Unique Peptide Maps of the Three Largest Proteins Specified by the Flavivirus Kunjin

Transcription

1 JOURNAL OF VIROLOGY, Nov. 1977, p Copyright 1977 American Society for Microbiology Vol. 24, No. 2 Printed in U.S.A. Unique Peptide Maps of the Three Largest Proteins Specified by the Flavivirus Kunjin PETER J. WRIGHT,* D. SCOTT BOWDEN, AND E. G. WESTAWAY Deparment of Microbiology, Monash University Medical School, Prahran, Victoria, Australia 3181 Received for publication 1 May 1977 Tryptic digests of four polypeptides found in Kunjin virus-infected Vero cells, NV5, NV4, V3, and NV3, were compared by peptide mapping. The polypeptides to be analyzed were labeled with radioactive methionine and separated by electrophoresis through polyacrylamide gels containing sodium dodecyl sulfate. Because infection of Vero cells by Kunjin virus does not inhibit host cell protein synthesis, radioactively labeled viral polypeptides prepared from infected cells migrate coincidentally during sodium dodecyl sulfate-gel electrophoresis with some of the labeled host proteins. Thus, the genuine viral methionine-containing peptides in tryptic digests of viral proteins have been identified by co-analyzing polypeptides from [3H]methionine-labeled uninfected cells and [3S]methioninelabeled infected cells and determining the 35S/3H ratio in the peptides resolved in two dimensions on thin-layer chromatography plates. The peptide map of NV3 demonstrated that it is host coded, whereas NV5, NV4, and V3 have unique peptide maps and, therefore, account for approximately one-half of the coding potential of Kunjin virus RNA. In cells infected with flaviviruses, virus-specified proteins with a total molecular weight of approximately 4, have been detected (11, 2, 22). If the amino acid sequences of each of these proteins are unique and there are no precursor-product relationships among them, then together they account for most of the coding potential of the viral RNA with a molecular weight of 4.2 x 16 (1). Since the RNA extracted from flaviviruses is infectious (17, 19) and no viral RNA species smaller than the genome have been detected in infected cells (R. W. Boulton and E. G. Westaway, Arch. Virol., in press), it appears that viral RNA acts as a polycistronic messenger in protein synthesis. However, evidence obtained so far suggests that post-translational cleavage may be of importance only in the synthesis of some small virus-specified proteins (12, 23a) and that the initiation of synthesis of individual polypeptides may occur independently at internal sites of the viral genome (21a). The identification in polyacrylamide gels of flavivirus-specified proteins against a background of host protein synthesis has been facilitated by treating infected cells with inhibitors of protein synthesis before labeling (11, 15), by a double-label and subtraction technique (2), and by analysis in slab gels (23a). Labeling infected cells and virions with radioactive carbohydrates has clearly identified two major glycoproteins, V3 and NV2 (2, 14); V3 is the envelope 651 protein of the virions, and NV2 is detected in immature visions and noninfectious hemagglutinating particles (12, 14, 21). Viral polypeptides have been further characterized in the present work. First, by analyzing maps of tryptic peptides derived from polypeptides radioactively labeled during synthesis in infected and in uninfected cells, we identified the peptides that are detected only in flavivirus-infected cells. Second, we sought further evidence on the possible occurrence of post-translational cleavage in flavivirus-infected cells and of precursor-product relationships among virus-specified proteins. This report is confined to the larger Kunjin virus-specified proteins, NV5, NV4, V3, and NV3. The tryptic peptide maps of the smaller proteins, NV2'/2, NV2, V2, NV1½/2, NV1, and V1 are presented in the accompanying paper (25). MATERIALS AND METHODS Cells and virus. The Kunjin virus (strain MRM61C) used to infect Vero cells was a 1% (wt/vol) suspension of infected suckling mouse brain (23). Vero cells were grown in medium 199 with 1% fetal calf serum and then maintained after infection in Eagle minimal essential medium containing.1% bovine serum albumin. Virus purification. Kunjin virus and its associated slower-sedimenting peak of hemagglutinin (SHA) were labeled with [3S]methionine and purified by sedimentation through a sucrose gradient as previously described (21, 23). The gradient fractions con- Downloaded from on November 3, 218 by guest

