in Cytoplasmic Extracts: I. Incorporation of Radioactively Labeled Amino Acids into Polypeptides

Polypeptide synthesis has been studied in cell-free systems prepared from vaccinia virus-infected and uninfected HeLa cells. Cytoplasmic extracts containing endogenous messenger ribonucleic acid were used. Amino acid incorporation into hot trichloroacetic acid-precipitable material was linear for 15 to 20 min at 37 C. The initial rate of protein synthesis was approximately 15% of the rate in intact cells. Optimal conditions for polypeptide synthesis were similar in cell-free systems prepared from infected or uninfected cells. Requirements for an energy source and Mg(++) were demonstrated. The optimal Mg(++) concentration was 4 to 5 mm. Ribonuclease, puromycin, and cycloheximide were inhibitory. The molecular weights of the polypeptides labeled in the cell-free systems, as determined by gel filtration in 5 m guanidine hydrochloride, ranged from 16,000 to above 68,000. Polyacrylamide gel electrophoresis indicated that the polypeptides labeled in cell-free extracts of uninfected and infected cells were different. The latter closely corresponded in electrophoretic mobility with the viral polypeptides made in intact, infected cells.     

Regulated, stable expression and nuclear presence of retrovirus double-stranded RNA-binding protein sigma3 in HeLa cells.

Reovirus genome segment S4 codes for polypeptide sigma3, a major outer capsid component of virions and a double-stranded RNA (dsRNA)-binding protein implicated in viral cytopathogenesis. We have constructed a stable HeLa cell line (S4tTA) that produces functional sigma3 under tetracycline transactivator control. In the absence of tetracycline, S4tTA cells synthesized stable dsRNA-binding sigma3 that accumulated in the nucleus as well as in the cytoplasm. However, in induced S4tTA cells also expressing reovirus outer shell polypeptide mu1/mu1C, migration of sigma3 into the nucleus was blocked, probably as a result of formation of a complex with mu1/mu1C which was exclusively in the cytoplasm. Mutant analyses indicated a correlation between dsRNA-binding activity and nuclear entry of sigma3, suggesting an additional role(s) for this capsid protein in virus-cell interactions.     

Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action.

Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.     

Genome RNAs and polypeptides of reovirus serotypes 1, 2, and 3.

The virus-specific double-stranded genome RNA and polypeptides present in virions and cells infected with the three mammalian reovirus serotypes have been examined by co-electrophoresis in several different polyacrylamide gel systems. The double-stranded RNA and polypeptide species previously described for type 3 Dearing were found to have corresponding species in the other serotypes examined. In each serotype several RNA and polypeptide species were found to have different electrophoretic mobilities from the corresponding RNA or polypeptide species of type 3 Dearing. The combination of electrophoretic variants among the RNAs and polypeptides of the reovirus serotypes gave electrophoretic markers in all 10 of the reovirus genes. The usefulness of these electrophoretic markers in "mapping" the reovirus genome is discussed.     

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.     

5''-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5''-thioadenosine (SAENTA), a novel ligand with high affinity for polypeptides associated with nucleoside transport. Partial purification of the nitrobenzylthioinosine-binding protein of pig erythrocytes by affinity chromatography.

Derivatives of N6-(4-aminobenzyl)adenosine (substituted at the aminobenzyl group) and 5'-linked derivatives of N6-(4-nitrobenzyl)adenosine (NBAdo) were evaluated as inhibitors of site-specific binding of [3H]nitrobenzylthioinosine (NBMPR) to pig erythrocyte membranes. Potent inhibitors were SAENTA [5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine] and acetyl-SAENTA (the 2-acetamidoethyl derivative of SAENTA). SAENTA was coupled to derivatized agarose-gel beads (Affi-Gel 10) to form an affinity matrix for chromatographic purification of NBMPR-binding polypeptides, which in pig erythrocytes are part of, or are associated with, the equilibrative nucleoside transporter. When pig erythrocyte membranes were solubilized with octyl glucoside (n-octyl beta-D-glucopyranoside) and applied to SAENTA-Affi-Gel 10 (SAENTA-AG10), polypeptides that migrated as a broad band on SDS/PAGE with an apparent molecular mass of 58-60 kDa were selectively retained by the affinity gel. These polypeptides were identified as components of the nucleoside transporter of pig erythrocytes by reactivity with a monoclonal antibody (mAb 11C4) that recognizes the NBMPR-binding protein of pig erythrocytes. Retention of the immunoreactive polypeptides by SAENTA-AG10 was blocked by NBAdo. The immunoreactive polypeptides were released from SAENTA-AG10 by elution under denaturing conditions with 1% SDS or by elution with detergent solutions containing competitive ligands (NBAdo or NBMPR). A 72-fold enrichment of the immunoreactive polypeptides was achieved by a single passage of solubilized, protein-depleted membranes through a column of SAENTA-AG10, followed by elution with detergent solutions containing NBAdo. These results demonstrate that polypeptide components of NBMPR-sensitive nucleoside-transport systems may be partly purified by affinity chromatography using gel media bearing SAENTA groups.     

