Arch Virol (1992) 125:53-69

_Archives

Vifrology © Springer-Verlag 1992 Printed in Austria

Coding assignments of the genome of adult diarrhea rotavirus*

Z.-Y. Fang 1' 2, S. S. Monroe 1, H. Dong 2, M. Penaranda 1, L. Wen 2, V. Gouvea 1, J. R. Allen 1, T. Hung 2, and R. I. Glass 1

1Viral Gastroenteritis Unit, Center for Infectious Diseases, Centers for Disease Control, Atlanta, Georgia, U.S.A.

2 Institute of Virology, Chinese Academy of Preventive Medicine, Beijing, China

Accepted December 19, 1991

Summary. Adult diarrhea rotavirus (ADRV) has caused epidemics of diarrhea in China since 1982 and remains the only group B rotavirus associated with widespread disease in humans. We recently characterized the proteins of ADRV and have now proceeded to identify the gene segments encoding each protein. Viral RNA transcripts were synthesized in vitro with the endogenous viral RNA polymerase and separated by electrophoresis in agarose. The individual tran- scripts were translated in a cell-free system using nuclease-treated rabbit reti- culocyte lysates. The translation products were compared with polypeptides found in purified virus and were characterized by SDS-PAGE, immunoprecip- itation, and Western blot analysis using antisera to double- and single-shelled virions, virus cores, and monoclonal antibodies. Furthermore, individual RNA transcripts were hybridized to total dsRNA to determine their genomic origin. Based on this analysis, the core polypeptides VP1, VP2 and VP3 are encoded by segments 1, 2, and 3, respectively. The main polypeptides in the inner capsid, VP6, and the outer capsid, VP4 and VP7, are encoded by segments 6, 4, and 8 respectively. Segments 5, 7, and 9 code for 60, 45, and 30 kDa nonstructural polypeptides. Two other nonstructural polypeptides (24 and 25 kDa) are derived from gene segment 11. Gene segment 10 codes for a 26 kDa polypeptide that is precipitated with serum to ADRV and may be a structural protein VP9. With this exception, gene coding assignments of ADRV are comparable to those of the group A rotaviruses. Our results have clear implications for filrther work in cloning, sequencing, and expression genes of ADRV and can provide direction towards understanding the origin and the evolution of this virus.

* Part of this work was presented at the International Congress of Virology, Berlin, Federal Republic of Germany, August 1990.

54 Z.-Y. Fang et al.

Introduction

Worldwide, rotaviruses have been associated with severe gastrointestinal disease in animals and children. Different groups of rotaviruses are morphologically identical but can be distinguished by RNA electropherotype, by in vitro hy- bridization, and by group-specific antigens [1-3]. The group B rotaviruses have been implicated as a cause of diarrhea in a variety of animal species [3-7] but have been found in association with human disease only in China. Since 1982, noncultivable group B rotavirus, called adult diarrhea rotavirus (ADRV), has been responsible for epidemics of diarrhea affecting tens of thousands of adults in the People's Republic of China [8-11]. These outbreaks have raised ~ many questions concerning the biology and origin of these strains, their relatedness to other rotavirus, and the prospect that group B rotaviruses might become epidemic in other parts of the world.

We recently characterized the structural proteins of ADRV and compared these with the proteins of a group A rotavirus, SA11 [12]. Removing the outer capsid of ADRV resulted in the loss of three polypeptides of 64 (VP5), 61 (VP5a), and 41 kDa (VP7). The 41 kDa polypeptide was a glycoprotein. One polypeptide of 47 kDa (VP6) composed the inner capsid and contained a com- mon group B antigen. Two major proteins of 136 (VP1) and 113 kDa (VP2) remained in the virus core, and several proteins in the t10 to 72kDa range might be VP3, VP4, or related proteins. The cloning of ADRV by Chen et al. [13-15] permitted characterization of genomic segments 5, 9, and 11. Segment 5 in this strain of ADRV shared amino acid sequence homology with VP6 of rotavirus of groups A and C and was therefore considered to encode for VP6. By similar comparisons, gene segments 9 and 11 shared sequence homology with genes encoding the VP7 and NS26 protein equivalents of group A rotavirus, respectively. Characterization of clones from another group B rotavirus derived from rats (IDIR) indicated that its sixth gene segment hybridized with gene 6 of bovine group B rotavirus but with gene 5 of ADRV demonstrating that strain differences in gene coding assignments do occur [16].