2 652 WRIGHT, BOWDEN, AND WESTAWAY training the hemagglutinin and radioactivity of either virus (ca. 2S) or SHA (65 to 8S) were pooled and centrifuged for 6 h at 3, x g in a Spinco SW56 rotor at 5VC. The pellets were suspended in 2% sodium dodecyl sulfate for electrophoresis. Preparation of labeled cells. Cells were infected at a multiplicity of 1 PFU/cell. At 2 h after infection, complete minimal essential medium was replaced with minimal essential medium lacking methionine and containing actinomycin D (3,g/ml); at 23 h after infection, [ns]methionine (Radiochemical Centre, Amersham, England; specific activity, 2 Ci/mmol) was added to a final concentration of 4 ttci/ml. Seven hours after the addition of label, the cells were washed twice with phosphate-buffered saline and dissolved in 2% sodium dodecyl sulfate. Uninfected cells were treated likewise throughout, except that they were mock-infected and labeled with [3H]methionine (Radiochemical Centre; specific activity, 5.5 Ci/mmol) at a concentration of 23 jici/ml. Gel electrophoresis. Labeled proteins were separated in polyacrylamide slab gels containing sodium dodecyl sulfate by two different methods. In the first, 8% (wt/wt) gels and a continuous sodium dodecyl sulfate-phosphate buffer system were used (2); in the second, polypeptides were subjected to electrophoresis in 13% (wt/wt), gels employing a discontinuous buffer system (7). Before electrophoresis, samples in 2% sodium dodecyl sulfate were adjusted to 1% dithiothreitol,.1% EDTA, and.5 M sodium phosphate (ph 7.2) (continuous system) or to 1% dithiothreitol and.5 M Tris-hydrochloride (ph 6.8) (discontinuous system) and heated at 1 C for 3 min. In preparative gels of mixed "H-labeled uninfected cells and 35S-labeled infected cells, the samples to be subjected to *coelectrophoresis were mixed, dithiothreitol was added, and they were then disrupted by heat. The final ratio of 15S counts per minute to :H counts per minute was 2.:1. After electrophoresis, the [3S]methionine-labeled polypeptides were located by exposing the gel to Kodirex X-ray film. Preparative gels were wrapped in thin plastic and exposed for 4 h; analytical gels were dried and then exposed for several days. Tryptic digests. The labeled viral proteins to be analyzed were eluted from the gels into 3 ml of.5 M NH4HCO3-.1% sodium dodecyl sulfate. Ovalbumin (2,ug) was added as a carrier, and the polypeptides were treated with iodoacetamide before digestion with trypsin as previously described (24). Samples were lyophilized and redissolved in high-voltage electrophoresis buffer (1% [wt/wt] acetic acid adjusted to ph 3.6 with pyridine). For peptide mapping in two dimensions, 2-cm2 thin-layer chromatography plates coated with cellulose to a thickness of.1 mm (Merck, Darmstadt, Germany) were used. The plates were pretreated before mapping by ascending chromatography in 1% (vol/vol) acetic acid (5). Standards in each map were 4,ug of DL-alanine and 4,ug of L-valine. Samples were applied to the thin-layer chromatography plates and subjected to electrophoresis in high-voltage electrophoresis buffer for 4 min at 2 C with a potential difference of 1,2 V and a current of approximately 4 ma. The plates were dried at 8 C for 5 min, and J. VIROL. the samples were then further separated by ascending chromatography in n-butanol-pyridine-glacial acetic acid-water (9:6:18:72) until the solvent front reached the top of the plate (about 6 h). The plates were dried, stained with ninhydrin to locate the spots of alanine and valine (positions are indicated in the figures), and then exposed to Kodirex X-ray film for 2 to 3 weeks. To determine the ratio of [35S]methionine to [3H]- methionine in