R- and M-p15(E) [data given in this report]) and compared

Transcription

1 JOURNAL OF VIROLOGY, Nov. 1984, p X/84/ $02.00/0 Copyright C) 1984, American Society for Microbiology Vol. 52, No. 2 Quantitative Separation of Murine Leukemia Virus Proteins by Reversed-Phase High-Pressure Liquid Chromatography Reveals Newly Described gag and env Cleavage Products LOUIS E. HENDERSON,* RAYMOND SOWDER, TERRY D. COPELAND, GARY SMYTHERS, AND STEPHEN OROSZLAN LBI-Basic Research Program, Laboratory of Molecular Virology and Carcinogenesis, Frederick Cancer Research Facility, National Cancer Institute, Frederick, Maryland Received 7 May 1984/Accepted 31 July 1984 The structural proteins of murine type C retroviruses are proteolytic cleavage products of two different precursor polyproteins coded by the viral gag and env genes. To further investigate the nature and number of proteolytic cleavages involved in virus maturation, we quantitatively isolated the structural proteins of the Rauscher and Moloney strains of type C murine leukemia virus (R-MuLV and M-MuLV, respectively) by reversed-phase high-pressure liquid chromatography. Proteins and polypeptides isolated from R-MuLV included plo, p12, p15, p30, p15(e), gp69, and gp71 and three previously undescribed virus components designated here as plo', p2(e), and p2(e)*. Homologous proteins and polypeptides were isolated from M- MuLV. Complete or partial amino acid sequences of all the proteins listed above were either determined in this study or were available in previous reports from this laboratory. These data were compared with those from the translation of the M-MuLV proviral DNA sequence (Shinnick et al., Nature [London] 293: , 1981) to determine the exact nature of proteolytic cleavages for all the structural proteins described above and to determine the origin of plo' and p2(e)s. The results showed that, during proteolytic processing of gp8oe"v from M-MuLV (M-gp 80env), a single Arg residue was excised between gp7o and p15(e) and a single peptide bond was cleaved between p15(e) and p2(e). The structure of M-gPr8Oe"v is gp70-(arg)-pl5(e)-p2(e). The data suggest that proteolytic cleavage sites in R-gp85e"v are identical to corresponding cleavage sites in M-gp8Oe"v. The p2(e)*s are shown to be different genetic variants of p2(e) present in the uncloned-virus preparations. The data for R- and M-p10's shows that they are cleavage products of the gag precursor with the structure p10-thr- Leu-Asp-Asp-OH. The complete structure of Pr65sag is p15-p12-p30-plo'. Stoichiometries of the gag and env cleavage products in mature R- and M-MuLV were determined. In each virus, gag cleavage products (p15, pl2, p30, and plo plus plo') were found in equimolar amounts and p15(e)s were equimolar with p2(e)s. The stoichiometry of gag to env cleavage products was 4:1. These data are consistent with the proposal that proteolytic processing of precursor polyproteins occurs after virus assembly and that the C-terminal portion of Prl5(E) [i.e., p15(e)-p2(e)] is located on the inner side of the lipid bilayer of the virus. Type C retroviruses are a morphologically distinct class of viruses composed of an RNA-protein inner core structure encapsulated in a lipoprotein envelope. Murine leukemia viruses (MuLVs) are among the most thoroughly characterized of mammalian retroviruses and have become prototypes for mammalian type C viruses. The protein structures of MuLVs have received a great deal of attention over the past decade, and the subject has been reviewed in detail (4). Briefly, the inner core proteins of MuLV are synthesized as a polyprotein, designated Pr65,""', and envelope proteins are synthesized as a glycosylated polyprotein, designated enm precursor (gpr8oe,,' or gpr85elll, depending upon the virus strain). Shortly after synthesis, the env precursor is proteolytically cleaved to Prl5(E) (C-terminal portion of the enm precursor), and an immature glycosylated protein that is destined to become the viral envelope glycoprotein (5). During or after virus assembly, both Pr65g'ag and Prl5(E) are proteolytically cleaved to yield viral structural proteins. Complete or partial amino acid sequences of structural proteins from the Moloney and Rauscher strains of MuLV (M-MuLV and R-MuLV, respectively) have been determined M- and R-p15 [11, 16], M- and R-p12 [21], R- and M- p30 [9, 16], (M- and R-plO [10], R-gp7l [8], R-gp69 [18], and * Corresponding author. R- and M-p15(E) [data given in this report]) and compared with the complete nucleotide sequence of M-MuLV (20). The comparison of these data proved the order of cleavage products for M-Pr651"' (p15-p12-p30-plo) and M-gPr80el' [gp7o-p15(e)] (products listed from N to C terminal) and showed that, in the case of M-Pr659'', no peptides were excised from between each of the fragments. Proteolytic cleavage sites in M-gPr80e?' were suggested by comparing available sequences for R-MuLV and M-env proteins with the nucleotide sequence, but it was unclear as to whether any peptide material was excised between M-gp7O and M- Prl5(E). The comparison of nucleotide and protein sequences also showed that predicted C-terminal peptide segments of both gag and env precursors were unaccounted for among the viral proteins. Specifically, translation of the gag gene nucleotide sequence predicted four residues at the C-terminal end of M-Pr65"'9 that were not found at the C-terminal end of M-plO. Translation of the env gene nucleotide sequence predicted residues at the C-terminal end of M-gPr80e'?l' that were not found at the C-terminal end of the mature viral transmembrane proteins, the pl5(e)s. In this report, we describe the quantitative isolation of structural proteins of R- and M-MuLV by reversed-phase high-pressure liquid chromatography (RP-HPLC). The separation revealed previously unknown proteolytic products of the gag and env precursors present in the viruses. Partial or 492