Temperature-Sensitive Mutants of Complementation Group E of Vesicular Stomatitis Virus New Jersey Serotype Possess Altered NS Polypeptides

In vesicular stomatitis virus New Jersey serotype polyacrylamide gel electrophoresis was unable to distinguish the polypeptides of the temperature-sensitive (ts) mutants of complementation groups A, B, C, and F from those of the wild-type virus. However, the NS polypeptide of the representative mutant of group E, ts E1, had a significantly greater electrophoretic mobility than that of the wild-type virus NS polypeptide. The electrophoretic mobilities of the NS polypeptides of the three mutants of complementation group E varied, being greatest in the case of ts E1, slightly less for ts E2, and only a little greater than that of wild-type virus NS polypeptide in the case of ts E3. Since the NS polypeptides of the revertant clones ts E1/R1 and ts E3/R1 have mobilities identical to that of wild-type NS polypeptide, the observed altered mobilities of the group E mutants are almost certainly the direct result of the ts mutations in the E locus. The electrophoretic mobilities of the intracellular NS polypeptides of the group E mutants were indistinguishable from those of their virion NS polypeptides. The electrophoretic mobilities of the NS polypeptides of the group E mutants synthesized in vitro using mRNA synthesized in vitro by TNP were identical to those of the NS polypeptides of their purified virions. The NS polypeptides of all three mutants were labeled with (32)P(i) to approximately the same extent as wild-type virus NS polypeptide, indicating that gross differences in phosphorylation of this polypeptide are unlikely to account for the altered mobilities. We propose a model in which the NS polypeptide consists of at least three loops held in this configuration by hydrophobic or ionic forces or both and stabilized by phosphodiester bridges. If a mutation affects one of the amino acids to which the phosphate is covalently linked, the phosphodiester bridge cannot be formed, and, as a result, in the presence of sodium dodecyl sulfate the affected loop opens and thus the NS polypeptide migrates further into the gel. Such a configuration may also explain the multifunctional nature of the NS polypeptide.     

Reovirus variants selected during persistent infections of L cells contain mutations in the viral S1 and S4 genes and are altered in viral disassembly.

Reoviruses isolated from persistently infected cultures (PI viruses) can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-capsid proteins during viral entry into cells. We used reassortant viruses isolated from crosses of wild-type (wt) reovirus strain, type 1 Lang, and three independent PI viruses, L/C, PI 2A1, and PI 3-1, to identify viral genes that segregate with the capacity of PI viruses to grow in cells treated with ammonium chloride. Growth of reassortant viruses in ammonium chloride-treated cells segregated with the S1 gene of L/C and the S4 gene of PI 2A1 and PI 3-1. The S1 gene encodes viral attachment protein sigma1, and the S4 gene encodes outer-capsid protein sigma3. To identify mutations in sigma3 selected during persistent reovirus infection, we determined the S4 gene nucleotide sequences of L/C, PI 2A1, PI 3-1, and four additional PI viruses. The deduced amino acid sequences of sigma3 protein of six of these PI viruses contained a tyrosine-to-histidine substitution at residue 354. To determine whether mutations selected during persistent infection alter cleavage of the viral outer capsid, the fate of viral structural proteins was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after treatment of virions of wt and PI viruses with chymotrypsin in vitro. Proteolysis of PI virus outer-capsid proteins sigma3 and mu1C occurred with faster kinetics than proteolysis of wt virus outer-capsid proteins. These results demonstrate that mutations in either the S1 or S4 gene alter acid-dependent disassembly of the reovirus outer capsid and suggest that increased efficiency of proteolysis of viral outer-capsid proteins is important for maintenance of persistent reovirus infections of cultured cells.     

Transformation-associated changes in nuclear-coded mitochondrial proteins in 3T3 cells and SV40-transformed 3T3 cells

Comparative two-dimensional gel electrophoretic studies were performed on mitochondrial proteins in nontransformed mouse 3T3 cells and in SV40-transformed 3T3 cells, SV-T2. Two polypeptides, of 58 and 40 kDa, were present in increased amounts in SV40-transformed cells. These polypeptides were demonstrated to be nuclear-coded mitochondrial proteins by their absence in mitochondrial preparations, when labeling was performed in the presence of a mitochondrial-specific inhibitor, Rhodamine 6G. Temperature-sensitive mutants for transformation were derived from 3T3 cells by transfection with cloned SV40 DNA containing the ts A58 mutation. Increased amounts of the 58 kDa protein were apparent in these cells at the permissive temperature (33 degrees C) compared to the restrictive temperature (39.5 degrees C).     

lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators

We have analyzed protein-DNA complexes formed between lac repressor and linear or differently supercoiled lac DNA (802 or 816 base-pairs in length), which carry all three natural lac operators (O1, O2 and O3) in their wild-type sequence context and spacing and compared them with constructs that contain specifically mutated "pseudo-operators" O2 or O3. We used gel retardation assays to identify the nature of the complexes according to their characteristic electrophoretic mobility and dissociation rate measurements to determine their stability. With linear DNA we found only indirect evidence for loop formation between O1 and O2. In covalently closed DNA minicircles the formation of a loop between O1 and O2 could be demonstrated by the observation that O1-O2 containing DNA with low negative supercoiling (sigma = -0.013 and less) is constricted by binding of lac repressor, resulting in an increased electrophoretic mobility. At elevated negative supercoiling (sigma = -0.025, -0.037, -0.05) O1-O2 containing DNA complexed with lac repressor migrates significantly slower than the corresponding O1-DNA, indicating loop formation. The dissociation of lac repressor-operator complexes is decreased with increasing negative supercoiling for all tested operator combinations of O1, O2 and O3. However, in the presence of at least two natural lac operators on the same DNA minicircle the enhancement of stability is particularly large. This indicates that a DNA loop is formed between these two lac operators, O1 and O2 as well as O1 and O3, since negative supercoiling is known specifically to promote the formation of looped structures. Additionally, we observe a dependence of dissociation rate on the spatial alignment of the operators as a result of changing helical periodicity in differently supercoiled DNA and consider this to be further evidence for loop formation between O1 and O2 as well as O1 and O3.     

Leucine-specific, functional interactions between human immunodeficiency virus type 1 Nef and adaptor protein complexes

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine- or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the mu subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the sigma1 plus gamma subunits of AP-1 or the sigma3 plus delta subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the mu subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The mu and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.     

Complete in vitro assembly of the reovirus outer capsid produces highly infectious particles suitable for genetic studies of the receptor-binding protein

Mammalian reoviruses, prototype members of the Reoviridae family of nonenveloped double-stranded RNA viruses, use at least three proteins--sigma1, mu1, and sigma3--to enter host cells. sigma1, a major determinant of cell tropism, mediates viral attachment to cellular receptors. Studies of sigma1 functions in reovirus entry have been restricted by the lack of methodologies to produce infectious virions containing engineered mutations in viral proteins. To mitigate this problem, we produced virion-like particles by "recoating" genome-containing core particles that lacked sigma1, mu1, and sigma3 with recombinant forms of these proteins in vitro. Image reconstructions from cryoelectron micrographs of the recoated particles revealed that they closely resembled native virions in three-dimensional structure, including features attributable to sigma1. The recoated particles bound to and infected cultured cells in a sigma1-dependent manner and were approximately 1 million times as infectious as cores and 0.5 times as infectious as native virions. Experiments with recoated particles containing recombinant sigma1 from either of two different reovirus strains confirmed that differences in cell attachment and infectivity previously observed between those strains are determined by the sigma1 protein. Additional experiments showed that recoated particles containing sigma1 proteins with engineered mutations can be used to analyze the effects of such mutations on the roles of particle-bound sigma1 in infection. The results demonstrate a powerful new system for molecular genetic dissections of sigma1 with respect to its structure, assembly into particles, and roles in entry.     

The extraction and characterization of bovine epidermal alpha-keratin.

1. The alpha-fibrous protein (alpha-keratin) component of bovine epidermis has been extracted and characterized. 2. Prekeratin, a multichain unit of the epidermal tonofilaments, was shown to consist of six different polypeptide chains on polyacrylamide-gel systems containing sodium dodecyl sulphate or sodium decyl sulphate with discontinuous gel buffers, but only three chains were seen when a gel system containing sodium dodecyl sulphate with a continuous gel buffer was used. 3. Extraction of the 'keratinized' stratum corneum and the living part of the epidermis with urea buffers at pH 7.6 or 9.0 released 60% of the total dry weight of the tissues in the form of alpha-helical polypeptides. 4. The numbers, relative amounts and properties of the extracted polypeptides were the same as the subunits of prekeratin and thus are derived from the tonofilaments in situ. 5. The subunits of prekeratin and the polypeptides extracted from the living cell layers contained an average of six cysteine residues, but those from the stratum corneum contained an average of three intrachain disulphide bonds. 6. The polypeptide chains aggregated through non-covalent interactions in vitro into filaments that were similar to the tonofilaments. 7. Since the polypeptides could be released from the stratum corneum without breaking covalent bonds, it is concluded that such bonds do not cross-link the tonofilaments and non-fibrous keratohyalin. It is suggested that the tonofilaments and keratohyalin of bovine epidermis are associated by secondary bonding forces.