peptides located by autoradiography, molten 2% agar was dropped onto each area of cellulose containing a peptide, the cellulose plus agar was scraped off, and the labeled material was eluted into.3 ml of water. The radioactivity in each sample was counted in toluene-triton X-based scintillation fluid. The spillover of 3H counts into the 5S channel was less than 1%; the spillover of 3S counts into the 3H channel was 7%. Only the peptides that, after elution from the cellulose, contained significant radioactivity (greater than twice the background count) are referred to in tables and figures. RESULTS Since host cell protein synthesis is not strongly inhibited by infection with flaviviruses (2), it is necessary to identify the peptides that are host coded in tryptic peptide maps of preparations of viral proteins derived from infected cells. These peptides result from the presence of unrepressed host polypeptides that are of molecular weights similar to those of viral proteins and that elute from the same segment of preparative polyacrylamide gel used to isolate the viral proteins. As described above, Kunjin virus-infected and uninfected cells were labeled under similar conditions with [35S]methionine and [3H]methionine, respectively, the cell extracts were subjected to coelectrophoresis in preparative gels, and the 35S-labeled viral proteins were located by autoradiography and then eluted from gel segments. The analyses of the 35S-labeled material used to prepare viral polypeptides are shown in Fig. 1 and 2. The nomenclature of the marked proteins that are virus specified is well documented (11, 2). Small portions of each eluted protein preparation were again subjected to electrophoresis in gels of both the continuous and discontinuous buffer type. Some of these analyses are shown in Fig. 2. The ratio of 35S counts per minute to 3H counts per minute in each preparation of separated viral protein was determined (Table 1). 3H-labeled cell protein comigrated to the greatest extent with NV3, NV11/2, and V3. The presence of host protein similar in size to that of V3 (HP51) has already been reported (2, 22). Approximately 6% of the total 35S-labeled infected material subjected to electrophoresis was recovered in the preparations listed in Table 1. As expected, these same preparations contained a Downloaded from on November 3, 218 by guest

3 VOL. 24, 1977 a b c d ro I -NVS V3mm V24.vie*i a i L-NV4 *- V3 F-NV 3 _:-NV 2i U-NV2.4 " -NV II.-NV 1*9 FIG. 1. Analyses of rs~methionine-labeled polypeptides by electrophoresis in 8%polyacrylamide gels with the continuous buffer system. (a) Purified Kunjin virus. (b) Kunjin SHA. (c) Kunjin virus. (d) Infected cells used to prepare samples for tryptic digestion. Samples in (a) and (b) were subjected to electrophoresis in the same gel, as were those in (c) and (d). different proportion (4%) of the total 3H-labeled uninfected material subjected to electrophoresis, reflecting the differences between uninfected and infected cells in the pattern of protein synthesis. The polypeptide compositions of purified Kunjin virus and SHA labeled with [35S]methionine and used as the source of virus structural proteins for peptide mapping are displayed in Fig. 1. The amount of NV2 found in SHA of flaviviruses labeled with radioactive amino acids varies (11, 16). In these experiments, we obtained an insufficient amount of NV2 from SHA for satisfactory peptide mapping. NV5. The tryptic peptide map of [35S]methionine-labeled NV5 is shown in Fig. 3A. The ratios of 3S counts per minute to 3H counts per minute were determined for the numbered peptides (Fig. 3B) and are listed in Table 2. The 35S/3H ratio in the total tryptic digest (3.4:1) was measured immediately before analysis to allow for 35S decay and any possible selective loss of 3H- or 35S-labeled protein during digest preparation. Any peptide with 3S/3H ratio of greater than 6.8:1 (i.e., twice the ratio in the PEPTIDES OF KUNJIN PROTEINS. I. 653 a 7,-e b ao_ im f -NVS -N V4 - V3 -NV3 -NV2& 4P -NV2 4 -NVJ~ FIG. 2. Analyses of rs]methionine-labeled polypeptides in 13% gels with the discontinuous