2 VOL. 52, 1984 complete amino acid sequence analysis of these newly discovered viral proteins and R- and M-p15(E) and M-gp7O are presented. These data account for the missing C-terminal portion of M-Pr659'9 and M-gPr80ehl' (described above) and prove the exact nature of the proteolytic processing events for M-gPr8Oenv. The stoichiometry of precursor cleavage products found in mature virus was also determined. The relationship of these data to mechanisms of proteolytic processing of gag and env precursors and membrane orientation of Pr15(E) are discussed. MATERIALS AND METHODS Virus strains. R-MuLV was grown in monolayer cultures of chronically infected BALB/c JLS-V9 cells. M-MuLV was grown in NIH/3T3 cells. Both R-MuLV and M-MuLV were purified by sucrose density gradient centrifugation and supplied by the Viral Resources Laboratory, Frederick Cancer Research Facility, National Cancer Institute, Frederick, Md Ċhemicals. All chemicals used in the liquid-phase spinning-cup sequenator were purchased from Beckman Instruments, Inc., Palo Alto, Calif. Polybrene was purchased from Aldrich Chemical Co., Inc., Milwaukee, Wis. Guanidine hydrochloride (enzyme grade) was purchased from Bethesda Research Laboratories, Inc., Gaithersburg, Md. Acetonitrile and 1-propanol were obtained from Burdick & Jackson Laboratories, Inc., Muskegon, Mass. Trifluoroacetic acid (Sequanal grade) and carboxypeptidase Y were purchased from Pierce Chemical Co., Rockford, Ill. Carboxypeptidase A was purchased from Worthington Diagnostics, Freehold, N.J. Gel electrophoresis. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed on 10 to 20% gradient gels by the method of Laemmli (14). Proteins were visualized by staining with Coomassie brilliant blue R- 250 (Bio-Rad Laboratories, Richmond, Calif.). Amino acid analysis. Samples for amino acid analysis were hydrolyzed in vacuo with 6 N HCl containing 0.1% liquid phenol for 24 h and then dried by vacuum desiccation. Analysis was performed with a Durrum D500 amino acid analyzer with ninhydrin detection of eluted amino acids. Carboxypeptidase digestions. Samples for C-terminal amino acid sequence analysis were prepared and digested with carboxypeptidases as previously described (16). The free amino acids released from the substrate protein were quantitatively determined with an amino acid analyzer. Liquid-phase sequencing. Semiautomated microsequencing was performed with an updated 890B Beckman sequencer as previously described (10). Phenylthiohydantoins (PTH) derivatives of amino acids were quantitatively identified as previously described (7). Separation of viral proteins by RP-HPLC. Concentrated suspensions of purified virus (1 mg/ml) in 0.1 M sodium phosphate (ph 7.0) were made ph 2.0 by the addition of trifluoroacetic acid and added to five volumes of saturated (23 C) guanidine hydrochloride. The resulting slightly turbid solution of disrupted virus was injected into a high-pressure liquid chromatograph (Waters Associates, Milford, Mass.) and separated by RP-HPLC on a,u-bondapak phenyl column (Fatty Acid Analysis Column; Waters Associates) or a,u-bondapak C18 column (Waters Associates) as previously described (12). Eluted proteins were detected by UV absorption at 206 nm with a model 450 variable-wavelength detector (Waters Associates) and collected manually to optimize separations and recoveries. Solvents were removed by lyophilization. Conditions of flow rate, elution solvent, and gradient conditions are given in the figure legends. After SEPARATION OF MuLV PROTEINS BY HPLC 493 each separation of viral proteins, the RP-HPLC column was washed at 50 C with 120 ml of 1-propanol-water-trifluoroacetic acid (90:10:0.1) at a flow rate of 0.2 ml/min. Columns were stored in 1-propanol or acetonitrile (0.1% trifluoroacetic acid). Columns were frequently checked for their ability to separate a standard mixture of proteins and peptides (50,ug each of bovine serum albumin, ovalbumin, lysozyme, RNase, methionine enkephalin, and leucine enkephalin) as previously described (13). When necessary, columns were cleaned and repacked as previously described (12). Molar ratios of gag and env proteolytic products (i). Method 1. Whole-virus suspensions were disrupted with SDS and separated by SDS-PAGE with phosphate buffers (22), and the proteins were visualized with Coomassie brilliant blue. Stained protein bands were cut from the gel, lyophilized, and hydrolyzed with 6 N HCI as was done for amino acid analysis. After hydrolysis, the sample was centrifuged (5,000 x g) to remove gel fragments and the liquid was transferred to another tube. The gel fragments were resuspended in fresh 6 N HCl and extracted overnight at room temperature; the second extract was then added to the first. The combined 6 N HCl extracts were analyzed as was done for amino acid analysis, except during elution of ammonia the ninhydrin flow was stopped to avoid precipitation in the reaction coil. The molar amount of viral protein in the gel slice could be calculated from the analysis results and the known amino acid composition of the respective protein. (ii) Method 2. The area (A) of the eluted protein peaks in the RP-HPLC separation of the viral proteins was determined by cutting out a tracing of the peaks and weighing the paper. The weight of the paper tracing of each peak was divided by the number of amino acid residues (r) in the respective protein to obtain a number (0) proportional to the molar amount of protein (Air = 0). Ratios of 0 were used to determine the molar ratios of the respective proteins. In separate experiments with standard proteins (bovine serum albumin, lysozyme, and RNase A), it was shown that the area under an eluted protein peak detected at 206 nm was proportional