Two glycoproteins are produced from the rotavirus neutralization gene

VIROLOGY

151,243-252

(1986)

Two Glycoproteins

Are Produced from the Rotavirus Neutralization

WAI-KIT

CHAN,* MARIA E. PENARANDA,* SUE E. CRAWFORD,* AND MARY K. ESTES**pl

Depurtments

of * Virology and Epidemiology One Baylor

and of ~Medicine, Baylor Plaza, Houston, Texas 77050

Received December 18, 1985; accepted Februry

Gene

College of Medicine,

11, 1986

The major neutralization antigen of rotaviruses is an outer capsid glycoprotein, VP7, with an apparent molecular weight of 38,096 (38K). The simian rotavirus SAll genome segment 9, which codes for VP’7, contains two in-phase initiation codons, each of which is followed by a sequence that codes for a region of hydrophobic amino acids. We have determined that this gene is functionally bicistronic by analyzing the synthesis of VP7 in SAll-infected cells and in cell-free translation systems programmed with hybrid-selected, segment 9 specific mRNA and dog pancreatic microsomes. The translation of hybridselected gene 9 mRNA in wheat germ extracts yielded two distinct polypeptides of molecular weights 37K and 35.3K. In vitro translation in the presence of microsomes yielded one diffuse band of 38K that was converted into the 37K and 35.3K precursor bands by digestion with endoglycosidase H. Studies with a variant of SAll that lacks the glycosylation site in VP7 confirmed these precursor-product relationships and extended them by indicating that the glycoprotein produced by translation from the first AUG contained a cleaved signal sequence whereas the glycoprotein produced by translation from the second AUG contained an uncleaved signal sequence. Immunoprecipitation with monospecific anti-VP7 serum and improved gel electrophoresis conditions allowed us to show that both VP7s were expressed at similar times in infected cells and both were found in purified virus particles of several different rotavirus strains. Whether these two VP7 glycoproteins are functionally distinct remains to be determined. 0 1986 Academic Press, Inc.

INTRODUCTION

In recent years, many animal virus genes have been recognized as bicistronic or polycistronic. This was first documented in viruses with nonsegmented genomes, such as adenovirus (Bos et al, 1981), simian virus 40 (Jay et al, 1981;Mertz et al, 1983), herpes simplex virus (Haarr et al., 1985), EpsteinBarr virus (Beisel et ah, 1985), Sendai virus (Giorgi et aL, 1983), and measles virus (Bellini et aL, 1985). Polycistronic genes also have been found in viruses with segmented genomes [e.g., influenza viruses (Lamb and Choppin, 1983) and bunyaviruses (Akashi and Bishop, 1983)]. In the last year, the reovirus Sl gene segment also has been found to be bicistronic; the Sl gene codes for the viral neutralization ani To whom dressed.

requests

for

reprints

should

be ad-

243

tigen (al) and for a second protein with an apparent mol wt of 12,000(12K) (Ernst and Shatkin, 1985). In most cases, the function(s) of the second gene product remains to be determined. The rotavirus SAll genome segment 9 codes for an outer capsid glycoprotein (VP?‘;Mason et al, 1983;Ericson et ah, 1983) which is the major neutralization antigen (Kalica et al, 1981). We previously reported that VP7 is synthesized as a precursor protein of mol wt 37K and that mature VP7 is produced by the cleavage of a signal sequence of mol wt 1.5K, followed by the addition and processing (trimming) of Nlinked high mannose residues at a single glycosylation site (Ericson et aL, 1983). These results were confirmed when the DNA sequence of cloned SAll genome segment 9 was published (Both et ak, 1983; Arias et ah, 1984). The DNA sequence also re0042-6822/86 Copyright All rights

$3.00

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

244

CHAN

ET AL.