buffer system. (a) Uninfected cells labeled with r3slmethionine (included for comparison only). (b) Infected cells used to prepare samples for tryptic digestion. (c) V3, (d) NV4, and (e) NV5 are samples of the preparations used for tryptic peptide mapping after separation as in (b) in preparative gels. c.1 d _ ap TABLE 1. Recovery of radioactivity in polypeptides eluted from a preparative gel in which f5s] methionine-labeled infected cells were subjected to coelectrophoresis with fihimethionine-labeled uninfected cells Viral polypeptide 'S cpm/3h cpm 'S cpm recovered NV NV V NV NV2A, NV NV1½, NV of [ns]methionine counts per minute a The ratio to [3H]methionine counts per minute in the sample measured before electrophoresis through the gel was 2.:1. total digest) was designated as viral and is included in the "net" map of NV5 (Fig. 3C). It is apparent from Table 2 that the 35S/3H ratio varies over a wide range for different peptides. The reason for this variation is plain when the composite nature of the peptide map is considered. Analyzed together are 35S-labeled viral Downloaded from on November 3, 218 by guest

4 654 WRIGHT, BOWDEN, AND WESTAWAY A a C NVS.J) Is J. VIROL. peptides, 35S-labeled host peptides with less radioactivity (from host proteins made after infection), and 3H-labeled host peptides from uninfected cells. A peptide with a high 35S/3H ratio is clearly viral. A low ratio indicates that either (i) a peptide is host coded or (ii) a peptide is possibly viral but has a low ratio because of an overlapping or comigrating 3H-labeled host peptide. These two possibilities are not resolved by our method, and so the requirement that we adopted, i.e., that a peptide must have at least twice the 35S/3H ratio of total digest, is quite stringent. It is possible that some genuine viral peptides are excluded by this criterion, as demonstrated by the peptide maps of V3 (see below). The total number of [35S]methionine-containing viral tryptic peptides of NV5 resolved was 34. If the exclusion ratio is increased to 2.5 times that of the total digest, the number of viral peptides is decreased by only five; if the ratio is decreased to 1.5 times that of the total digest, the number of viral peptides is increased by only four. These figures indicate that there is not a large number of "borderline" peptides where grouping as either host or viral is very sensitive to the choice of exclusion ratio. The majority of peptides are easily designated. Cal- 3 culated ratio of 35S/3H of greater than 2 are not listed since the exact figure is meaningless in the presence of comparatively small to negligible amounts of tritium. NV4. The tryptic peptide map of NV4 is shown in Fig. 4, and the 35S/3H ratios of the peptides counted are listed in Table 2. The number of viral peptides was 22, again excluding those peptides for which the 35S/3H ratio was less than twice that of the total digest. If the exclusion ratio is increased to 2.5 times that of the total digest, the number of viral peptides is decreased by only four; if the ratio is decreased to 1.5 times that of the total digest, the number of viral peptides is increased by four. Thus, as for NV5, there are not a large number of borderline peptides. V3. Much interest lay in comparing the peptide maps of the glycoprotein V3 from cytoplasm and V3 from virion and SHA. These maps presented an opportunity to check the method of designating viral peptides and to determine whether the V3 incorporated into SHA, although migrating in gels in a position similar to that of V3 of virions, possesses detectable struc- Downloaded from on November 3, 218 by guest FIG. 3. (A) Tryptic peptide map of I5S]methionine-labeled NV5. Electrophoresis was from left to right; chromatography was from top to bottom. The open circles mark the positions of alanine and valine markers. (B) Peptides for which the ratio of35s counts per minute to 3H counts per minute was calculated as described in the text. (C) Net viral map showing peptides for which the ratio was more than twice the initial ratio of the total digest. Actual ratios are listed in Table 2.