to the mass of protein in the peak. The experimentally determined values of 0 for the standard proteins agreed with the known mass of protein within ±+10%. RESULTS Separation of MuLV proteins by RP-HPLC. In earlier publications, we described the partial separation of MuLV proteins by RP-HPLC (12, 13). We have since optimized conditions for the separation of all known structural proteins of R-MuLV by RP-HPLC on a,u-bondapak phenyl column (Fig. 1). Positive identifications of viral proteins labeled in Fig. 1 were made by amino acid sequence analysis. We have previously published the partial or complete amino acid sequences of plo (10), p12 (21), p30 (9, 16), p15 (13, 16), gp69 (18), and gp7l (8). The identity of p2(e), plo', and p15(e) [previously designated pl2(e)] will be established by the data given in this report. Material in unlabeled peaks (Fig. 1) was tentatively identified by SDS-PAGE and amino acid analysis or gas chromatography-mass spectral analysis (data not shown) as described in the legend to Fig. 1. Recovery of the viral proteins from the RP-HPLC column appeared to be nearly quantitative. The total viral protein eluted from the column was determined by amino acid analysis of recombined aliquots of all column fractions. In three separate experiments (Fig. 1), at least 90% of the applied protein was recovered in the total column eluate. The area under a protein peak detected at 206 nm is proportional to the mass of eluted protein. Therefore, the areas of the various viral protein peaks in Fig. 1 (shaded

3 494 HENDERSON ET AL. areas) may be used to determine stoichiometric relationships. The structural proteins of M-MuLV were separated by RP-HPLC on a,u-bondapak C18 column by the same procedure as described above for R-MuLV proteins. Portions of the chromatogram showing the separation of M-p10, M-plO', M-p2(E), M-p2(E)*, and M-p15(E) are shown in Fig. 2. The partial amino acid sequence of M-plO (10) was reported previously, and the identities of M-p10', M-p2(E), M-p2(E)*, and M-p15(E) will be established by the data given in this report. plo'. Both R- and M-MuLV contain a previously unidentified protein which we designated p10'. The following data are presented to support the conclusion that p10' is a cleavage product of the gag precursor and that it differs from plo by having an additional C-terminal tetrapeptide (Thr- Leu-Asp-Asp-OH). Figure 3 shows the results of SDS- PAGE analysis of unfractionated R-MuLV proteins (lane V) compared with R-plO (Fig. 1, fraction A) and R-plO' (Fig. 1, fraction B). The SDS-PAGE mobilities suggest a slightly greater molecular weight for R-plO' than for R-plO. The amino acid compositions of R-plO and R-plO' (Table 1) were identical, except R-plO' contains four additional residues (ASP2, Thr, Leu). The N-terminal amino acid sequence of R- plo', determined by Edman degradation for 15 residues (data not shown), was identical to that of R-plO (10). The C- terminal amino acid sequence of R-plO (Ala-Ser-Leu-Leu- OH) was previously determined (10, 16) by carboxypeptidase A digestions. R-plO' was resistant to the action of carboxypeptidase A but susceptible to the action of carboxypeptidase Y. Residues released from p10' by digestion with J. VIROL. carboxypeptidase Y (1 h) are shown in Table 1. Four of these residues (Asp2, Thr, Leu) were shown to be present in the amino acid composition of R-plO' but absent in R-plO (Table 1). The remaining four residues (Ser, Ala, Leu2) are identical to the C-terminal residues of R-plO. Digestions of R-plO' with carboxypeptidase Y for shorter time periods (10 min) gave reduced yields of all eight residues but did not give significant differences in the molar ratios of released amino acids, suggesting that release of the ultimate C-terminal residues of R-plO' is rate limiting for digestion by carboxypeptidase Y. Since it is known that an Asp-Asp-OH C- terminal sequence retards the action of carboxypeptidase Y (6) and inhibits the action of carboxypeptidase A (17), our results are consistent with a C-terminal sequence of Asp- Asp-OH for R-plO'. Since R-plO and R-plO' had a common N-terminal sequence and amino acid composition (Table 1) except for four additional residues that were shown to be located in the C-terminal sequence of R-plO' (Table 1), we conclude that these residues are contiguous at the C-terminal end of R-plO' and the remainder of the protein is identical to R-plO. The data thus support the structure R-plO-(Thr, Leu)-Asp-Asp-OH for R-plO'. The p10' of MuLV prepared as shown in Fig. 2A was further purified by rechromatography on a,u-bondapak C18 column, and 10 cycles of N-terminal Edman degradation were performed on the purified protein (data not shown). Despite repurification, the analysis showed that the sample contained two independent sequences. About 80% of the total protein analyzed had an N-terminal amino acid sequence identical to that of M-plO, and the remainder of the protein had an N-terminal sequence of a fragment of M-p12 p50' 1.0BmsHigt p2(e)~~~~~~~~~~~~~~~~~~~~~~~~~cc 00 F-~~~~~~~m gp ml FIG. 1. Separation of R-MuLV proteins by RP-HPLC. R-MuLV (5.0 mg) disrupted with guanidine hydrochloride was injected onto a p.- Bondapak phenyl column (Waters Associates) and separated at ph 2.0 (trifluoroacetic acid) with linear gradients of acetonitrile --- -) at 230C and 1-propanol (. ) at 500C. All labeled protein peaks were identified by amino acid sequence analysis. Shaded areas were used to determine molar ratios of proteins in mature virus. Peaks eluting between p15 and p15(e) were UV-absorbing lipids together with disulfide cross-linked viral proteins, accounting for about 5% of the total protein applied to the column. The small peaks eluting between p30 and pls are believed to be Pr659ag and uncleaved p15-pl2. A206, Absorbance at 206 nm.