We have now identified the gene segments coding for each protein using a different strategy. Viral RNA transcripts were synthesized in vitro with the endogenous viral RNA polymerase and separated by electrophoresis in agarose gels. Individual transcripts were translated in a cell-free system. The translation products were compared with polypepfides found in purified virus and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), by immunoprecipitation, and by Western blot analysis using polyclonal anfisera to double- and single-shelled virions, virus cores, as well as monoclonal antibodies. To determine the genomic origin of individual RNA transcripts, we hybridized them to total dsRNA. These procedures allowed us to identify the proteins coded by each of the viral genomic segments.

ADRV gene-coding assignments 55

Materials and methods Virus and antisera

Fecal specimens containing ADRV were collected from hospitalized patients in a single outbreak that occurred in Chengde, Hebei Province, The People's Republic of China, in 1987. Feces were pooled before further analysis. ADRV was purified directly from stool specimens by methods described previously [12]. Purified single-shelled virus particles were obtained by treating purified double-shelled virions with 10mM EDTA (20 min at 37 °C), and core particles of ADRV were obtained by treating single-shelled particles with 1 M CaC12 (20 rain at room temperature).

Hyperimmune guinea pig antisera to ADRV and hyperimmune mouse arltisera to single- shelled virus and to virus core particles were prepared using cesium gradient purified antigen as described previously [12]. Despite our efforts to purify these fractions, some fragments of VP6, the most abundant viral protein, were present in the core preparation. Monoclonal antibodies (MAbs) to ADRV were provided by Wei-Wei Ye (Ym-1), (Institute of Virology, Beijing, The People's Republic of China) and Mary K. Estes (10G10) (Baylor College of Medicine, Houston). Ym-1 has been used previously as a detector in a blocking EIA to test for antibodies to ADRV [11]. By immunoelectronmicroscopy, 10G10 is specific for double-shelled particles (M. K. Estes, pets. comm.).

Synthesis of transcripts Rotavirus mRNA was synthesized in vitro from purified virus [-17]. The RNA polymerase in double-shelled virus particles was activated by preincubation at 37 °C for 30 min after the additon of 10 mM EDTA. The standard reaction mixture (1 ml) contained 100 mM Tris hydrochloride (pH 8.0), 10 mM magnesium acetate, 0.1% bentonite prepared according to the method of Fraenkel-Conrat et al. [18], 2.5 mM each of ATP, CTP, UTP, and GTP, 0.5 mM S-adenosylmethionine, 8 mM phosphoenolpyruvate, 50 gg/ml pyruvate kinase, and about 100 gg of purified rotavirus. The reactions were incubated at 41 °C for 2 h. After the bentonite was spun down at 12,000 x g for 30rain, the supernatant containing the syn- thesized RNA was centrifuged at 27,000 x g for 40 min to remove virus particles and extracted with an equal volume of water-saturated phenol after adding 0.5% SDS. The synthesized RNA was precipitated with 2 volumes of ethanol and stored at - 2 0 °C. To produce more mRNA, the same reaction mixture without additional virus was added to the pelleted bentonite to initiate ~a new cycle of transcription.

Preparation of individual mRNAs Individual mRNA transcripts were obtained by separation on a large 1.5% agarose gel (13 × 45cm) prepared in TBE buffer (89mM Tris base, 89mM boric acid, 0.2mM EDTA [pH 8.0-8.3] and electrophoresed at 100V for 45 h. Bands containing individual mRNAs were cut out of the gel and electroeluted at 80 V for 40 min with a unidirectional electroelutor (International Biotechnologies, Inc., New Haven, CT). The mRNA was drawn down in running buffer (20raM Tris-hydrochloride [pH 8.0], 0.2mM EDTA, and 5 mM NaC1), trapped in a small volume of high-salt buffet (7.5 M ammonium acetate with 0.01% bromo- phenol blue), and precipitated by adding 2 volumes of ethanol at - 2 0 °C

in vitro translation mRNA was translated in vitro by using nuclease-treated rabbit reticulocyte lysates (Pro- mega, Madison, WI). For a standard translation assay, 0.25 to 1.0gg of RNA and 15 gCi of L-[35S]methionine were added to 10 gl of the nuclease-treated rabbit reticulocyte lysate to make a final volume of 15 ~tl. RNasin was included in the assay mixture to inhibit RNase activity. In some experiments, canine pancreatic microsomal membranes (Promega, Ma- dison, WI) were added to the assay mixture for glycosylation. Since many independent

56 Z.-Y. Fang et al.

translations of single mRNAs were required to provide a complete set of results, we prepared a composite of the best gels, scaling them by using size markers that were coelectrophoresed in each run.

PAGE of proteins Electrophoresis of polypeptides was performed in 12% polyacrylamide gels using Laemmli's discontinuous buffer system and 0.5 M urea as described previously [ 12, 19]. The translation mixture was treated with RNase A and precipitated with 80% cold acetone before being loaded onto gels.