5 VOL. 24, 1977 PEPTIDES OF KUNJIN PROTEINS. I. 655 tural differences from virion V3. SHA lacks RNA, and its polypeptide composition differs from that of virions (11, 16). The importance of A NV4 SHA in the assembly of infectious virus is at present uncertain, but, since glycoprotein V3 is the major polypeptide present in both virion and SHA, more information on its structure in both particles is needed. The tryptic peptide map of 36S-labeled V3 O.R derived from infected cells is displayed in Fig. 5. Using a cut-off ratio (3.:1) twice that of the tryptic digest (1.5:1), we designated half of the resolved peptides as host coded (Table 2). Identifying viral peptides in this protein is a more severe test of the double-labeling technique than identification of viral peptides of NV4 or NV5 because of the presence of a cell polypeptide of similar molecular weight (2). The number of peptides designated as viral in V3 preparations from infected cells is 17. All but three of these B were found in V3 of virions (see below); if the exclusion ratio is increased to 2.5 times that of the total digest, three peptides also found in I 2 virion V3 (no. 18, 2, and 25) are excluded. If 1 3 the ratio is decreased to 1.5 times that of the IIOo C) total digest, three peptides found in virion V3 6 are included (no. 23, 24, and 26). 14o The maps of V3 from 3S-labeled virions and W (:7 CD SHA are presented in Fig. 6A and C, respec- 8~ ) tively. The peptides in Fig. 6B and D are given 27 c:, >22 the same numbers as those counted in Fig. 5B, and, thus, are found in the total map of V3 from 24 infected cells. It is obvious that the maps of V3 25C of virions and V3 of SHA are virtually identical and contain, respectively, 24 and 23 of the peptides found in the total maps of the V3 preparations from infected cells. The most noticeable peptides present in V3 of virions (Fig. 6A and B) but lacking in V3 of 1 C infected cells (Fig. 5) are one immediately above peptide 31 and one below peptide 24. The peptide directly below peptide 5 and to the right of peptide 9 was resolved in some preparations of cellular V3 (not shown). The viral peptides present in V3 of infected cells that are conspicuously o absent from virion V3 are peptides 29 and 11. With these exceptions and one minor, poorly ) resolved peptide (no. 17), all of the peptides in O C the total V3 map (Fig. 5A and B) that were designated as viral (Fig. 5C) are present in V3 of purified virions. This indicates that the double-label technique successfully discriminates viral from host peptides in a digest containing a comparatively high proportion (Table 1) of host polypeptide. The exclusion ratio chosen (i.e., FIG. 4. (A) Tryptic peptide map of frjsmethio- twice the ratio of the total digest) is conservative nine-labeled NV4. (B) and (C) were ddetermined as and not unexpectedly excludes some genuine described in the legend to Fig. 3. viral peptides because of the presence of comi- Downloaded from on November 3, 218 by guest

6 656 WRIGHT, BOWDEN, AND WESTAWAY TABLE 2. Ratios of eluted radioactivity in methionine-labeled tryptic peptides after separation in the cellulose layer on thin-layer chromatography plates NV5 initial ratio,b 3.4:1 NV4 initial ratio, 4.2:1 V3 initial ratio, 1.5:1 Pep- 35S/3H Pep- 35S/:H Pep- 35S/ H Pep- 35S/3H Pep- 35S/-"H Pep- 35S/3H tide tide tide tide tide tide c c > > > c c c c 6 4.3c c 6 2.7c c 7 > c 7 4.9c c >2 29 > c c c 9 > c 27.5c 1 4.4c c 1.3c 28 Li c 32 > >2 29 > > c 12.5c 3 1.3c > c c 14 > ' c 15 > > c c 16 > c 17 > > >2 2 6.c 41 > >2 a The peptides are numbered as in Fig. 3 to 5 for viral polypeptides NV5, NV4, and V3, respectively. b Initial ratio, Ratio of 'S counts per minute to 3H counts per minute in the tryptic digest immediately before peptide separation. c Peptides with a 3S/3H ratio equal to or less than twice the initial ratio. grating or overlapping host peptides. The numbers of net viral peptides resolved for NV5, NV4, and V3 from infected cells are 34, 22 and 17, respectively; the molecular weights of these proteins