4 VOL. 52, 1984 E 0 CM 1, ml FIG. 2. Separation of M-MuLV proteins by RP-HPLC. M- MuLV (1.64 mg) was disrupted and separated as described in the legend to Fig. 1. (A) Portion of the chromatogram showing the separation of plo, plo', p2(e), and p2(e)'. All components in panel A were further purified by rechromatography by RP-HPLC before sequence analysis and identification. (B) Portion of the chromatogram showing the elution of p15(e). M-MuLV p12, glycoproteins, p30, and p15 were separated as described for R-MuLV in the legend to Fig. 1 (data not shown). The shaded area was used to determine molar ratios of proteins in mature virus. (residues 24 through 84 in the sequence of M-p12). The amino acid composition of the fraction, which was 80% pure for M-plO', could thus be corrected for known contamination by the p12 fragment. The resulting amino acid composition of M-plO' was compared with that of M-plO (9) (Table 2). As found in R-MuLV described above, these results showed that M-p1O' contains four additional residues (Asp2, Thr, Leu) not found in the composition of M-plO. The results of N-terminal sequence analysis and amino acid analysis (Table 2) suggest that M-plO' has the same relationship to M- plo as R-plO' has to R-plO. p2(e). R-p2(E) was isolated as shown in Fig. 1. Another peptide, designated R-p2(E)*, coeluted with R-plO' (Fig. 1) and was subsequently separated from R-plO' by rechromatography on a,u-bondapak C18 column (data not shown). R- p2(e)* represented less than 10% of the total protein in the peak designated R-plO' in Fig. 1. Two similar peptides were isolated from M-MuLV (see Fig. 2A) and designated M- p2(e) and M-p2(E)*. The M-p2(E) peptides eluted from the RP-HPLC column in the trailing edge of the M-plO' peak, as indicated by the shading in Fig. 2A. The M-p2(E) peptides from Fig. 2A were separated from residual M-plO' by rechromatography on a,u-bondapak C18 column (data not SEPARATION OF MuLV PROTEINS BY HPLC 495 shown). The amino acid compositions of R- and M-p2(E) and R- and M-p2(E)* are shown in Table 3. The amino acid sequence of each p2(e) was determined by Edman degradation of about 10 nmol of peptide. The experimentally determined amino acid sequence of each p2(e) is shown in Fig. 4. The amino acid sequences of R- and M-p2(E) and M-p2(E)* obtained were complete, since all residues indicated by their respective amino acid compositions in Table 3 were identified as PTH derivatives of amino acids in the subsequent sequential Edman degradations. In the Edman degradation of R-p2(E)*, no PTH derivatives of amino acids could be assigned at step 10. Except for lysine, all amino acid residues indicated by the amino acid composition of R-p2(E)* were identified as PTH derivatives of amino acids in the sequenator run; therefore, lysine was tentatively assigned to position 10. The amino acid sequences of the p2(e)s were compared with the C-terminal amino acid sequence predicted for the M-MuLV env gene translational product (20) (Fig. 4). The amino acid sequence of M-p2(E) was identical to that obtained by translation of the last 16 codons of the M-env gene (see Fig. 4). R-p2(E) was identical to M-p2(E), except for substitutions of leucine for isoleucine and histidine for tyrosine at positions 12 and 14, respectively. R-p2(E)* was identical to M-p2(E), except for a single substitution of leucine for isoleucine at position 12. Except for an additional N-terminal residue (alanine), M-p2(E)* was identical to M- p2(e). p15(e). The purification of R-p15(E) is shown in Fig. 1 (fraction C), and the purification of M-p15(E) is shown in Fig. 2B. These very hydrophobic proteins were eluted from the reversed-phase support by gradient elution with 1- propanol at an elevated temperature (50 C) after an isocratic elution at 47% 1-propanol to remove viral lipids. Figure 5 (lane C) shows the result of SDS-PAGE analysis of R-p15(E) (fraction C, Fig. 1) compared with unfractionated R-MuLV gp \ Pr65gagN3 Prl 5(E) p30- p15 5 p15(e) p12- p10f p1o- V A B _ FIG. 3. SDS-PAGE analysis of R-plO (Fig. 1, fraction A, lane A) and plo' (Fig. 1, fraction B, lane B) compared with whole R-MuLV (lane V).

5 496 HENDERSON ET AL. (lane V). The MuLV p15(e)s obtained from the initial chromatographic separation (Fig. 1 and 2) were at least 80% pure (Fig. 5). Both R- and M-plS(E) were repurified to apparent homogeneity (by SDS-PAGE) by rechromatography on a,u-bondapak C18 column at 50 C eluting with a 1- propanol gradient (data not shown). The SDS-PAGE mobilities of R- and M-p15(E) were identical (data not shown). MuLV p15(e)s migrated slightly faster than R-pl5g'g, but, as will be shown, the polypeptide chain of p15(e) (180 residues) was longer than that of R-p159'9 (130 residues). The N-terminal amino acid sequences of R- and M-p15(E) were determined by Edman degradation. The quantitative data for the PTH derivatives of amino acids recovered at each Edman cycle and assigned to the sequence of each p15(e) are shown in Table 4. At positions where identical PTH derivatives of amino acids were identified in both R- and M-p15(E), the PTH derivative of the amino acid is listed only once. The N-terminal amino acid sequence of M-p15(E) (33 residues; Table 4) was identical to that obtained by translation of 33 codons of the M-MuLV proviral DNA sequence, starting with codon 470 of the env gene. The N- terminal amino acid sequence of R-p15(E) (50 residues; Table 4) differed from the translation of the M-MuLV nucleotide sequence by substitutions at five positions: 23 (valine for isoleucine), 31 (valine for methionine), 35 (glutamic acid for glutamine), 40 (histidine for glutamine), and 48 (lysine for arginine). The C-terminal amino acid sequences of R- and M-p15(E) were determined by digestion of the denatured proteins with carboxypeptidase A. Quantitative data for the rate of carboxypeptidase-catalyzed release of C-terminal residues from R- and M-p15(E) are shown in Table 5 and show that the TABLE 1. Differences between amino acid compositions of R- MuLV plo' and plo are accounted for by additional residues at the C terminal end of p10' Residues per No. of dif- mleased-b Composition ferences in carboxypep- Amino acid cops ciasebdigestion (p10 - tione igs plo) plo' plob plo' plob Aspartic acid + asparagine 5.8 (6) d Threonine 1.7 (2) Serine 2.1 (2) Glutamic acid + glutamine 8.4 (8) 8 Proline 5.8 (6) 6 Glycine 6.4 (6) 6 Alanine 4.3 (4) Cysteine 3.3 (3) 3 Valine 2.1 (2) 2 Methionine 0 0 Isoleucine 0 0 Leucine 3.8 (4) Tyrosine 1.0 (1) 1 Phenylalanine 0 0 Histidine 1. 1 (1) 1 Lysine 4.9 (5) 5 Arginine 8.5 (9) 9 Tryptophan NDe 1 ND a Number of residues detected by amino acid analysis; rounded-off values are given in parentheses. b Amino acid sequence data taken from reference 10. c Digestion for 1.0 h with carboxypeptidase Y. d Detected as aspartic acid. ' ND, Not detected. TABLE 2. Difference between amino acid compositions of M- MuLV plo' and plo No. of differ- Amino acid ploa plob ences in composition (plo'-plo) Aspartic acid 5.7 (6) 4 2 Threonine 2.7 (3) 2 1 Serine 2.8 (3) 3 Glutamine 8.0 (8) 8 Proline 5.4 (5) 5 Glycine 6.0 (6) 6 Alanine 2.9 (3) 3 Cysteine NDC 3 Valine 1.9 (2) 2 Methionine 0 0 Isoleucine 0 0 Leucine 3.9 (4) 3 1 Tyrosine 1.0 (1) 1 Phenylalanine 0 0 Histidine 1.0 (1) 1 Lysine 5.7 (6) 6 Arginine 8.3 (8) 8 Tryptophan ND 1 a Values corrected for 24.6% contamination of p12 fragment (residues 27 through 84). Numbers represent number of residues determined by amino acid analysis; rounded-off values are given in parentheses. b Number of residues determined by sequence analysis. c ND, Not detected. J. VIROL. common C-terminal amino acid sequence of both R- and M- p15(e) is Val-Val-Gln-Ala-Leu-OH. This amino acid sequence is identical to the translation of the M-MuLV nucleotide sequence immediately upstream from the codon for the N-terminal residue of M-p2(E) (codon 650; see Fig. 4). Thus, M-p15(E) is encoded by 180 codons (codons 470 to 650). The amino acid composition of M-p15(E) (data not shown) is in TABLE 3. Amino acid compositions of p2(e) determined by amino acid analysis and sequence Amino acid residues (mol/mol of peptide)a R-MuLV M-MuLV Amino acid p2(e) p2(e)* p2(e)b p2(e)* A S A S A S A S Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan NDC ND 0 ND 0 a Values are expressed as moles of amino acid residue per mole of peptide. Values less than 0.2 are expressed as 0. A, Analysis; S, sequence. b Values corrected for 20% contamination of a peptide consisting of residues 26 through 84 of M-MuLV p12. c ND, Not detected.

6 VOL Transl ati on of Nucleotide Sequence M-p2(E) R-p2(E) SEPARATION OF MuLV PROTEINS BY HPLC 497 pl 5(E) p2(e) val gin ala leu val leu tyr gin gin tyr his gin leu lys pro ile glu tyr glu pro Stop Val -Leu-Thr-Gl n-gl n-tyr-hi s-gl n-leu-lys-pro-i1 e-gl u-tyr-gl u-pro-oh Val-Leu-Thr-Gl n-gl n-tyr-hi s-gl n-leu-lys-pro-leu-gl u-hi s-gl u-pro-oh M-p2(E)* R-p2(E)* Al a-val -Leu-Thr-Gl n-gl n-tyr-hi s-gl n-leu-lys-pro-il e-gl u-tyr-gl u-pro-oh Val -Leu-Thr-Gl n-gl n-tyr-hi s-gl n-leu(lys) Pro-Leu-Gl u-tyr-gl u-pro-oh FIG. 4. Amino acid sequences of R- and M-p2(E) and R- and M-p2(E)* compared with the translation of the 3' end of the M-env showing codons for the C-terminal residues of p15(e) (Table 5) and coding regions for p2(e). Residues that differ from the predicted sequence are underlined. The lysine residue at position 10 of R-p2(E)* is tentatively assigned on the basis of amino acid composition (see text). excellent agreement with the predicted composition of the protein. Glycoproteins. The glycoprotein of M-MuLV