In vitro transcription and translation of rotavirus mRNA. Preparation of rotavirus mRNA and translation of the mRNA in RRL followed the procedure of Mason et al. (1980). Nuclease-treated wheat germ extracts were purchased from Bethesda Research Laboratories (Gaithersburg, Md.). For cell-free translations, hybrid-selected mRNA (50-150 ng) or total viral mRNA (1 pg) was added to the system; all translation reactions were supplemented with RNAsin (1000 units/ml; Promega Biolab, Madison, Wise.) and sometimes with microsomes (l2 equivalents; Walter et a& 1981). Hybrid selecticmof mRNA. A cDNA clone of SAll genome segment 9, constructed in our laboratory as previously described (Cohen et ah, 1983), was used to obtain hybrid-selected segment 9 transcripts. The hybrid-selection protocol was a modification of that described by Maniatis et al. MATERIALS AND METHODS (1982). Briefly, 50 pg of CsCl-EtBr purified Cells and viruses. SAll virus stocks [A/ plasmid DNA containing the segment 9 inSI/S. Africa/SA11/58/N3, S1, HsAll; des- sert (which was approximately 20% of the ignated as proposed by Graham and Estes total plasmid sequence) was denatured by (1985)] were plaque purified three times sonication and boiling and was immobiand propagated at low multiplicity (0.1 lized on aminophenyl-thioether paper PFU/cell) in MA104 cells as described (Schleicher & Schuell, Inc., Keene, N.H.). (Estes et ah, 1979). Different strains of Rotavirus mRNA (100 pg), synthesized SAll which code for a nonglycosylated VP7 from purified single-shelled virus partior for VP7s of different sizes (Estes et al., cles with endogenous polymerase, was 1982) and other rotavirus strains (canine added to the DNA-containing aminoA/CN/USA/CU-1/76/N3,S1,Hcu-r; bovine phenyl-thioether paper in 1 ml of hybridA/BO/USA/NCDV/69/N5,S1 ,HNcuv; rhe- ization buffer (50% formamide, 0.1% SDS, sus A/SI/USA/RRV-2/79/N3 ,S1,Hsav) 0.6 M NaCl, 80 mM Tris, pH 7.8, 4 mlM were cultivated in MA104 cells as described EDTA). Hybridization was carried out in (Dimitrov et al, 1985). this buffer at 37” for 18 hr. The paper was Preparation of dog pancreatic microscrmal then washed 10 times with buffer containmembranes. The preparation of rough dog ing 50% formamide, 0.3 MNaCl, 0.4 MTris, pancreatic microsomes for in vitro glyco- pH 7.8, 20 mM EDTA and 0.1% SDS. The sylation using rabbit reticulocyte lysates RNA was eluted in 99% formamide at 65”, (RRL) followed the method of Ericson et precipitated with ethanol, and used for al. (1983). However, these microsomes translation. This protocol routinely seinhibited the translation of rotavirus lected 0.5-1.0 pg of segment 9 mRNA. AfmRNAs in wheat germ extracts. Therefore, ter elution, the paper was washed three cell-free glycosylation in wheat germ ex- times in water, dried, and stored at room tracts was performed with dog pancreatic temperature for subsequent reuse. The pumicrosomes kindly provided by Dr. R. Gil- rity of the RNA was checked by in vitro more (Rockefeller University, New York, translation, by electrophoresis of RNA in N.Y.); these microsomes were purified ac- 4% polyacrylamide gels containing 6 M cording to the procedure of Walter and urea (Cohen et aZ.,1983), or by both. Blobel (1983) and lacked the inhibitory Analysis and immunoprecipitation of the factor(s). viral glycoprotein VP7 from infected cells. vealed other interesting features of this genome segment. Most striking was the presence of two in-phase initiation codons with the second codon having the optimal sequence for efficient initiation of translation (Kozak, 1984). In addition, each of the initiation codons was followed by sequences that code for a stretch of hydrophobic amino acids which theoretically could function as signal sequences. We now report studies which reveal that the SAll genome segment 9 is functionally bicistronic. Although both regions of hydrophobic amino acid sequences function as signal sequences, the processing of each of the two gene 9 products is distinct; one contains an uncleaved signal sequence and the other contains a cleaved signal sequence.