are 98, and 7,5, and 51,3 respectively (2). If, for convenience, we assume that each labeled peptide contains only one methionine residue, then these polypeptides contain three to four methionine residues per 1, daltons. Amino acid analyses of flavivirus proteins are not available, but this approximate figure for the methionine content is consistent with those reported for other viral proteins, e.g., Semliki Forest virus, 1.85 to 2.55 mol of methionine per 1 mol of amino acids (6); Sindbis virus, 1.81 to 4.18 mol per 1 mol (3); and Newcastle disease virus, 3.41 to 7.91 mol per 1 mol (8). The relative numbers of [35S]methionine-containing tryptic peptides found in J. VIROL. Kunjin virus-specified NV5, NV4, and V3 are also consistent with estimates of the relative methionine content reported for Japanese encephalitis virus-specified polypeptides, the ratio of NV5/NV4/V3 being 1.4:1.2:1. (15). NV3. The polypeptide that was designated NV3 (Fig. 1) in infected cells was examined by the double-label technique and by peptide mapping (Fig. 6E and 6F). The 35S/3H ratio in the digested preparation before analysis on thinlayer chromatography plates was.51:1, indicating that it contained a high proportion of material from uninfected cells. The 35S/3H ratios for the 12 peptides eluted and counted (Fig. 6F) were between.38:1 and.58:1. Thus, none reached the ratio of 1.2:1 required for designation as viral. The narrow range of ratios close to the initial loading ratio is strong evidence that the protein in Kunjin virus-infected Vero cells that has been designated NV3 (2) is host coded. This result lends no support to the suggestion that glycoprotein NV3 is a naturally occurring fragment of the glycoprotein V3 (13). NV5 compared with NV4 and V3. To determine whether the smaller proteins, NV4 and V3 (from infected cells), contain amino acid sequences in common with the larger protein, NV5, the tryptic digest of each was subjected to coelectrophoresis and co-chromatography with the tryptic peptides of NV5 (Fig. 7A and C). In the accompanying diagrams (Fig. 7B and D) are indicated some of the viral peptides that belong to the smaller protein (NV4 and V3, respectively) and that had been previously identified (Table 2). Those marked are the viral peptides of NV4 and V3 that are resolved from those of NV5 in the mixed run. In the area of the maps immediately to the right of the markers, it is difficult to assign peptides to either Downloaded from on November 3, 218 by guest

7 VOL. 24, 1977 S A V3e.ii B FC B O 2 iq 9 O C 17 34C 19C3 Q21 35Q 25Q24 I 32QD 27QQ:Z 3) C (D g FIG. 5. (A) Tryptic peptide map of [35S]methionine-labeled V3 derived from infected cells. (B) and (C) were determined as described in the legend to Fig. 3. O b PEPTIDES OF KUNJIN PROTEINS. I. 657 component polypeptides of the mixed analyses because of the large number of spots. However, the majority of peptides of V3 (9 of 17) and NV4 (16 of 22) are resolved in the mixtures from a majority of the peptides of NV5 (ca. 25 of 34 in each map). It seems reasonable to assume that the peptides resolved in the mixtures are representative of the whole polypeptides rather than being confined to only a minor segment of each protein, and thus V3 and NV4 could not possibly be products of cleavage of NV5. The molecular weights of NV5, NV4, and V3 are, respectively, 98,, 7,5, and 51,3 (2); hence, the differences in molecular weight between NV5 and NV4 and between NV5 and V3 are insufficient to represent all of the noncoincident peptides. NV4 and V3. An analysis of a mixture of NV4 and V3 (from infected cells) is presented in Fig. 7E. Nineteen viral peptides of the smaller protein V3 that had been identified in preparations from infected cells (Fig. 5C) or virions (Fig. 6A) were resolved from peptides of NV4 in the mixture (Fig. 7E) and are shown in Fig. 7F. Because the difference in molecular weight between NV4 and V3 is only 19,, and by reasoning similar to that in the above paragraph, we concluded that V3 is not a product of any hypothetical cleavage of