was also isolated by RP-HPLC (data not shown) under the same conditions of chromatography as those used for R-gp71 (Fig. 1). The N-terminal amino acid sequence of M-gp7O was determined for 33 residues by Edman degradation (data not shown). These data were in complete agreement with the sequence obtained by translation of the M-MuLV nucleotide sequence starting codon 34 of the M-eni' gene. Digestion of M-gp7O at ph 9.0 with carboxypeptidase A gave 0.99 mol of lysine and 0.41 mol of histidine per mol of protein. Translation of the M-MuLV nucleotide sequence from codons 466 through 470 of the enm gene predicted Arg-His-Lys-Arg-Glu. The glutamic acid residue in this sequence was shown to be the N-terminal residue of M-pl5(E) (Table 4). The results of exopeptidase digestion of M-gp7O are consistent with lysine in the above sequence being C-terminal on the glycoprotein of mature virus. Thus, M-gp7O is encoded by 435 codons (codons 34 to 469), and the arginine residue predicted by codon 469 is excised during proteolytic processing of M- gpr80e'l'. We previously reported tyrosine as the C-terminal residue of R-gp7l on the basis of results of carboxypeptidase A digestion (8). Digestion of R-gp7l with carboxypeptidase B yielded arginine, lysine, and histidine in addition to tyrosine. Quantitative results suggest that about 60% of our R-gp7l preparations has C-terminal tyrosine and the remainder has (Lys, His)-Arg C-terminal. R-gp7l thus appears to have a ragged C-terminal end, possibly resulting from partial proteolysis occurring after initial cleavage at an Arg-Glu bond to generate the glycoprotein and R-Prl5(E). Stoichiometry. The stoichiometry of gag and emll protein in mature R-MuLV was determined by two methods. The proteins of disrupted virus were separated by SDS-PAGE (Fig. 3, lane V) and visualized by Coomassie brilliant blue. The stained bands corresponding to plo plus p10', p12, and p30 were sliced from the gel and hydrolyzed with 6 N HCI, and the molar amount of protein in each band was determined by amino acid analysis. These results showed that plo plus plo', p12, and p30 were present in the virus in equimolar amounts within an experimental error of ±10%. Incomplete resolution of p159l`1 from p15(e) by SDS-PAGE (Fig. 5) prevented quantitation of these proteins by this method. M- MuLV proteins were not analyzed by this method. The second method for determining the stoichiometry of gag and en' proteins of R- and M-MuLVs was based on integration of the area under the various protein peaks eluted in the RP-HPLC separation (see above). This method of quantitative analysis was applied to all major protein peaks in Fig. 1 and 2 except the p30 peak, because SDS-PAGE analysis of column fractions (data not shown) indicated that p30 showed a considerable tendency to trail from the column, making it difficult to determine the amount of p30 in the virus from the area of its peak. The area of each peak taken for integration is indicated by the shading in Fig. 1 and 2. The apparent rising baselines seen in Fig. 1 and 2 were mostly due to UV absorption caused by increasing concentrations of organic solvents and accompanying changes in the extent of ionization of trifluoroacetic acid. In addition, minor amounts of UV-absorbing substances present in the virus preparations were also eluted from the column and contrib- C.l R g V Prl 5(E) FIG.5.Analysis of SDS-PAGE C..g.,. # of R-p15(E) (Fig. 1. fraction 0 p 15(E) FIG. 5. Analysis of SDS-PAGE of R-p15(E) (Fig. 1, fraction C. lane C) compared with whole R-MuLV (lane V).

7 498 HENDERSON ET AL. uted to the apparent baseline. Because of uncertainties as to the actual baseline levels for each peak, all peaks were integrated from valley to valley as indicated in the figures. This method of integration makes the least assumption about baseline levels. Normalizing the molar amount of R-p12 to 1.0, the estimated molar amounts of R-MuLV proteins were 0.78 (plo), 0.30 (p-10'), 1.1 (p15), 0.25 [p15(e)], 0.26 [p2(e)], 0.20 (gp7l), and 0.03 (gp69). Normalizing the molar amount of M-p12 to 1.0, the molar amounts of other M-MuLV proteins were 0.9 (plo plus plo'), 1.0 (p15; not shown in Fig. 2), 0.27 [p15(e)], 0.20 [p2(e)], and 0.08 [p2(e)*]. We conclude from the above results that the molar ratios of Pr659'9 cleavage products found in mature MuLV (pl5/ p12/p30/plo plus p10') are 1:1:1:1 (± 10%). The molar ratio of p15(e) to p2(e) is 1:1, and that of p12 (gag product) to p15(e) (env product) is 4:1. The molar ratio of the glycoproteins to p15(e) was near 1:1 in the preparation of R-MuLV separated in Fig. 1. Other preparations of R-MuLV, however, showed smaller amounts of glycoproteins relative to other gag and env cleavage products. The molar amount of M-gp7O was also variable from one preparation to another. DISCUSSION In this report we describe the quantitative separation of structural proteins of MuLV by a single chromatographic procedure with RP-HPLC (Fig. 1). Newly described viral proteins, designated here as plo', p2(e), and p2(e)*, were isolated, in addition to all the previously known structural proteins. All viral structural proteins isolated by this procedure have been unequivocally identified by partial or complete amino acid sequence analysis. When compared with TABLE 4. Edman degradation of R- and M-plS(E)" PTH amino acid deriva- PTH amino acid deriva- Cycle tives (nmol) Cycle tives (nmol) no. no. R-MuLV M-MuLV R-MuLV M-MuLV 1 Glu (0.8) (3.0) 26 Gly (0.3) (0.1) 2 Pro (0.8) (1.2) 27 Thr (0.6) (0.1) 3 Val (2.0) (1.8) 28 Thr (1.0) (0.2) 4 Ser (0.3) (0.4) 29 Ala (1.0) (0.2) S Leu (1.1) (0.9) 30 Leu (0.7) (0.1) 6 Thr (0.7) (1.1) 31 Val (1.4) Met (0.1) 7 Leu (1.0) (0.7) 32 Ala (1.0) (0.1) 8 Ala (1.8) (0.7) 33 Thr (0.7) (0.1) 9 Leu (0.8) (0.5) 34 Gln (0.4) 10 Leu (1.2) (0.5) 35 Glu (0.4) (Gln)b 11 Leu (1.2) (0.5) 36 Phe (0.5) 12 Gly (0.4) (0.3) 37 Glnb 13 Gly (0.8) (0.3) 38 Glnb 14 Leu (1.0) (0.4) 39 Leub 15 Thr (0.8) (0.3) 40 His (0.3) (Gln)' 16 Met (0.7) (0.2) 41 Ala (0.6) 17 Gly (0.5) (0.2) 42 Ala (0.7) 18 Gly (1.0) (0.3) 43 Val (0.8) 19 Ile (1.2) (0.2) 44 Glnb 20 Ala (1.3) (0.2) 45 Aspb 21 Ala ti.3) (0.3) 46 Asp (1.0) 22 Gly (0.7) (0.1) 47 Leu (0.4) 23 Val (1.3) Ile (0.2) 48 Lys (0.2) (Arg)' 24 Gly (0.5) (0.1) 49 Glu (0.3) 25 Thr (1.1) (0.2) 50 Val (0.5) a Glu, Glutamic acid; Pro, proline; Val, valine; Ser, serine; Leu, leucine; Thr, threonine; Ala, alanine; Gly, glycine; Met, methionine; Ile, isoleucine; Gln, glutamine; His, histidine; Asp, aspartic acid; Lys, lysine. bpth derivatives of amino acids qualitatively but not quantitatively identified. ' Residues indicated by translation of M-MuLV nucleotide sequence. J. VIROL. TABLE 5. C-terminal amino acid sequence of R- and M-MuLVpl5(E): results of carboxypeptidase A digestion' Source of p15(e) Residues released (mol/mol of protein) and digestion time (min) Valine Glutamine Alanine Leucine R-MuLV M-MuLV " Data for both proteins are consistent with the C-terminal amino acid sequence Val-Val-Gln-Ala-Leo-OH. the nucleotide sequence of M-MuLV, these data confirm the nucleotide sequence and identity of the viral proteins and also show the exact nature of proteolytic cleavages leading to production of the viral proteins. The results show that mature virions contain proteolytic products accounting for all portions of M-Pr659'9 and all portions of M-gPr8O"' except for a single arginine residue. Previous studies have shown that a single peptide bond is cleaved between each of the fragments of M-Pr659'9 to produce the mature viral structural proteins (p15, p12, p3o, and p1o). These viral core proteins account for all portions of M-Pr659"I except for a C-terminal tetrapeptide (Thr-Leu- Asp-Asp-OH) predicted by the nucleotide sequence but not found in the primary structure of M-plO. Partial structural analysis indicated that plo' of R- and M-MuLV, described for the first time in this report, may be partial cleavage products that are derived by normal cleavage between the p30-plo portion of the gag precursors but that retain the C- terminal tetrapeptide predicted for M-PrOgag. Thus, all portions of the predicted structure of M-PrOgag are accounted for in the assembled virus by p15, p12, p30, and plo'. To determine the exact portions of the env precursor incorporated into the mature virus and to prove proteolytic processing events for M-gPr8O'", we compared the translation of the M-env gene with the N- and C-terminal amino acid sequences of M-gp7O and M-p15(E) and the complete amino acid sequence of the viral peptide designated M-p2(E) described in this report. This comparison showed the exact nature of the proteolytic cleavages that took place during processing of M-gPr80en'. A single peptide bond was cleaved between M-p15(E) and M-p2(E) (see Fig. 4), and an arginine residue (codon 469) was excised between M-gp7O and M- p15(e) (Table 6). The complete structure of M-gPr8Oe""" was gp7o-(arg)-p15(e)-p2(e). The proteolytic cleavage sites in R-gPr85en"" for generating R-p15(E) and R-p2(E) appear to be identical to the corresponding sites in M-gPr80e?l'l. The N-terminal amino acid sequence of R-p15(E) (Table 4) was identical to that of M- p15(e) for the first 22 residues and showed only five amino acid substitutions in 50 compared residues. The C-terminal amino acid sequence of R-p15(E) (Table 5) was identical to that of M-p15(E) in the compared region. The amino acid sequence of R-p2(E) was identical to that of M-p2(E) except for substitutions at positions 12 and 14 (Fig. 4). Both R- and M-MuLV preparations contained peptides

8 VOL. 52, 1984 TABLE 6. Proteolytic cleavage sites in M- and R-MuLV gag and env precursor Proteolytic cleavage be- Gene tween X and Y Bond Probable location at product cleaved time of cleavage" gag Initiator Met Pr659'9 Met-Gly Cytoplasm p15 p12 Tyr-Pro Intravirion p12 p30 Phe-Pro Intravirion p30 plo Leu-Ala Intravirion plo C-terminal Leu-Thr Undetermined tetrapeptide* env, Leader pep- gpr80env C Thr-Ala Rough endoplasmic tide reticulum Glycoprotein Prl5(E) Lys-(Arg)- Rough endoplasmic Glud reticulum p15(e) p2(e) Leu-Val Intravirion See text for discussion and references. b Thr-Leu-Asp-Asp-OH. ' Proteolytic cleavage probably takes place before synthesis and glycosylation of gpr8oe" is complete (20). d Arginine residue is excised during proteolytic processing of M-gPr8O""' The C-terminal of R-gp7l is ragged (see text). that were shown to be variants of p2(e). We have tentatively designated these variants as p2(e)*s. M-p2(E)* has an additional N-terminal alanine residue not found on or predicted for M-p2(E) (12). R-p2(E)* differs from M-p2(E) by a single amino acid substitution at position 12 (leucine for isoleucine). Thus, M-p2(E) is more closely related to R- p2(e)* than to R-p2(E). The origins of R- and M-p2(E)* are unknown, but they probably reflect genetic variants of the parent viruses present in the uncloned-virus preparations. A complete list of all known peptide bonds cleaved during proteolytic processing of the gag and env gene products of R- and M-MuLV is given in Table 6. The probable cellular or viral location of the substrate and cleavage enzyme at the time of proteolytic cleavage is also listed. Three proteolytic processing events are known to occur before virus assembly: (i) the initiator methionine residue of the primary translational product of the gag gene is removed before myristylation [CH3(CH2)12-CO-] of the N-terminal glycine amino group (11); (ii) the primary translational product of the env gene is synthesized with a leader peptide (codons 1 through 33 of the env gene) that is removed by cellular enzymes in the rough endoplasmic reticulum accompanying glycosylation of the env precursor (1, 4); and (iii) the env precursor is cleaved in the rough endoplasmic reticulum to the glycoprotein and Pr15(E) [i.e., pl5(e)-p2(e)] (15). This cleavage is probably accomplished by cellular trypsin-like enzymes acting on the Arg-Glu peptide bond between the glycoprotein and the N- terminal residue of p15(e) (see Table 6). The resulting C- terminal end of the glycoprotein (Arg-His-Lys-Arg-OH) may be attacked by proteases (probably exoproteases) at some later stage of development. This conclusion is based on the observation that the glycoprotein of Friend MuLV retains the C-terminal arginine residue (3), whereas most M-gp70 has the C-terminal arginine residue removed, and that R- gp7l appears to have a ragged C-terminal end resulting from partial proteolysis. Four proteolytic processing events are believed to occur soon after virus budding during the maturation process and are listed in Table 6 as intravirion events [i.e., cleavages between p15 and p12, p12 and p30, p30 and plo, and p15(e) and p2(e)]. As pointed out by Bolognesi et al. (2), products SEPARATION OF MuLV PROTEINS BY HPLC 499 of proteolytic cleavage that take place after budding and closure of the lipid bilayer of the virus should be found in the virion in equimolar stoichiometry. Cleavage products of Pr65gag (p15, p12, p30, and plo plus p10') were found in mature MuLVs in equimolar amounts (within an experimental error of + 10%). Cleavage products of Prl5(E) [R-pl5(E) and R-p2(E) for R-MuLV; M-p15(E) and M-p2(E) plus M- p2(e)* for M-MuLV] were also found in equimolar amounts. These data are consistent with the earlier proposal that proteolytic cleavage of Pr65gag takes place after virus budding during the maturation process (15). The fact that p2(e) is quantitatively retained in the virus to give 1:1 stoichiometry with p15(e) suggests that proteolytic cleavage of Prl5(E) also takes place after budding and that the C- terminal portion of Prl5(E) including p2(e) is located on the inner side of the lipid bilayer of the virus. Intravirion proteolysis is believed to be catalyzed by one or more virusencoded proteases. A virus-encoded protease has recently been isolated from M-MuLV (Y. Yoshinaka and S. Oroszlan, unpublished data) and is currently under study to determine its peptide specificity. Proteolytic cleavage between the C-terminal residue plo and the C-terminal tetrapeptide of p10' or gag precursor is only suggested by the data given here. No direct precursor product relationships have been shown, and the resulting tetrapeptide was not found among the virus products. Thus, the probable cellular or viral location of the substrate and cleavage enzyme for this suggested cleavage remain unclear: Virus assembly occurs on the inner side of the cell membrane and probably involves a specific complex of viral RNA with Pr65gag and Prl5(E). The data presented here indicate that, in the immature virion, the ratio of Pr65gag to Prl5(E) is 4:1. This may suggest stoichiometric relationships in the initial RNA-Pr65gag_Prl5(E) complex. Intravirion proteolytic processing during virus maturation appears to be necessary for the virus to gain full infectivity (15). Schwartzberg et al. (19) have recently shown that failure to cleave between p15 and p12 results in decreased virus infectivity. The biological significance of other intravirion cleavages, however, is unknown. Now that the nature and products of proteolytic cleavages of the gag and env precursor polyproteins are known, it may be possible to determine the biological significance of all individual proteolytic cleavage events. ACKNOWLEDGMENTS This research was sponsored in part by the National Cancer Institute under contract NO1-CO with Litton Bionetics, Inc. LITERATURE CITED 1. Blobel, G Intracellular protein topogenesis. Proc. Natl. Acad. Sci. U.S.A. 77: Bolognesi, D. P., R. C. Montelaro, H. Frank, and W. Schafer Assembly of type C oncornaviruses: a model. Science 199: Chen, R Complete amino acid sequence and glycosylation of glycoprotein gp71a of Friend murine leukemia virus. Proc. Natl. Acad. Sci. U.S.A. 79: Dickson, C., R. Eiseman, H. Fan, E. Hunter, and N. Teich Protein biosynthesis and assembly, p In R. A. Weiss, N. M. Teich, H. E. Varmus, and J. M. Coffin (ed.), Molecular biology of tumor viruses: RNA tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 5. Fitting, T., and D. Kabat Evidence for a glycoprotein ''signal" involved in transport between subcellular organelles. J. Biol. Chem. 257: Hayashi, R Carboxypeptidase Y. Methods Enzymol.

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