Immunoprecipitation Viral structural proteins in translation mixtures were immunoprecipitated to determine their identity [20]. The translation mixture (8 ~tl) was diluted 1 : 1 with 2 × radioimmune precipitation (RIP) buffer (2% [vol/vol] Nonidet-P40, 1% [wt/vol] sodium deoxycholate, 0.2% [wt/vol] SDS, and 2raM phenylmethylsulfonyl fluoride [PMSF] in 10mM Tris [pH 8.0], 100 mM NaC1, and 1 mM EDTA [pH 8.0] [TNE]). Hyperimmune sera (5 lal or MAb to ADRV diluted 1 : 1 with 2 x RIP buffer was added, and the mixture was incubated at 37 °C for 1 h and stored at 4 °C overnight. Subsequently, 100 ~1 of a 10% suspension of staphylococci carrying protein A (BRL Life Technologies Inc., Gaithersburg, MD) in RIP buffer were added to the mixture, with occasional mixing for 1 h at 4 °C. Bacterial cells were then washed three times with 0.5% Nonidet-P40 and 0.5% sodium deoxycholate in TNE, and pellets were suspended in 30 ~tl of sample buffer (5 mM Tris-hydrochloride [pH6.8], 8% glycerol, 4% SDS, 0.5M urea, 5% [3-mercaptoethanol, 0.01% phenol red), boiled for 2 min, and spun down before being loaded onto gels.

Western blotting Viral polypeptides from purified virus were separated by SDS-PAGE as described above, except that no urea was used in the electrophoretic buffer. The polypeptides were electro- transferred onto a nitrocellulose membrane. The resulting blots were probed with hyper- immune sera to ADRV double- or single-shelled virus particles and to virus core [12].

Trypsin and Endo-fl-N-acetylglucosaminidase H digestion Synthesized polypeptides from whole mRNA were treated with trypsin (6 ~tg/ml) at 37 °C for 30 rain before being precipitated with acetone. Digestion with Endo-[3-N-acetylglucos- aminidase H (Endo H) was performed at a final enzyme concentration of 0.5 U/ml for 1.5 h at 37 °C after the synthesized polypeptides were denatured by boiling for 2 min.

Electrophoresis and transfer of rotavirus dsRNA Virus dsRNA was prepared from virus particles trapped in the bentonite after in-vitro transcription by suspending the bentonite in TBS and 1% SDS and extracting it with water- saturated phenol, dsRNA was electrophoresed in a 1.5% low-melting-point agarose gel (SeaPlaque, FMC BioProducts, Rockland, ME) or in a 10% polyacrylamide gel for sep- arating RNA segments 5 and 6. Ethidium bromide (0.5 ~tg/ml) was incorporated both into the agarose gel and in the running buffer (TBE). Separated RNA segments were denatured in 0.2 M NaOH for 30min and transferred to a nylon membrane (Zeta-Probe, Bio-Rad Co., Richmond, CA) in 10mM NaOH overnight with the Blot Transfer System (BRL, Gaithersburg, MD). For polyacrylamide gels, an acrylamide-to-methylenebisacrylamide ratio of 1 : 37.5 was used in the separating gel (10%), and a 4% polyacrylamide gel was used in the stacking gel with the Laemmli buffer system [19]. Separated RNA segments were electrophoretically transferred to the nylon membrane by using the procedure of Tanaka et at. [21]. In brief, each gel was immersed for 20min in 0.1 M NaOH containing

ADRV gene-coding assignments 57

0.25 M NaC1 to denature the RNA followed by two 20-rain immersions in 4 x TAE (1 x TAE is 10mM Tris, 5mM sodium acetate, 0.5mM EDTA [pH7.8]) and one 20-min treatment in 1 x TAE. Etectrophoretic transfer was performed in 1 x TAE buffer at 0.8 A for 4h at 10 °C.

Partial fragmentation of ssRNA and kinasing with [~35S]A TP Partial fragmentation of single-strand RNA (ssRNA) was performed by hydrolysis [22]. mRNA samples (0.5 gg) were dissolved in 12.5 gl water and preheated to 50 °C for 10min, and 12.5 gl of 0.1 M Na2CO3 (also preheated to 50°C) were added. After heating 20min, the hydrolysis was stopped by the addition of Tris-hydrochloride (pH 7.5) to 0.1 M and 2 volumes of ethanol. End labeling of fragmented mRNA with [73sS]ATP was performed in a kinasing mixture: 60 mM Tris hydrochloride (pH 7.4), 10 mM MgC12, 5 mM dithioth- reitol, 25 gCi ['/35S]ATP, and 5 units T4 polynucleotide kinase (Boehringer Mannheim) per hour (total amount = 20 units) at 37 °C for 4 h. The mixture was boiled for 5 min, a half volume of 7.5 M NHgOAc was added and the mixture was kept on ice for 5 rnin to pellet the proteins. Unincorporated label was removed by ethanol precipitation.