NV4. DISCUSSION Our most important result is the evidence that NV5, NV4, and V3, the three largest virus-specified proteins detected in flavivirus-infected cells, each have a unique tryptic peptide map. Their total molecular weight of 22, (2) accounts for approximately one-half of the coding potential of Kunjin virus RNA, which has a molecular weight of 4.2 x 16 (1). They are stable during pulse-chase experiments, and no polypeptides larger than NV5 have been detected in infected cells despite efforts to inhibit any hypothetical post-translational cleavage (23a). The above peptide maps have confirmed that NV5 is not a precursor of NV4 and V3 and that NV4 is not cleaved to produce V3. In infected cells the times required for the translation of these proteins are proportional to their molecular weights, and the synthesis of each is reinitiated within 1 min after reversal of the inhibition of initiation of translation by hypertonic salt (21a). Together these findings indicate that each of the three polypeptides, representing at least half the genetic information of Kunjin, is not produced by post-translational cleavage of a precursor molecule translated from a polycistronic mrna containing a single initiation site, and Downloaded from on November 3, 218 by guest

8 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~k 658 WRIGHT, BOWDEN, AND WESTAWAY A v 3.,.. Dl~~1 B J. VIROL. I C' c sh.- V E NV 3 *1 & _ Oq *1* _o ~ ol Am.. IV! D F Downloaded from on November 3, 218 by guest FIG. 6. Tryptic peptide maps of f36s~methionine-labeled V3 prepared from (A) Kunjin virions and (C) SHA. (B) Peptides present in V3 in infected cells (Fig 5B) which are also found in (A). (D) Peptides present in Fig. 5B which are also found in (C). (E) Peptide map of r3s]methionine-labeled NV3 from infected cells. (F) Peptides of NV3 for which the ratio of35s counts per minute to 3H counts per minute was calculated.

9 9 A NYV5 +NV4 C B c NV5 +V3Cell S E NV4+ V3ceii a -,4 SL do it coo F D C F o a 4z2)o S coo o a co L) C Downloaded from on November 3, 218 by guest FIG. 7. Analyses of mixtures of tryptic digests of O5S~methionine-labeled polypeptides. (A) NV5 (19, cpm) and NV4 (13, cpm). (B) Viral peptides of NV4 (Fig. 4C) that were resolved in the mixture in (A). (C) NV5 (19, cpm) and V3 from infected cells (12, cpm). (D) Viral peptides of V3 from infected cells (Fig. 5C) that were resolved in the mixture in (C). (E) NV4 (13, cpm) and V3 from infected cells (12, cpm). (F) Viral peptides of V3 that were resolved in the mixture in (E). The net viral map of V3 from infected cells (Fig. 5C) and the total map of V3 from virions (Fig. 6A) were used in identifying V3 viral peptides shown in (F). 659

10 66 WRIGHT, BOWDEN, AND WESTAWAY J. VIROL. yet no species of viral RNA smaller than that of the genome which might act as messengers for the synthesis of the three proteins have been detected in infected cells (Boulton and Westaway, in press). Accordingly, the results of the peptide mapping experiments are consistent with the suggestion that internal initiation of protein synthesis on viral mrna does occur in flavivirus-infected cells (21a). So far, the functions of the nonstructural viral proteins detected in flavivirus-infected cells have not been determined. The sizes of NV5 (98, daltons) and NV4 (7,5 daltons) are constant in cells infected by Kunjin or by each of 1 other flaviviruses, whereas the sizes of other virus-specified proteins found in cells infected by these flaviviruses vary much more, depending on the virus species used for infection (2-22). It has been suggested that NV4 and NV5 may be polymerase proteins (21a), since the two viral polypeptides comprising purified Semliki Forest virus polymerase (4) are similar in size to NV5 and NV4 of flavivirus. If NV5 (or NV4) is a polymerase protein, then some common amino acid sequences might be expected in NV5 (or NV4) derived from cells infected with different flaviviruses. It is interesting, therefore, that Qureshi and Trent (1), by immunodiffusion and complement