ROTAVIRUS

BICISTRONIC

Radioactively labeled viral polypeptides were prepared according to the method of Ericson et al. (1982). [?S]Methionine was added at 3.5 hr postinfection, and virusinfected cells were harvested between 6 and 7 hr postinfection. To identify and characterize the genome segment 9 products, we used monospecific guinea pig antiserum to the glycoprotein (VP7; Ericson et aZ., 1982). Our immunoprecipitation procedure was modified according to Anderson and Blobel (1983) to improve detection of the glycoprotein. In brief, a radiolabeled cell lysate in RIPA buffer (0.15 1M NaCl, 1% sodium desoxycholate, 1% Triton X-100, 0.1% SDS, 0.01 M Tris-HCl, pH 7.2, 100 units Trasylol per ml) was clarified by centrifugation at 100,000 g, adjusted to 1% SDS, and heated for 4 min in boiling water. Four volumes of dilution buffer (1.25% Triton X-100, 190 mM NaCl, 6 mM EDTA, 50 mM Tris-HCl, 10 units Trasylol per ml) were added before incubation with one-tenth volume of a 10% Formalin-fixed Staphylococcus aureus A (strain Cowan I) suspension for 15 min at room temperature. After the bacteria were removed by centrifugation, 5 ~1 of antiserum was added to 30 ~1 of the adsorbed lysate, and the mixture was incubated at 4” overnight. A second portion (50 ~1) of bacteria was added to each sample and incubated at 4” for 15 min. The bacteria with the adsorbed immune complexes were then collected by centrifugation and washed as described (Ericson et ah, 1982). After removal of the bacteria, the polypeptides were analyzed in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The conditions for SDS-PAGE and V8-protease peptide mapping have been described (Ericson et ab, 1983).

Endoglycosidase H digestion of puti$ed virus, infected cell lysates, and cell-free translation products. Purified virus particles, infected cell lysates, or cell-free translation samples in RIPA buffer (radiolabeled with [35S]methionine, approximately 50,000 counts) were digested with endo-P-N-acetyl-glucosaminidase H (Endo H; 0.2 U/ml final concentration; Miles Biochemicals, Elkhart, Ind.) for 1 hr at 37”. The reaction was stopped by adding an

NEUTRALIZATION

GENE

equal volume of 2X electrophoresis buffer and boiling for 2 min.

245

sample

RESULTS

Cell-free translation of mRNA hybrid selected with genome segment 9 cDNA yields two protein products. Translation of hybridselected, genome-9-specific mRNA in a wheat germ extract cell-free system (Fig. 1, lane b) produced two distinct polypeptide bands with apparent mol wt of 3’7K and 35K. These two bands also were present in the translation products made from total viral mRNA (Fig. 1, lane a), and they were clearly resolved after immunoprecipitation. In contrast, only one band of the correct molecular weight was seen when hya aQ -

bc

d

53 41 37 35.1

16

20

FIG. 1. In vitro translation of hybrid-selected mRNA. Rotavirus polypeptides synthesized in cellfree translation systems derived from wheat germ extracts (lanes a-d) or RRL (lanes e, f) as described under Materials and Methods were analyzed by 12% SDS-PAGE (a-d) or 10% SDS-PAGE (e and f). The [36S]methionine-labeled polypeptides were made in cell-free systems programmed with total viral mRNA isolated from in vitro transcription reactions (lanes a and e) or with hybrid-selected gene 9 mRNA (lanes b and f), hybrid-selected gene 10 mRNA (lane c), or no exogenous RNA (lane d). Asterisks indicate the polypeptides synthesized from endogenous RNA in RRL. The 34K band seen in RRL was synthesized with all hybrid-selected transcripts (see Fig. 3).

246

CHAN

brid-selected mRNA from genome segment 10 (which codes for a 20K precursor to a SAll glycoprotein, Fig. 1, lane c) or when genome segment 6 mRNA was translated (data not shown). Changes in magnesium and potassium concentrations did not affect the appearance of the two distinct genome segment 9 bands, indicating that they did not result from suboptimal cation concentration in the cell-free system (data not shown). Translation of the hybrid-selected genome segment 9 mRNA in RRL produced a major band on SDS-PAGE of mol wt 35K and longer exposure of the gel showed that the 37K product also was made although to a much lesser extent (Fig. 1, lane f). The running of longer polyacrylamide gels allowed better resolution of the polypeptides with mol wt 34K to 41K. Direct comparative analyses of samples on short and long gels demonstrated that the 35K translation product made from the hybrid-selected genome segment 9 mRNA in wheat germ extracts and the 35.3K product noted previously in RRL (Mason et al., 1980) were the same. Therefore, for consistency we refer to the smaller genome segment 9 product as 35.3K. We (Mason et ah, 1983) had previously shown that the 37K glycoprotein precursor was translated poorly in RRL, and postulated that this was because RRL contain endogenous signal recognition particles (SRP) that can arrest the synthesis of secretory polypeptides. Other studies have now suggested that the two protein products of genome segment 9 may interact differently with the SRP so that less 37K is made in RRL (W. K. Chan, B. L. Ericson, and M. K. Estes, manuscript in preparation). Because of this, we used the wheat germ system in most experiments to further characterize the two products of genome segment 9. We hypothesized that the two proteins (37K and 35.3K) translated in wheat germ extracts from segment 9 mRNA were derived from initiation of translation at the first and the second in-phase initiation codons, respectively. The relatedness of the two segment 9 products was confirmed by V8-protease partial mapping (data not