RNA-RNA blot hybridization Transcript RNA was hybridized to genomic dsRNA immobilized on a nylon membrane [23]. The blotted membranes were first incubated in hybridization buffer (5 x SSC, 5 x Denhardt's, 50 mM phosphate buffer [pH 6.5], 500 gg/ml salmon sperm DNA, 0.1% SDS) at 42°C for 3 to 18h. After adding mRNA probe (abom 5 x 104cpm), hybridization was carried out at 55 °C for 16 to 50 h, and was terminated by washing the membrane in 2 x SSC-0.1% SDS at room temperature (four times) followed by two 10-rain washes at 55 *C in 0.1 x SSC-0. 1% SDS. The membranes were rinsed in 0.1 x SSC, dried in a vacuum oven at about 40 °C for 30 rain, and then exposed to X-ray film for 1 to 10 days.

Results

Comparison of viral polypeptides synthesized in vitro and in vivo

Our previous studies identified the structural polypeptides of A D R V [12]. The polypeptides synthesized in vitro were labeled with [35S]methionine by cell-free translation of unfractionated A D R V transcripts in the rabbit reticulocyte lysates (Fig. 1, lane B) and compared with polypeptides found in purified virus (lane A) that were stained with Coomassie brillant blue. Although lanes A and B of Fig. 1 come from different gels, the spacing of proteins according to size markers (not shown) made them comparable. After staining with Coomassie blue, the gel was dried, exposed to X-ray film, and aligned so that the migration patterns of viral polypeptides could be compared with the translation products. An mRNA-free control for the in vitro translation system was also run and only showed two bands of 43 and 44 kDa (see control track, Fig. 2). The structural polypeptides are designated VP1 through VP7, and the polypeptides synthesized in vitro are designated by molecular mass (in thousands). According to our results, thirteen polypeptides synthesized in vitro are identified as viral proteins or their precursors and the other bands (except for the 43/44 kDa bands) are probably degraded polypeptides or partial glycosylated products. The poly- peptides of 136, 113, 84, 47, and 41 kDa appear to be comparable with VP1, VP2, VP4, VP6, and VP7 (glycosylated), respectively. The 30 kDa and 26 kDa viral bands which appear weakly in this figure were more clearly visualized as

58 Z.-Y. Fang etal.

Fig. 1. Comparison of polypeptides found in virus purified from infected feces and stained with Coomassie blue (A) with polypeptides synthesized in vitro (B) using unfractionated ADRV transcripts and a cell-free translation system derived from nuclease-treated rabbit reticulocyte lysates. During synthesis, pro- teins were labelled with L-[35S]methionine. After staining the gel with Coomassie blue, the gel was dried and exposed to X-ray film. The developed film and the dried gel were aligned to compare migration patterns of the viral polypeptides versus the translation products. Numbers at fight indicate apparent molecular masses (kDa). Of note, a small quantity of the abundant VP7 precursor (35 kDa) was apparently glycosylated to a 41 kDa protein

in vitro translation products (Fig. 2). The 26 kDa polypeptide was very reactive by immune precipitation (Fig. 3).

In vitro translation of fractionated transcripts

To determine the translation product of each viral RNA transcript, 200 ~g of viral RNA was fractionated on a 1.5% agarose gel, and individual R N A bands electroeluted from the gel were translated in the cell-free reticulocyte lysate system in the presence of [35S]methionine. After translation, the polypeptides were analyzed in 12% polyacrylamide gels. The translation products of each of the 11 fractionated transcripts (Fig. 2, lanes m l - m l 1) were compared with the products translated from unfractionated virus RNA. The individual tran- scripts produced an array of small translation products, perhaps from frag- mentation of the transcript or incomplete elongation.

With only a few exceptions, the largest polypeptide was interpreted to be the product of the complete transcript. Hence, the 136kDa polypeptide was synthesized from the largest transcript (ml). The second message, m2, was translated into the 113 kDa polypeptide. An inversion was seen in the size of the polypeptides encoded by the next two transcripts: the third message, m3, encoded a polypeptide of 78 kDa, whereas the fraction containing m4 coded for the 84 kDa polypeptide. For m3, some larger translation products of m4

ADRV gene-coding assignments 59

Fig. 2. Polypeptides synthesized in vitro from 11 fractions of viral mRNA. The mRNA was separated in agarose gels, electroeluted, and added to the translation system from the largest mRNA (ml) through the smallest mRNA (m11). Total Unfractionated viral mRNA.