fixation, analyzed purified antigen corresponding in size to NV5 and derived from cells infected with four of the eleven flaviviruses studied by Westaway et al. (22) and found no common antigenic determinants. To further examine the relationships among the flaviviruses, we are comparing the tryptic peptide maps of NV5 derived from cells infected with different members of the genus. The envelope protein V3 was found in infected cells, virions and SHA. The peptide maps presented above do not indicate any major differences between the primary structure of vision V3 and that of V3 from SHA and provide no further clues on a possible role for SHA in vision assembly. However, there are differences between the peptide map of V3 from virions and the net viral map of V3 from infected cells. If allowance is made for the viral peptides in cellular V3 preparations that are excluded by the presence of overlapping host peptides but are found in virion V3 and for host peptides in the cellular V3 preparation, then cellular V3 contains at least two viral peptides not found in virion V3, and the latter possesses at least two peptides not found in the former. This implies that the major species of V3 found in infected cells differs slightly in structure from the molecules of V3 incorporated into virions and SHA. Experiments in which V3 is labeled with radioactive glucosamine or mannose indicate that not all V3 molecules in infected cells (and in virions) are glycosylated to the same extent (23a). Since the length and composition of oligosaccharides affect the migration of tryptic peptides to which they are attached during electrophoresis and chromatography, the differences that we noted between maps of methionine-containing tryptic peptides of V3 probably reflect differences in glycosylation between V3 from infected cells and V3 from virions and SHA. Other possible structural variations altering the mobilities of peptides of V3 are sulfation and phosphorylation (9, 18). Further studies are needed to determine when V3 is structurally modified, since addition of carbohydrate (or sulfate or phosphate groups) to the molecule may occur during polypeptide synthesis, after release of the protein from the polyribosome and before assembly of SHA and virions, or after assembly of the two particles. Such modifications may be necessary to allow completion of the assembly process. For example, V3 is synthesized in Kunjin virus-infected Vero cells during treatment with puromycin (16 Lg/ml) but is not incorporated into virions, even after removal of the drug when virus assembly is resumed (23a). Blockage by puromycin of the postulated modifications) normally occurring (say) during or shortly after completion of translation may prevent the formation of fully functional V3 molecules. More information on the basis of the effect of puromycin and on the variation in glycosylated species of V3 found in infected-cell membranes (2) will aid in understanding the assembly of the flaviviruses. ACKNOWLEDGMENT This work was supported by a grant from the National Health and Medical Research Council of Australia. LITERATURE CITED 1. Boulton, R. W., and E. G. Westaway Comparisons of togaviruses: Sindbis virus (group A) and Kunjin virus (group B). Virology 49: Boulton, R. W., and E. G. Westaway Replication of the flavivirus Kunjin: proteins, glycoproteins and maturation associated with cell membranes. Virology 69: Burke, D. J., and K. Keegstra Purification and composition of the proteins from Sindbis virus grown in chick and BHK cells. J. Virol. 2: Clewley, J. P., and S. I. T. Kennedy Purification and polypeptide composition of Semliki Forest virus RNA polymerase. J. Gen. Virol. 32: Hindennach, I., and H. Jockusch Peptide mapping of phage QB proteins using cell-free synthesis. Virology 6: Kennedy, S. I. T., and D. C. Burke Studies on the structural proteins of Semliki Forest virus. J. Gen. Virol. 14: Laemmli, U. K Cleavage of structural proteins Downloaded from on November 3, 218 by guest

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