ET AL.

shown) and by immunoprecipitation with monospecific VP7 antiserum (see below) or monoclonal VP7 antibody (data not shown). These data suggested that both products were synthesized in the same reading frame and strengthened the hypothesis that the SAll genome segment 9 is functionally bicistronic. Glycosylation

of the two genome segment

9 proteins, To determine whether both translation products were glycosylated, we added microsomes to the wheat germ and rabbit reticulocyte systems (Figs. 2,3). The addition of microsomes to wheat germ extracts resulted in the disappearance of the 37K and 35.3K bands and the appearance of a new band migrating with an approximate mol wt of 38K in SDS-PAGE (Fig. 2, lanes a and b). Occasionally, two bands

37

.

.

41

.

38

.

35

.

34

.

28

35.3 .

. 20

ENDO

H

-

-

+

MlCROSOMES

-

+

+

+

FIG. 2. Glycosylation of the 3’7K and 35.3K proteins in wheat germ extracts and in infected cells. The two gene 9 products translated in wheat germ extracts programmed with hybrid-selected gene 9 mRNA (lane a) were converted to a 38K band after the addition of microsomes to the extracts (lane b). Lane c shows the results of Endo H digestion of the samples analyzed in lane b. Lanes d and e show the virus-specific polypeptides in infected cells, and lane d shows the resulting bands seen after Endo H digestion of lane e. The arrowheads highlight the 38K glycosylated VP7 proteins, and the double arrowheads show the two deglycosylated VP7 polypeptides. All the radioactively labeled polypeptides were analyzed by 10% SDSPAGE.

ROTAVIRUS

cd

ab

BICISTRONIC

e

f

35.3 34 .28

20

MiCROSOMES

-

+

-

+

-

+

FIG. 3. Translation and glycosylation of hybrid-selected gene 9 mRNA in RRL. The cell-free translation products were made in RRL programmed with total SAll mRNA (lanes a and b), hybrid-selected gene 9 mRNA (lanes c and d), or hybrid-selected gene 10 mRNA (lanes e and f). Lanes b, d, and f show the products made in the presence of microsomal membranes. Translation and glycosylation of the gene 10 product (lanes e and f) were used as internal controls for the activity of the lysates and the microsomal membranes. The translated products were analyzed by 12% SDS-PAGE.

were resolved in the 38K region. To verify that the 38K band was the glycosylated product from both precursors, the highmannose oligosaccharides were removed by Endo H digestion. Two deglycosylated bands were seen (Fig. 2, lane c), indicating that the 38K protein was indeed derived from both precursor proteins. Neither band comigrated with the original primary translation products, which was consistent with the retention of one N-acetylglucosamine residue on a polypeptide after Endo H digestion of the glycoproteins. Similar results were seen following addition of mi-

NEUTRALIZATION

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247

crosomes to RRL; a 38K glycoprotein was synthesized (Fig. 3, lane d), and digestion with Endo H resulted in the appearance of two bands that comigrated with those from the infected cells (data not shown). The appearance of both deglycosylated bands in the RRL supplemented with microsomes was expected, because the translational arrest of the 37K band (seen in RRL in the absence of microsomes) was released by the addition of microsomes (W. K. Chan, B. L. Ericson, and M. K. Estes, manuscript in preparation). We interpreted the appearance of the two bands produced by Endo H treatment as representing two translation products initiated at the different in-phase AUG codons in genome segment 9. An alternative interpretation that the addition of microsomes caused preferential translation and glycosylation of one product and that Endo H treatment produced two deglycosylated forms of one protein was refuted by obtaining identical results with a nonglycosylated virus variant (see below). Therefore, both genome segment 9 products are glycosylated and both regions of hydrophobic amino acids probably function as signal sequences for translocation. Both genome segment 9 glycoprotein products are present in infected cells. To

show that the two genome segment 9 products were not artifacts of in vitro translation, we identified these polypeptides in infected cells. [%]Methionine-labeled, SAll-specific polypeptides were analyzed on the longer SDS-polyacrylamide gels (Fig. 2, lane e), and the glycoprotein precursors were characterized further by digestion with Endo H. Two deglycosylated bands were clearly observed among the infected cell proteins (Fig. 2, lane d), and these bands comigrated with the genome segment 9 products synthesized in the in vitro wheat germ system. IdentiJication of two VP7s in other rotavirus strains. We used both immunopre-

cipitation and Endo H digestion to facilitate interpretation of the migration pattern for glycoproteins of other rotavirus strains. The two genome segment 9 products were observed with all rotavirus strains tested (canine, bovine, and rhesus;