Control Endogenous control for in vitro translation with no viral RNA

were also present indicating the incomplete separation of mRNAs. The m5 made at 60 kDa polypeptide. The sixth and seventh fractions were difficult to separate and were always slightly contaminated by each other. The 47 kDa and 45 kDa polypeptides were synthesized from fraction m6 and m7, respectively, although for m7, some larger products of m6 were also present° While the 47 kDa polypeptide was present in the translation products of all 3 messages, m5, m6, and mT, it was assigned to m6 because m5 yielded a most distinct, larger band at 60 kDa and m7 yielded a distinct smaller band at 45 kDa. The m8, m9 and ml0 fractions coded for the 35 kDa, 30kDa, and 26kDa poly- peptides, respectively. The final two polypeptides, 25 kDa and 24 kDa, were both synthesized from the smallest transcript (ml 1).

60 Z.-Y. Fang etal.

Fig. 3. Polypeptides synthesized in vitro from unfractionated total mRNA (Total) were immu- noprecipitated with guinea-pig anti-ADRV serum (Gp) or MAb to ADRV (Ym-1, IOGIO). Virus Purified virus control. Numbers indicate the molecular masses (kDa) of viral structural polypeptides or their precursors

Immunoprecipitation of polypeptides synthesized in vitro

To determine which polypeptides synthesized in vitro were structural proteins or their precursors, the translated products from unfractionated total transcripts were immunoprecipitated with guinea pig anti-ADRV serum and two mono- clonal antibodies (MAbs) to ADRV, 10G10 which was specific for double- shelled particles and Ym-1 which was not (Fig. 3). We also have immunopre- cipitated polypeptides translated from each of the fractionated transcripts, ml through ml 1, with guinea pig anti-ADRV serum and these results confirm the data from unfractionated transcripts (data not shown). The viral structural proteins or their precursors in translation products were immunoprecipitated with apparent molecular mass 113, 84, 78, 47, 4t, 35 and 26kDa (Fig. 3). Two polypeptides of 136 and 41 kDa that were immunoprecipitated with guinea pig anti-ADRV serum in other experiments (data not shown) were not seen in Fig. 3 (lane Gp). MAb 10G10 immunoprecipitated an 84 kDa polypeptide in the outer capsid and Ym-1 reacted with the 47 kDa polypeptide, the main component of the inner capsid. These immunoprecipitated polypeptides were comparable with those found in purified virus although polypeptides 35 and 26kDa were not

ADRV gene-coding assignments 61

detected in purified virus (Fig. 3, lane virus). The other translation products with molecular masses of 60, 45, 30, 25 and 24 kDa were nonstructural poly- peptides. Translation products from unfractionated transcripts were immu- noprecipitated with preimmune mouse and rabbit normal sera and nonspecific bands were not found (data not incorporated in Fig. 3).

Localization of polypeptides translated in-vitro in the virion

Since ADRV cannot yet be cultivated in vitro, comparative peptide mapping of in-vitro and in-vivo synthesized polypeptides could not be carried out. To confirm the identification of the viral structural proteins or their precursors in translation products, the localization of these polypeptides in the outer-capsid, inner-capsid, and virus core was analyzed by immunoprecipitation of the trans- lated products from unfractionated total mRNA with antisera raised against purified double-shelled (DS) particles, single-shelled (SS) particles, and virus core, respectively (Fig. 4). These results were then compared with those obtained by Western blot analysis in which purified virus protein were probed with the same antisera. By immunoprecipitation, the 84, 35, and 26 kDa polypeptides reacted only with antiserum to DS particles (lane C) and not with. antisera to SS particles (lane D). When antisera to the virus core were prepared, the cores contained some adherent fragments of protein from the DS and SS particles. Consequently, antisera to the core did not react with the 41, 35, or 26 kDa polypeptides in the DS particles but did react weakly with the 84 kDa poly- peptide (lane E). By Western blot, the 84, and 35 kDa polypeptides as well as the 41 kDa polypeptide were observed, but the 26kDa polypeptide was not present, and a 64 kDa polypeptide was seen (lane G). These four polypeptides (84, 64, 41, and 35 kDa) were not detected with antisera to SS particles (lane H) or core (lane I); however there was a weak reaction with the 84 kDa polypeptide. By Western blot, the 84 kDa reacted weakly with antisera to the DS particle, presumably because most of it had been digested into a 64 kDa (VP5) and a 20 kDa (VP8) component (not shown) that was not visualized in purified virus. These results indicate that the 84, 64, 41, and 35 kDa polypeptides were localized in the outer capsid (noted by arrows in Fig. 4, lane G).

To further test the hypothesis that the 84 kDa polypeptide was in the outer capsid, translation products from unfractionated total mRNA were digested with trypsin (Fig. 4, lane A). Some of the 84 kDa polypeptide, like VP4 of group A rotavirus, was digested into a 64 kDa polypeptide consistent with VP5. The remaining fragment may correspond to VP8 but was not consistently visualized.

By both immunoprecipitation and Western blot, the 47 kDa polypeptide band was localized to the inner capsid and reacted strongly with antisera to SS particles (Fig. 4, la~es D and H). This band reacted with antisera to core as well (lanes E and I), probably due to some contamination of the core preparation used for immunization with some SS particles. Our previous study indicated that no 47 kDa polypeptide was located in the core. Nonetheless, the reaction

62 Z.-Y. Fang et al.

Fig. 4. Localization of polypeptides from the translated products in the outer-capsid, inner- capsid, and virus core. The polypeptides synthesized in vitro from unfractionated total mRNA (B) were then immunoprecipitated with guinea pig or mouse antisera raised against purified double-shelled (DS) (C) and single-shelled (SS) particles (D), and virus cores (E). For comparison, Western blots of ADRV polypeptides were probed with these antisera against DS particles (G), SS particles (H) and virus cores (/). Arrows highlight the poly- peptides discussed in the text. FPurified virus as control. Trypsin digestion of the translation

products of total viral mRNA is indicated (A)

was extremely intense with antisera to SS particles, and very little material was required to visualize the band.

By immunoprecipitation, two polypeptide bands of 113 and 78 kDa reacted strongly with antisera to core (Fig. 4, lane E), and one band of 136 kDa reacted weakly. By Western blot, bands of 136, 113, and 92 kDa polypeptides were seen (laneI). The two larger bands (136 and l l 3 k D a ) were consistently identified using both methods and were localized to the virus core. Since no 92kDa polypeptide was detected by immunoprecipitation of the translation product,

ADRV gene-coding assignments 63

the 78 kDa polypeptide which reacted strongly may be a related polypeptide such as a precursor to the mature core protein.

Identification of glycoproteins in translation products

Our previous studies identified the 41 kDa polypeptide (VP7) in purified virus to be a glycoprotein. After endo H digestion, the 41 kDa polypeptide shifted to be a 35 kDa polypeptide. To determine if the 41 kDa polypeptide in the translation products was a glycoprotein and the 35 kDa polypeptide was its precursor, canine microsomal membranes were added to the translation system. While a small amount of the 41 kDa polypeptide was synthesized without canine microsomal membranes in the translation system (e.g., Fig. 1, lane B), much more was synthesized when they were present (Fig. 5, lane B vs. lane A). After more 41 kDa polypeptide was synthesized, the translation products were di- gested with endo H. The 41 kDa polypeptide shifted to be a 35 kDa deglyco- sylated polypeptide (lane C). The 30 kDa nonstructural polypeptide appeared to be digested by endo H and was present in greater quantity when translated in the presence of canine microsomal membranes suggesting that it may be glycoprotein as well.

Fig. 5. Glycosylation of the outer shell 41 kDa polypeptide of ADRV. The polypeptides were synthesized from total mRNA in rabbit reticulocyte lysates with (B and C) or without (A) canine microsomal membranes. In C, the syn- thesized polypeptides were digested with endo-[3-N-acetyl- glucosaminidase H. The arrow indicates the migration posi- tion of deglycosytated polypeptides

64 Z.-Y. Fang et al.

Hybridization of fractionated transcripts to A D R V genome segments

To determine which RNA segment encoded which RNA transcript, each 35S- labeled fractionated transcript was hybridized to the total genome segments immobilized on a nylon membrane (Fig. 6). Each mRNA species hybridized to a corresponding dsRNA segment in the general order of electrophoretic mobility (i.e., ml hybridized to segment 1, m2 hybridized to segment 2, and so on). The large mRNAs hybridized cleanly to the larger dsRNA segments, and the label could be detected by autoradiography within hours with little or no background. The small mRNAs incorporated less label and.the hybridization signal required up to 10 days exposure for visualization of bands. For these hybridizations, some non-specific bands appeared in most lanes, probably from contaminated small pieces of each transcript in the fractionated probe. Of note, no cross

Fig. 6. Hybridization of fractionated mRNA to the dsRNA of ADRV. mRNA fractionated by agarose gel electrophoresis (m1-m11) was labeled with 35S and then hybridized to the viral dsRNA segments (indicated by numbers at left) immobilized on nylon membranes by electrophoretic transfer. Unfractionated total mRNA (Total) was hybridized to the

dsRNA as a control

ADRV gene-coding assignments 65

reactions were visible for those small bands immediately adjacent to the mRNAs being specifically examined indicating that each transcript was cleanly separated from its neighbors.

Summary of the gene assignment map of ADR V

A map of the ADRV gene coding assignment, based on these results, shows the gene segment that codes for each mRNA transcript, the corresponding polypeptide product of each transcript, and the designation of each transcript after post-translational modification (Fig. 7). The virus core polypeptides VP1, VP2, and VP3 were encoded by gene segments 1, 2, and 3, respectively. The inner-capsid polypeptide VP6 was most likely encoded by segment 6 and the outer-capsid polypeptides VP4, VP7, and possibly VP9 were encoded by seg- ments 4, 8, and 10. The 35 kDa translated polypeptide was a precursor of the 41 kDa glycoprotein. As VP9 was not yet identified in purified virus, the 26 kDa polypeptide may be the viral protein VP9 or its precursor. Nonstructural poly- peptides with apparent molecular masses of 60, 45, and 30 kDa were encoded

Fig. 7. Summary of the gene assignment map of ADRV

66 Z.-Y. Fang et al.

by segments 5, 7, and 9, respectively. The other two nonstructural polypeptides, with molecular masses of 25 and 24 kDa, were both encoded by segment 11. They may be two different proteins or one may be the precursor of the other.

Discussion

We have mapped the gene-coding assignments of the noncultivable human group B rotavirus ADRV. Our method was based on the strategy for mapping the SA11 genome developed by Mason [24], in which small amounts of virus were used to synthesize larger amounts of rotavirus transcripts in vitro with endogenous viral RNA polymerase. We combined this procedure with other immunologic methods to identify the translation products from each fraction- ated transcript and to avoid partial proteolysis mapping of viral proteins used for cultivatable rotavirus [24, 25]. In addition, our method used blot hybrid- ization to identify the gene segment coding for each fractionated transcript. Because segments 3 and 4, and segments 5 and 6 could not be separated adequately on an agarose gel, we separated segments 3 and 4 by using a pro- longed running time in an extra large gel and segments 5 and 6 by running dsRNA in a polyacrylamide gel (data not shown). For fractionation of the transcripts, we also used an extra large gel and a prolonged running time (see Fig. 7, lane mRNA). Even so, each fractionate mRNA was not completely pure and probably contained some smaller mRNA fragments trapped or broken during separation and extraction. Consequently, while the largest protein was translated from the principal mRNA fraction, many smaller polypeptides were seen as contaminants representing the translation products of these smaller entrapped mRNA fragments. This was observed previously in translation studies with reovirus and has been attributed to problems with translation conditions such as the presence of nucleases which preferentially reduce the yield of large proteins, the concentration of salt needed to facilitate completion of elongation, and the f'mal etectrophoretic conditions needed to resolve translation products [26, 27]. In our experiments, some mRNA translated very well in vitro (e.g., m6) while others could only be translated with difficulty (e.g., m5). Also, some of the small, contaminating mRNA fragments translated in vitro better than the larger full length segments.

Several groups have mapped the genome of the group A rotavirus [17, 20, 21, 24, 25, 28, 29, 30] and the structure and function of the genome have recently been reviewed [31]. Our previous study demonstrated that the migration pattern of viral structural proteins on SDS-PAGE and localization of the proteins in the virion were comparable between group A and group B rotaviruses. This study shows major similarities and some differences in the gene-coding assign- ments between these two groups. The viral core proteins VP1, VP2, VP3 and the inner capsid protein VP6 are encoded by gene segments 1, 2, 3 and 6, respectively, in both the group A and B rotaviruses. The outer capsid protein, VP4 also is the same and is encoded by gene segment 4. VP7, a glycoprotein, is coded by gene segment 8 for human group B rotavirus in this outbreak (i.e.,

ADRV gene-coding assignments 67

Chengde, Hebei Province, 1987), but by segment 9 for the strain studied recently by Chen etal. [14] detected in specimens from Chingdao, Shandong Province, four years before. This difference is not unlike the pattern seen for the group A rotavirus in which VP7 is encoded by segment 9 of some strains (e.g., SA11) but by segment 8 for others (e.g., NCDV). mRNA of gene segments 8 and 9 were easy to separate making this an unlikely source of the difference. The absolute order of migration of a gene coding for a particular protein (cognate gene) can differ among various virus strains, and this might be expected for other variants of group B rotavirus as well. The other possible capsid protein, VP9, is coded by segment 10. We are not sure of the identity of VP9 which was not detected by SDS-PAGE in purified virus. The 26 kDa polypeptide in the translation products from both unfractionated total mRNA transcripts and fractionated individual transcripts ml0 was precipitated with guinea pig anti- serum to ADRV, but not with preimmune sera which could be explained either as it being a structural protein or merely a nonspecific immunoprecipitated nonstructural protein. For group A rotavirus, a similar 26kDa polypeptide coded by segment 10 or 11, depending on different virus strains, was also seen in translation products and it now appears to be a structural or nonstructural protein [30]. The final gene coding assignments could be confirmed with further studies of gene cloning and expression [13].

Using distinct methods, we observed 2 differences in gene-coding assignments from the results of cloning studies by Chert etal. [14, 15]. VP7 appeared to be encoded by gene segment 8 in our analysis but by gene segment 9 in the study of Chen based upon the 28 % homology of amino acid sequence to gene segment 9 of the group A rhesus rotavirus. The mRNA transcribed by gene segments 8 and 9 in our study were significantly distinct to provide for clean separation and translation, a procedure that was repeated in order to confirm the results. VP7 in the group A rotaviruses is encoded in different strains by either gene segment 8 or 9 so similar variability may well occur with different strains of ADRV. Secondly, Chen concluded that the major inner capsid protein, VP6, was encoded by gene segment 5 versus our assignment to segment 6. Because segments 5 and 6 migrated together, we separated their mRNAs on a long electrophoresis apparatus specially prepared to distinguish these bands and used polyacrylamide instead of agarose. These results were repeated to confirm the assignments which were consistent throughout. While it is possible that subtle differences in the migration of these mRNAs might have occurred, another explanation relating to differences in the parent strain appears possible. Eiden et al. [16] has observed that clones of gene segment 6 of the rat group B rotavirus that encodes for VP6 hybridizes with segment 6 of the bovine group B strain but gene 5 of ADRV, The VP6 of ADRV is likely encoded by segment 5 in some strains and difference in our separation system led to a difference in our results.

Identification of the gene coding assignments of ADRV has clear implica- tions for understanding the biology of the rotavirus and prospects for the disease

68 Z.-Y. Fang et al.

control. Knowledge of the specific genes encoding the capsid proteins should facilitate cloning, sequencing, and expression of these genes [13]. Sequence information will help assess the relatedness of A D R V to animal strains of group B rotavirus and human strains of group A rotavirus and suggest a possible origin for this strain. Since A D R V has not yet been cultivated, expression of inner capsid proteins from gene segment 6 would be a key antigen for immune diagnosis. Similarly, based on parallel work with the group A rotaviruses, outer capsid proteins expressed from gene segments 4 and 8 could be immunogenic in animal models of group B rotavirus and provide a direction for future work with vaccines.

Acknowledgements We thank Larry Anderson, Mary K. Estes, Jon Gentsch, Joseph Eiden, Maria Penaranda, Patty Woods, Olen Kew, Bill Bellini, Marie Morgan, Anthony Sanchez, Wei-Wei Ye, John O'Connor, and Laveme Tucker for assistance.

References 1. Pedley S, Bridger JC, Brown JF, McCrae MA (1983) Molecular characterization of

rotavirus with distinct group antigens. J Gen Virol 64:2093-2101 2. Pedley S, Bridger JC, Chasey D, McCrae MA (1986) Definition of two new groups of

atypical rotaviruses. J Gen Virol 67:131-137 3. Bridger JC (1987) Novel rotaviruses in animals and man. Ciba Found Syrup 128: 5-

23 4. Chasey D, Banks J (1984) The commonest rotaviruses from neonatal lamb diarrhoea

in England and Wales have atypical electropherotypcs. Vet Rec 115:326-327 5. Snodgrass DR, Herring AJ, Campbell I, Inglis JM, Hargreaves FD (1984) Comparison

of atypical rotaviruses from calves, piglets, lambs, and man. J Virol 65:909-914 6. Vonderfecht SL, Huber AC, Eiden J J, Mader LC, Yolken RH (1984) Infectious diarrhea

of infant rats produced by a rotavirus-like agent. J Virol 52:94--98 7. Vonderfecht SL, Eiden JJ, Tortes A, Miskuff R, Mebuys CA, Yolken RH (1986)

Identification of a bovine enteric syncytial virus as a non-group A rotavirus. Am J Vet Res 47:1913-1918

8. Hung T, Chen G, Wang C (1984) Waterbourne outbreak of rotavirus diarrhea in adults in China caused by a novel rotavirus. Lancet i: 1139-1142

9. Hung T (1988) