Proteolytic Cleavage of Subunits of the Nucleocapsid of the Paramyxovirus Simian Virus 5

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1 JouRNAL of VOLOy, Nov. 1974, p Copyright American Society for Microbiology Vol. 14, No. 5 Printed in U.S.A. Proteolytic Cleavage of Subunits of the Nucleocapsid of the Paramyxovirus Simian Virus 5 WALTR. MOUNTCASTL, RCHARD W. COMPANS, HNRY LACKLAND AND PURNLL W. CHOPPN The Rockefeller University, New York, New York Received for publication 29 July 1974 The protein subunits of the nucleocapsid of the parainfluenza virus simian virus 5 isolated from infected cells after dispersion with trypsin, chymotrypsin, or ficin are cleaved proteolytically. The molecular weights of the subunits which result from cleavage depend on the enzyme used, but are around 43,000, compared to the native subunit of 61,000. n most instances cleavage of the subunit appears to be due to the protease used to disperse the cell, and follows cell disruption. Nucleocapsids composed of native, uncleaved subunits can frequently be obtained from infected cells dispersed without a proteolytic enzyme; however, cleavage occasionally occurs even under those conditions, indicating that cellular proteases can at times cleave this protein. Nucleocapsids containing uncleaved subunits can be isolated from cells persistently infected with simian virus 5, indicating that persistent infection is not invariably associated with intracellular cleavage of this protein. Nucleocapsids composed of native subunits are hydrophobic, whereas those composed of the cleaved subunit can be dispersed in aqueous solution. t is suggested that the portion of the molecule removed by cleavage may be responsible for a specific interaction during virus assembly between the nucleocapsid and those areas of plasma membrane which contain the non-glycosylated viral membrane protein, which is also hydrophobic. An amino acid analysis of native and cleaved subunits has been done. The portion of the subunit removed by cleavage does not have a high proportion of hydrophobic residues, suggesting that those present are arranged together to form a hydrophobic domain. The N termini of both the native and cleaved subunits are blocked. This suggests that the portion of the molecule which is externally disposed and removed by cleavage contains the C terminus, and the cleaved subunit which reacts with the viral RNA contains the N terminus. The nucleocapsids of paramyxoviruses are single-helical structures approximately 1,m in length and are composed of single-stranded RNA and protein subunits with a molecular weight of approximately 60,000 (6, 9, 21). Previous studies of nucleocapsids of simian virus 5 (SV5), Newcastle disease (NDV), and Sendai viruses showed that the nucleocapsids can exist in two forms depending on the size of their protein subunits (21). One form of SV5 nucleocapsid, isolated from virions or from infected cells dispersed without trypsin, is a very flexible helix, and contains the native protein subunits with a mol wt of 61,000. The other form, obtained from cells dispersed with trypsin, or produced by trypsin treatment in vitro, is composed of subunits with a mol wt of approxi- Present address: Worthington Biochemical Corp., Freehold, N.J mately 43,000 which are derived from the native subunit by proteolytic cleavage. This nucleocapsid forms a more stable and relatively rigid helix. These results suggested (21) that there is a portion of the protein subunit which is extremely sensitive to proteolytic cleavage that results in the removal of 18,000 daltons of protein, and that the remainder of the subunit is resistant to cleavage. The RNA in the nucleocapsid composed of the smaller cleaved subunit is resistant to ribonuclease digestion as is that found in the nucleocapsids composed of the native subunit (7). These findings suggested that the 43,000-dalton-cleaved subunit contains those portions of the molecule to which the RNA is bound and possesses the necessary configuration for subunit-subunit bonding which results in a stable helical structure (21). On the other hand, the