CHAN

248 cu-1

abc

R RV

NCDV

def

gh

i

-++

-+

+

ET AL.

were removed during processing. We studied a variant of SAll (clone 28) that contains a nonglycosylated VP7 (Estes et al,, 1982) to address this question. We had previously used this variant to show that the 37K segment 9 product precursor contains a cleaved signal sequence (Ericson et ah, 1983). DNA sequencing has shown that the glycosylation site of clone 28 is changed; this presumably explains its nonglycosylated VP7 phenotype (unpublished data). The translation of hybrid-selected clone 28 segment 9 specific mRNA in wheat germ extracts yielded both segment 9 polypeptides (Fig. 6, lane a), and the addition of microsomes caused a shift in the migration of the 37K band to a position of 35.5K (Fig. 6, lane b). In contrast, the migration of the 35.3K precursor protein was not affected by the addition of microsomes. Both 35.5K

CL 3 VP7 ENDOH--t

-++

-

-

+

-

-

a

CL 28

bc

d

CL 37

ef

a

hi

t

FIG. 4. Two VP’i’s were found in different strains of rotavirus. The viral polypeptides in cells infected with different strains of rotavirus were labeled with [?S]methionine. Lanes a, b, and c show proteins from cells infected with canine CU-1 rotavirus; lanes d, e, and f were from cells infected with bovine NCDV; and lanes g, h, and i were from cells infected with RRV. Lanes b, e, e, f, h, and i show the proteins that were immunoprecipitated by anti-VP7 polyclonal serum. The lysates in lanes c, f, and i were digested with Endo H before immunoprecipitation. The double arrowheads show the two deglycosylated VP7 polypeptides in each virus.

Fig. 4, lanes c, f, and i) and with two additional variants of SAll that possess glycosylated or nonglycosylated VP7s of different sizes (Fig. 5, lanes c, f, and i). Therefore, the production of two VP7s appeared to be a general phenomenon for rotaviruses. Processing of the two genome segment 9 glycoprotks. The demonstration that both segment 9 product precursors were glycosylated (suggesting that both precursors must contain signal sequences) raised the question of whether both signal sequences

94 88

VP7-

+

+

-

+

+

-

+

+

ENDOH-

-

+

-

-

+

-

-

+

FIG. 5. Two VP7s were found in different clones of SAll. The viral polypeptides in cells infected with different clones of SAll were labeled with [%S]methionine. Lanes a, b, and c were from cells infected with clone 3; lanes d, e, and f were from clone 28; and lanes g, h, and i were from clone 37. Lanes b, c, e, f, h, and i show the proteins that were immunoprecipitated by anti-VP7 polyclonal serum. The double arrowhead in lane c highlights the two VP7s.

ROTAVIRUS

BICISTRONIC

NEUTRALIZATION

and 35.3K also were observed in clone 28 infected cells (Fig. 6, lane d), and Endo H digestion did not affect the migration of either band (Fig. 6, lane c). These results suggest that the biosynthesis of the segment 9 protein products proceeds as follows: translation from the first AUG produces a 37K product which contains a cleavable signal sequence. Translation from the second AUG produces a 35.3K product, and this protein contains an uncleaved signal sequence. Both 37K and 35.3K precursor proteins can be glycosylated (providing the glycosylation signal is present in the gene), and both precursors contribute to the mature 38K gly-

IN

VITRO

b

(I

: I

INFECTED

c

GENE CL 3

249 CL 28

CL 37

a

b

c

d

e

f

-

+

-

+

-

+

CELl

d

END0

H

FIG. ‘7. Detection of both gene 9 proteins in purified virus. Different clones of SAll were labeled and purified as described under Materials and Methods. The purified virus particles were disrupted by boiling in sample buffer for 2 min and were analyzed by 10% SDS-PAGE. Lanes b, d, and f show the Endo H digestion products of the samples analyzed in lanes a, c, and e, respectively. The polypeptides in lanes a and b are from clone 3 virus, lanes c and d are from clone 28, and lanes e and f are from clone 37. The double arrowheads show the two deglycosylated VP7 polypeptides in each virus.

37 33.3

cosylated band. The reason for the different forms of signal sequences remains unknown. END0

H

MICROSOMES

.-

-

+

-

+

-

FIG. 6. In vitro translation and glycosylation of clone 28 gene 9 mRNA. Lanes a and b show the in vitro translation products from wheat germ extracts programmed with hybrid-selected gene 9 mRNA of SAll clone 28. Lanes c and d show the polypeptides in clone 28 virus-infected cells. Microsomes were added to the wheat germ extract products shown in lane b. Lane c is the Endo H digestion profile of the samples from lane d. The proteins were analyzed by 10% SDSPAGE.

Possible functions of the two genome segment 9 glycoproteins. To determine if the

two segment 9 glycoproteins had different functions, we first asked whether both proteins were assembled into virus particles. Analysis of [35S]methionine-labeled polypeptides in purified SAll particles showed that both proteins were present in virions (Fig. 7, lanes b, d, and f). Both proteins were directly evident in the clone 28 variant that lacked a glycosylated VP7, but they were only seen in clone 3 and clone 37 particles

GHAN

250

ET AL.

following Endo H digestion of the glycosylated VP7s of these viruses. In this experiment, the Endo H digestion was incomplete so both the glycosylated and deglycosylated forms were seen (lanes b and f). Exhaustive digestion with Endo H converted the entire 38K band to the two deglycosylated bands (data not shown). Secondly, we investigated the kinetics of expression of the two VP7s in infected cells. Pulse-label experiments showed that both segment 9 polypeptides were initially expressed at the same time (l-2 hr postinfection). Quantitation of the relative amounts of each protein in infected cells was not possible because the 35.5K and 35.3K proteins migrated too closely to each other. DISCUSSION

We previously reported that the SAll genome segment 9 codes for a 37K precursor to VP7 which contains a cleavable signal sequence. This conclusion was based on cell-free translation studies performed with viral mRNA representing all 11 segments of rotavirus RNA. Our current results using hybrid-selected mRNA demonstrate that the SAll segment 9 (the major neutralization gene) codes for two primary products of 37K and 35.3K. These results resolve the apparent discrepancy between our previous report that the pre-

cursor to VP7 made in wheat germ extracts was 37K and the report of Both et al. (1983) that the precursor made in RRL was 35.5K. The identification of two VP7s raises some problems in nomenclature. We propose calling the glycoprotein with the 37K precursor VP7(1) to indicate that this protein arises from translation from the first initiation codon; VP7(2) would be used to denote the 35.3K protein translated from the second initiation codon. The observation that a viral gene can be functionally bicistronic is not unique. However, the two rotavirus segment 9 products are particularly interesting because (i) these two proteins are produced from two in-phase initiation codons, (ii) the difference between the two mature glycoproteins is likely to be only four or five amino acid residues, and (iii) the processing pathways of the two glycoprotein products appear distinct (summarized in Fig. 8). The strict conservation of the segment 9 sequence (including the two initiation sites followed by the sequences that code for the two hydrophobic regions) in different rotavirus strains and the synthesis of both proteins by all rotavirus strains tested strongly argue that both proteins perform some essential function(s). Whether these two genome segment 9 products are functionally distinct remains unknown, but precedence for independent functions of two overlapping proteins (that

37K -

Met -CTMet

35.3K

-CIB,L

I

Glycosylation

Met--Met

-&----+

37K

Met V+

Glycorylation

v

38K

Met&/-

38K

I

Endo H

wk

v

I

I -Met

35.3K

Endo H

35.5K

Met-k

35.3K

FIG. 8. Schematic drawing of the biosynthesis of the two gene 9 VP%. The top lines show the two apparent primary precursor products (37K and 35.3K) made from gene 9 mRNA. After processing in the presence of microsomes and glycosylation, both precursors migrate with apparent molecular weights of 38K in SDS-PAGE.~Symbolises the carbohydrate. During this processing, an aminoterminal signal sequence on the 3’7K precursor protein is cleaved, but the signal on the 35.313 precursor is not cleaved. Endo H digestion removes the high-mannose oligosaccharides and leaves one sugar residue on the backbone, as shown (1). The approximate molecular weight of each protein is indicated.

ROTAVIRUS

BICISTRONIC

NEUTRALIZATION

GENE

251

from one specific synthetic mRNA generare synthesized by a different mechanism) ated by transcription of full-length gene 9 has been documented for the adenovirus cDNA in the SP6 riboprobe system (unElA 243 a.a. and 289 8.8. proteins (Spindler published data). et cd, 1985). Finally, the rotavirus system should One question raised by our results is whether both hydrophobic amino acid re- provide a useful model to study the differential use of in-phase AUG codons for the gions or only the second uncleaved hydroinitiation of protein synthesis as a mechphobic amino acid sequence (that is present in both precursor proteins) function as anism for the translational control of eusignals for translocation. Our demonstrakaryotic gene expression (Haarr et al., tion that VP’7(2) is glycosylated and studies 1985). of the expression of a series of genome segment 9 deletion mutants (Poruchynsky et ACKNOWLEDGMENTS al., 1985) both provide evidence that the second hydrophobic domain contributes to We thank Ron Loosle for his excellent technical astranslocation of VP7 into the rough endosistance. This research was supported by Grant AMplasmic reticulum. The role of the first hy30144 from the National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases, by a grant from drophobic sequence is less clear; presumably it functions as a signal sequence, since the Diarrhoeal Disease Programme of the World it is cleaved, and we are unaware of any Health Organization, and by a grant from the Health (Shelton, Conn.). cleaved hydrophobic sequences that do not Care Division of Richardson-Vicks We also thank Dr. Joseph L. Melnick for his continued function as translocation signals. encouragement and support. It is interesting to consider a few possible functions for the two genome segment 9 REFERENCES proteins. First, the different signal sequences on the proteins or the expected difAKASHI, H., and BISHOP, D. H. L. (1983). Comparison ferent amino termini may influence protein of the sequences and coding of La Crosse and transport or processing or virus morphosnowshoe hare bunyavirus S RNA species. J. Viral genesis. Preliminary experiments indicate 45,1155-1158. the cleaved and uncleaved signal sequences ANDERSON, D. J., and BLOBEL, G. (1983). Immunopreon VP7(1) and VP7(2) interact differently cipitation of proteins from cell-free translations. with the SRP (W. K. Chan, B. L. Ericson, In “Methods in Enzymology” (S. Fleischer and B. Fleischer, eds.), Vol. 96, pp. 111-121. Academic and M. K. Estes, manuscript in preparaPress, New York. tion). It is possible that VP7(1) may transARIAS, C. F., LOPEZ, S., BELL, J. R., and STRAUSS, locate more efficiently than VP7(2) and J. H. (1984). Primary structure of the neutralization that VP7(1) might facilitate the transloantigen of simian rotavirus SAll as deduced from cation of VP7(2) alone or in association cDNA sequence. J. ViroL 50,657-661. with other viral proteins. In this case, the BEISEL, C., TANNER, J., MATSUO, T., THORLEY-LAWSON, two segment 9 products may perform difD., KEZDY, F., and KIEFF, E. (1985). Two major outer ferent functions required for assembly of envelope glycoproteins of Epstein-Barr virus are the outer capsid of particles, a process that encoded by the same gene. J. Viral. 54,665-6’74. occurs at or inside the endoplasmic reticBELLINI, W. J., ENGLUND, G., ROZENBLATT, S., ARNHEITER, H., and RICHARDSON, C. D. (1985). Measles ulum. virus P gene codes for two proteins. J. Viral. 53, Although we have not definitively proven 908-919. that the two VP7s are synthesized from Bos, J. L., POLDER, L. J., BERNARDS, R., SCHRIER, only one bicistronic mRNA during viral P. I., VAN DEN ELSEN, P. J., VAN DER EB, A. J., and infection, analysis of our segment 9 hybridVAN ORMONDT, H. (1981). The 2.2 kb Elb mRNA of selected transcripts on gels showed only human Ad12 and Ad5 codes for two tumor antigens one discrete band of mRNA; this result arstarting at different AUG triplets. CeU 27,121-131. gues for the mRNA being bicistronic. FurBOTH, G. W., MA~ICK, J. S., and BELLAMY, A. R. (1983). ther support for this mechanism of synSerotype-specific glycoprotein of simian 11 rotavithesis also comes from preliminary data rus: Coding assignment and gene sequence. Proc. Nat/. Acad Sci. USA 80,3091-3095. showing that both VP7s are translated

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