2 1254 MOUNTCASTL T AL. J. VROL. 18,000-dalton portion which is removed by cleavage appears to confer flexibility on the helical structure. Further, since this region of the molecule is apparently externally disposed, it was suggested that it may be involved during virus assembly in the recognition between the nucleocapsid and those regions of the cell membrane that contain viral envelope proteins. Such recognition is a necessary step in assembly (4, 5, 8), and there is evidence which suggests that the nucleocapsid recognizes the non-glycosylated membrane proteins (5, 18, 23). These observations raised the possibility that if intracellular cleavage could occur, then virus assembly might be inhibited for two reasons: (i) the loss of flexibility of the helix which would make folding into the budding virion difficult; and (ii) the loss of the site on the nucleocapsid which specifically interacts with areas of membrane-containing envelope proteins (21). Such a block in assembly, if it occurred, might play a role in the accumulation of nucleocapsids which occurs in paramyxovirus-infected cells, particularly in persistently infeeted cells. The present report describes further studies with SV5 designed to investigate the cleavage of the nucleocapsid by proteolytic enzymes of different specificities, to determine the amino acid composition of the native and cleaved subunits and which terminus of the molecule was removed and which was associated with the RNA, and finally to explore whether cleavage could occur intracellularly and whether it occurred in cells persistently infected with SV5. MATRALS AND MTHODS Cells. Monolayer cultures of a variant of the MDBK line of bovine kidney cells were grown on plastic surfaces in reinforced agle medium (RM) (1) with 10% fetal calf serum as described previously (3). The HKCC line of adult hamster kidney cells, and the BHK21-F line of baby hamster kidney cells were grown in RM with 10% calf serum and 10% tryptose phosphate broth as described previously (14). Virus. The W3 strain of SV5 was grown in the above cell lines in RM with 2% calf serum (2). Cells were inoculated at a multiplicity of 2 to 10 PFU/cell. Chemicals and isotopes. n the previous study (21) the solution used to disperse infected cell monolayers was 0.25% pancreatic extract (NBCo , Nutritional Biochemicals Corp., Cleveland, Ohio) plus 0.05% DTA. The manufacturer's analysis of this enzyme preparation revealed that it contains roughly equal amounts of trypsin and chymotrypsin plus other pancreatic enzymes in small amounts. For convenience, this preparation will be referred to as crude trypsin in this communication, and the purified, crystallized, enzyme preparations used will be referred to as crystallized. These enzymes and their sources were as follows: trypsin, 2X crystallized and chymotrypsin, 2X crystallized, from Worthington Biochemical Corp., Freehold, N.J.; ficin, 2X-crystallized, from Sigma Chemical Co., St. Louis, Mo. Soybean-trypsin inhibitor was obtained from Worthington Biochemical Corp. Components for sodium dodecyl sulfate-polyacrylamide gels were obtained from Canalco, Rockville, Md.; [3H ]-leucine and [14C ]amino acid mixture from New ngland Nuclear Corp., Boston, Mass., or Schwarz/Mann, Orangeburg, N. Y.; and Nuclear Chicago Solubilizer from Amersham/Searle Corp., Des Plaines, ll. Purification of nucleocapsid. Cells containing SV5 nucleocapsid were harvested for 24 to 44 h postinfection. After washing with phosphate-buffered saline (10) lacking Ca2+ and Mg2+, the infected cells were dispersed with one of the following solutions, each in phosphate-buffered saline without Ca'+ and Mg2+: (i) 0.05% DTA and 0.25% crude trypsin 1-300; (ii) 0.1% trypsin (2X crystallized); (iii) 0.1% chymotrypsin (2X crystallized); (iv) 0.1% ficin (2X crystallized); (v) 0.07% DTA; or (vi) combinations of the above as described. After addition of one of the above solutions, the monolayers were dispersed by incubation at 37 C for 5 to 20 min. An equal volume of RM containing 10% fetal calf serum was added, and the cells were then pelleted, resuspended in approximately 40 volumes of deionized distilled water, and disrupted by Dounce homogenization. n some experiments, monolayers were scraped directly into deionized distilled water and homogenized immediately. The released nucleocapsid was then purified as described previously by density gradient centrifugation in CsCl (6). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ten-centimeter-cylindrical-7.5% polyacrylamide gels were used, and procedures for electrophoresis and determination of incorporated isotopes by scintillation counting were as described previously (21). Amino acid analysis. Amino acid analysis was performed according to the method of Spackman et al. (25) on a modified Beckman 120C amino acid analyzer. Protein for analysis was hydrolyzed in 6 N HC at 110 C for 24, 48, and 72 h in vacuo (20). Half-cystine and methionine were nmeasured as cysteic acid and methionine sulfone after performic acid oxidation by the method of Moore (19). Tryptophan was measured after hydrolysis with p-toluenesulfonic acid by the method of Liu and Chang (17). N-terminal analysis was done as described by Kindt et al. (16). RSULTS The effect on the nucleocapsid protein of different enzymes used to disperse infected cells. Figure 1 shows the results obtained when crystallized trypsin, which cleaves at lysine and arginine residues (15), was used to disperse SV5-infected cells. The purified nucleocapsids obtained from cells dispersed with this enzyme possess protein subunits which consistently and

3 VOL. 14, ri ,l PROTOLYTC 2 CLAVAG OF SUBUNTS C., 250 ) cl 1255 U-t 750 t t Ux tl m,o,-9,w -a&pa%g Froction 000 r F Froction i T 200 ) Q Froction FG. 1. Co-electrophoresis in a polyacrylamide gel of protein subunits of nucleocapsids isolated from SV5- infected cell monolayers dispersed with crystallized trypsin or crude trypsin-dta. Subunits of nucleocapsids labeled with ['H]leucine from cells dispersed with crystallized trypsin (0). Subunits of nucleocapsids labeled with ['4CJamino acids from cells dispersed with crude trypsin-dta (0). n this and subsequent figures, the origin is on the left and the anode on the right. FG. 2. nhibition of proteolytic cleavage of the SV5 nucleocapsid subunits by trypsin inhibitor. nfected cell monolayers labeled with ['HJleucine were dispersed with crystallized trypsin. An equal volume of 0.1% soybean-trypsin inhibitor was added, and the cells were pelleted, disrupted osmotically, and nucleocapsids were isolated as described. These nucleocapsids were mixed with nucleocapsids labeled with [14C]amino acids from cells dispersed with crude trypsin-dta without trypsin inhibitor to provide marker cleaved subunits, and the proteins were analyzed by co-electrophoresis. Symbols: (0) 'H-labeled subunits; (0) "4C-labeled cleaved subunits. FG. 3. Subunits of SV5 nucleocapsids obtained from cells dispersed with crystallized chymotrypsin. nfected cell monolayers labeled with ['H]leucine were dispersed with crystallized chymotrypsin, and nucleocapsids were isolated as described. Nucleocapsids labeled with [14CJamino acids isolated from cells dispersed with crude trypsin-dta were added, and the protein subunits were analyzed by co-electrophoresis. Symbols: (0) 'H-labeled subunits; (0) "4C-labeled subunits. FG. 4. Subunits of SV5 nucleocapsids from cells dispersed by combined crystallized trypsin and chymotrypsin. qual amounts of crystallized trypsin and crystallized chymotrypsin were used to disperse 'H-labeled infected cell monolayers before nucleocapsid isolation. "4C-labeled nucleocapsids from cells dispersed with crude trypsin-dta were added, and the protein subunits were analyzed by co-electrophoresis. Symbols: (0) 'H-labeled subunits; (0) "4C-labeled subunits. uniformly migrated slightly slower in sodium chymotrypsin also, yields a smaller subunit dodecyl sulfate-polyacrylamide gels than the which was shown previously to have a mol wt of subunit obtained with the crude trypsin DTA. 43,000 (21). The addition of soybean-trypsin The latter enzyme preparation, which contains inhibitor to the cells, before their disruption in Froction 750

4 1256 MOUNTCASTL T AL. J. VMOL. the presence of crystalline trypsin, prevents almost completely the cleavage of the protein subunit (Fig. 2). The two small peaks migrating faster than the major component in Fig. 2 may result from incomplete inhibition by the soybean product, or from the action of undefined cellular enzymes which may produce cleavage of the nucleocapsid proteins in cells not exposed to exogenous proteases. These results suggest that the bulk of the cleavage observed when cells are disrupted in the presence of trypsin is due to the action of that enzyme on the protein subunits after cell disruption, rather than to another proteolytic enzyme released by trypsinization of cells. Figure 3 shows the results obtained when infected cells were dispersed and disrupted in the presence of crystallized chymotrypsin, which cleaves preferentially at aromatic residues, but also can cleave at asparagine, glutamine, methionine, and leucine residues (13). With chymotrypsin, a nucleocapsid subunit was obtained which is larger than that obtained after dispersal with crude trypsin-dta. From its migration in the gel this cleaved subunit appears to be similar in size to that of the crystallized trypsin-cleavage product (Fig. 1). However, if crystallized trypsin and crystallized chymotrypsin were combined in a solution containing DTA, the dispersed infected cells yielded nucleocapsids with a cleaved subunit indistinguishable by gel migration from that produced in infected cells dispersed by crude trypsin-dta (Fig. 4). Therefore, it appears that the combination of the two crystallized proteases produced results similar to the crude trypsin-dta preparation. Thus, the two proteases acting in concert are able to degrade the subunit slightly further than either enzyme alone. Presumably, one of the enzymes exposes a site not otherwise available for digestion by the other, possibly because of steric hindrance. t is clear, however, that beyond that point, the remainder of the molecule is resistant to further degradation by either protease alone or in combination. The presence of DTA apparently has no significant effect on the cleavage process, since its inclusion in the dispersing solution produced no discernible change in the migration of the cleavage product produced by crystallized trypsin alone, or crude trypsin (data not shown). Crystallized ficin has also been used (without DTA) to disperse SV5-infected MDBK cells. Ficin is a heterogeneous enzyme capable of cleaving at basic amino acid residues, as well as at leucine or glycine (12). The size of the cleavage product produced by ficin appears to be very nearly the same as that of the mixture of crystallized trypsin and chymotrypsin or the crude trypsin product (Fig. 5). These results indicate that the protein subunit of SV5 nucleocapsids is extremely sensitive to three proteases of differing specificities, but that the sensitive portion of the subunit molecule is strictly limited. The remaining portion is very resistant to further cleavage by any of these enzymes. t should be emphasized that these studies were carried out on the intact nucleocapsid structure, i.e., the helical structure composed of the RNA with associated protein subunits. As discussed below, disruption of this helical structure with urea allowed preparation of tryptic peptides of the resistant portion. This suggests that bonding of subunits to adjacent subunits as well as to the RNA may play a role in conferring resistance to proteolytic attack. Lack of effect of the host cell on cleavage of the nucleocapsid subunits. To determine if the host cell affects the size of nucleocapsid subunits obtained after dispersion with enzymes, MDBK, HKCC, and BHK21-F cells were infected with SV5, and intracellular nucleocapsids were isolated after dispersal with crude trypsin-dta. All three cell types yielded nucleocapsids with cleaved subunits indistinguishable by co-electrophoresis. A comparison of the subunits from HKCC and MDBK cells is illustrated in Fig. 6. These results suggest that -i 1800 Fraction FG. 5. Nucleocapsid subunits obtained from infected cells dispersed by ficin. 'H-labeled infected cell monolayers were dispersed by ficin, and the nucleocapsids were isolated as described. "4C-labeled nucleocapsids from cells dispersed with crude trypsin- DTA were added, and the protein subunits were analyzed by co-electrophoresis. Symbols: (0) 'Hlabeled subunits; (0) 14C-labeled subunits. 6

5 VOL 14, 1974 PROTOLYTC CLAVAG OF SUBUNTS r ' O Fraction FiG. 6. Comparison of nucleocapsid subuni different cell lines dispersed with the same oenzyme cleavage of some nucleocapsid subunits raised preparation. SV5 was grown in monolayer cullstures of the question of whether disruption of the cell is MDBK or HKCC cells with medium containinw either a requirement for cleavage or whether dispersal 'H-leucine or 14C-amino acids, respectively. Bloth cell of the cells alone could result in intracellular types were dispersed with crude trypsin-d7'a, and cleavage. This question was investigated in the nucleocapsids isolated as described. After connbining following experiment. MDBK cells persistently the isolated nucleocapsids, the protein subunjits were infected with SV5 for over 60 cell generations analyzed by co-electrophoresis. Symbols: (*) 'Hlabeled subunits from MDBK cells; (0) 14Csubunits from HKCC cells. persistently infected cells in the presence of one were used. t was reasoned that growth of these labeled amino acid label, followed by trypsinization, passage and growth in medium containing an- other label would yield nucleocapsids to answer when proteolytic enzymes are used to dlisperse infected cells, the cleavage of the S5 V5 nu- the question of whether dispersal by trypsin cleocapsid subunits is not dependent on the cell without disruption would result in cleavage. type. Accordingly the persistently infected cells were Dispersal of infected cells without added enzyme. All the data presented thus ftir have suggested that the enzymes used to dispetrse the infected cell monolayer are the agents re-sponsi- However, as pointed out above, it was impor- ble for cleavage of the nucleocapsid subunits. tant to determine whether cleavage of intracel- lular nucleocapsids does occur during the> course of paramyxovirus infection in the abstence of exogenous enzymes. As described previously (21) (Fig. 3), the use of DTA alone to disperse infected cells iallowed recovery of nucleocapsids whose subuniits were indistinguishable from the virion subuniit. This would suggest that intracellular cleavalge does not invariably occur. However, in some experiments of this type, in which no ex( Dgenous enzymes were added, varying degrees oif cleavage of the nucleocapsid subunit hav,e been found. One such experiment is illustriated in Fig. 7. The polypeptide with the slowest migra- (21), tion represents uncleaved native subuniits the fastest component is similar in migriation to 2500 the crude trypsin-cleavage product, and two other polypeptide species of intermediate mobility are present. This indicates that some of the subunits within the cell were cleaved, presumably by cellular enzymes. Similar variation in the degree of cleavage has also been seen when monolayers were scraped directly into cold distilled deionized water before cell disrup <)tion, indicating that the presence of DTA was not necessary for the cleavage process. The a occurrence of cleavage without added enzymes has been unpredictable under all conditions so far used. As a result, the protein of each nucleocapsid preparation must be examined by co-electrophoresis with marker viral nucleocapsid protein to demonstrate the presence or absence of even partial cleavage. Lack of enzymatic cleavage in the absence of cell disruption. The observation that cell its from disruption alone could occasionally initiate Fraction FiG. 7. Cleavage of nucleocapsid protein subunits in the absence of added enzyme. nfected MDBK monolayers labeled with ['HJleucine were dispersed with 0.07% DTA in PBS without Ca'+ or Mg'+. ntracellular nucleocapsids were isolated as described. "4C-labeled whole virus was added as a marker, and the polypeptides were analyzed by coelectrophoresis. Symbols: (0) 'H-labeled polypeptides; (0) "4C-labeled SV5. SO o o al

6 1258 MOUNTCASTL T AL. J. VROL. grown in the presence of [14C ]amino acid mixture, dispersed from the monolayer with crude trypsin-dta, and passed 1:2 into medium containing [3H]leucine. After growth to confluence, the cells were scraped into cold distilled deionized water, disrupted, and the nucleocapsids were isolated as described. f trypsinization alone, without cell disruption, could initiate intracellular cleavage, then a major portion of the [14CJlabeled subunits would be cleaved at that step, and disruption after growth in [3H)leucine would yield a population of 'Hlabeled uncleaved or partially cleaved subunits with a different electrophoretic pattern. However, the distribution of radioactivity counts revealed several species of polypeptides with essentially identical ratios of 'H to "4C (i.e., 3:1) (Fig. 8). This indicates that the cleavage process was initiated at one point only; namely, at the time of cell disruption when both labels had been incorporated into protein subunits, and not at the time of trypsinization without disruption when only the [t4c]label was present. These results thus suggest that trypsinization of cells alone is not sufficient to cause cleavage and that disruption of the cells is required. n addition, isolation of nucleocapsids from cells dispersed with DTA alone yielded uncleaved L 500 _ t0oo Fraction FG. 8. vidence for cell disruption as a requirement for cleavage of nucleocapsid subunits. MDBK cell monolayers persistently-infected with SV5 for greater than 60 generations were grown in medium containing ['4C]amino acids. The cells were dispersed with crude trypsin-dta and passed into medium containing ['H]leucine. When confluent, the cells were scraped into cold distilled deionized water, homogenized, and nucleocapsids were isolated as described, and the nucleocapsid subunits obtained were analyzed by polyacrylamide gel electrophoresis. Symbols: (0) 'H-labeled polypeptides; (0) 14Clabeled polypeptides. nucleocapsid subunits, indicating that nucleocapsids within persistently infected cells are not invariably cleaved. Hydrophobic behavior of the native nucleocapsid subunit. A striking feature of nucleocapsids containing the native protein subunits obtained uncleaved either from virions or cells is their strong hydrophobic behavior. After purification of nucleocapsids in a CsCl gradient and removal of CsCl by dialysis against distilled deionized water, the native nucleocapsid aggregates cannot be freely suspended in the aqueous environment and tend to adhere to the dialysis membrane. n contrast, the nucleocapsids which contain the cleaved subunits, when purified and dialyzed in the same manner, are readily suspended in water, suggesting that a hydrophobic portion of the native subunit which is extemally disposed is removed by the cleavage process, resulting in a nucleocapsid which is dispersable in aqueous solution. The possible biological significance of a hydrophobic nucleocapsid is discussed below. Amino acid composition of native and cleaved nucleocapsid protein subunits. To obtain information regarding the composition of the native and cleaved subunits with the hope of eventually establishing a correlation between composition and the properties of the nucleocapsid, amino acid analyses were done on native subunits and those obtained by cleavage with crude trypsin. The results of such analyses are shown in Table 1. The composition of that portion of the molecule removed by proteolytic digestion (18,000 daltons) has been calculated from the difference between the composition of the native, 61,000-dalton subunit, and the cleaved 43,000-dalton subunit. Regarding the hydrophobic behavior of the native versus the cleaved subunits, the overall amino acid composition of the portion removed is not highly hydrophobic, and thus the basis for the hydrophobic behavior of the native nucleocapsids is not obvious from the composition alone, and the amino acid sequence is not yet known. However, the hydrophobicity could be explained if a sizable portion of the hydrophobic residues were located near the exposed surface of the native subunit, and were within that portion of the molecule removed by enzymatic cleavage. Such a hypothetical hydrophobic domain might also be involved in the interaction during virus assembly of the nucleocapsid with that region of plasma membrane which contains viral membrane proteins. The 18,000-dalton portion removed is relatively rich in aspartic acid/asparagine, threonine, and alanine, and relatively poor in lysine,

7 VOL. 14, 1974 PROTOLYTC CLAVAG OF SUBUNTS 1259 TAaz 1. Amino acid composition of SV5 nucleocapsid protein subunits Residues per molecule' Residues per 100 residues" Amino acidspotnprin Native" Cleavede Poretved Native', Cleavedc Poreovedn Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine soleucine Leucine Tyrosine Phenylalanine Tryptophane athe data were calculated using the molecular weight estimations determined by migration in sodium dodecyl sulfate polyacrylamide gels. b Nucleocapsid protein subunits with a mol wt of 61,000 isolated from cells dispersed without enzymes. These are similar to the subunits in mature virions. cnucleocapsid protein subunits with a mol wt of 43,000 isolated from cells dispersed with crude trypsin-dta. (See Methods). d The portion removed by proteolytic cleavage has a mol wt of 18,000. This has not been recovered as an intact polypeptide. These data are calculated from differences between the native and cleaved subunits. tyrosine, and phenylalanine (Table 1). That portion also contains only one or two half-cystine residues. The latter suggests that there is little or no disulfide bonding in this region, which may be of importance with regard to the marked sensitivity of this region to enzymatic digestion. N-terminal analyses were done on both the native and cleaved subunits and the N termini were found to be blocked in each case. This suggests that the portion of the molecule removed by the enzymes must contain the C terminus of the molecule. This would mean that the portion of the subunit molecule first synthesized (N-terminal portion) is contained in the smaller cleaved molecule, and also that this is the part of the subunit which interacts with the viral RNA. DSCUSSON The data presented indicate that when proteolytic enzymes are used to disperse cells infected with the paramyxovirus SV5, and the cells are then disrupted, the subunits of intracellular nucleocapsids are invariably and uniformly degraded to smaller molecules, the exact size of which depends upon the enzyme(s) used. The native subunit appears to be very sensitive to such cleavage, since the cleavage is complete and the amount of enzyme present at the time of homogenization must be small, since homogenization follows washing of the cells. The evidence suggests that in most instances cleavage is due to the added enzyme and follows cellular disruption rather than occurring intracellularly. This conclusion is based on the findings that the addition of trypsin inhibitor to the cells before disruption essentially prevents the cleavage (Fig. 2) and that trypsinization and passage of infected cells without disruption does not lead to cleavage of the nucleocapsid contained within these cells (Fig. 8). Further, as demonstrated by experiments in which no protease was added, i.e., only DTA or mechanical scraping was used to dislodge the infected cells, intact subunits have been found. However, it should be pointed out that at times cleaved subunits are seen after such treatment, indicating that cleavage may occasionally occur in the absence of added enzyme. ither crystallized trypsin or chymotrypsin used alone produces a cleavage product with a slightly larger molecular weight than that produced by the two enzymes acting together, or by

8 1260 MOUNTCASTL T AL. J. VROL. the heterogeneous enzyme ficin. The native protein subunit which has a mol wt of 61,000 is very sensitive to cleavage by several proteases of different specificities to yield subunits in the 43,000-dalton range. The 18,000-dalton portion removed has not been recovered as a single polypeptide, and is presumably degraded. However, the remainder of the molecule in the nucleocapsid structure is very resistant to further cleavage. As reported previously (21), the cleaved subunits of SV5, NDV, and Sendai viruses are very similar in size, i.e., 41,000 to 43,000 daltons. This raises the possibility of some similarities in the structure of the portion of these molecules which is resistant to proteolysis, and which binds to RNA and forms a tightly bonded helix. t has been found that disruption of the helix by 8 M urea allows tryptic digestion of the nucleocapsid, so that peptide mapping can be done. The amino acid composition of the native and cleaved subunits has indicated that the portion removed by proteolysis is not highly hydrophobic in its overall content, thus concentration of the hydrophobic residues in a region of the polypeptide seems to be the most likely explanation for the hydrophobic behavior of the native subunit in contrast to the cleaved one. Determination of the amino acid sequence of this portion of the molecule would be necessary to establish this. Knowledge of that portion of the molecule is of particular interest because both it and the non-glycosylated membrane (M) protein of the virion are hydrophobic (18, 23, 24), and the available evidence suggests that during virus assembly the viral nucleocapsid recognizes the viral membrane protein associated with areas of modified plasma membrane (18). Thus the interactions between these two proteins, which appears to be essential for virus assembly, may be hydrophobic in nature. The sensitivity of the nucleocapsid subunits of paramyxoviruses to proteolytic cleavage to yield a product of a certain size, and the resistance of these subunits to further proteolysis, provides another example of specific cleavage of virion proteins at limited sites. The biological significance of the cleavage of paramyxovirus nucleocapsids is not yet clear. One possibility which had been considered previously (21) was that intracellular cleavage might occur and result in failure of virion assembly due either to loss of the site on the nucleocapsid responsible for recognition of modified areas of plasma membrane or by inability of the more rigid nucleocapsids which contain the cleaved subunits to be coiled up and incorporated into budding virions. Such a block in assembly could play a role in persistent infections which are frequently characterized by intracellular accumulations of nucleocapsids. The accumulation of nucleocapsids in the brain cells of patients with subacute sclerosing panencephalitis represents an example of this, and in fact, two morphological types of subacute sclerosing panencephalitis nucleocapsid have been described (22). Our efforts to date to isolate subacute sclerosing panencephalitis nucleocapsids have been unsuccessful due to a paucity of material. n the present study, examination of nucleocapsid subunits from MDBK cells which had been continuously infected with SV5 for 66 cell passages suggests that at least in this case, the nucleocapsids present in the undisrupted infected cells were not composed of cleaved subunits. These results thus provide no support for the above hypothesis. However, there is not sufficient evidence to completely exclude this possibility. t is important to point out that in any study of viral nucleocapsids isolated from cells, the possibility must be considered that disruption of the cell may provoke cleavage of the nucleocapsid subunit which may be very sensitive to such cleavage. t is of interest that with another helical virus, tobacco mosaic virus, it has also been found that a particular region of the subunit is susceptible to cleavage by several proteolytic enzymes including trypsin (11), whereas the remainder of the subunit is resistant. This cleavage results in stronger intersubunit bonds than is the case with the native subunit. As a result, the cleaved subunits form stacked disk rods which are irreversible aggregates, in contrast to the reversible polymerization which the native subunits undergo. This reversible association is necessary for the assembly of the virus into the normal single-helical structure. The formation of stacked disk rods was initially observed on storage of TMV protein solutions, and later explained on the basis of cleavage by small amounts of contaminating enzymes. There is an obvious similarity between these findings with tobacco mosaic virus and the present and previous (21) work with SV5, NDV, and Sendai virus nucleocapsids in which the cleaved subunits form a more rigid structure than the native ones. Further study is required to determine whether it is a general property of helical viruses that limited proteolytic cleavage of the nucleocapsid subunits may occur, accompanied by changes in the bonding properties of the protein which affect the process of virus assembly.

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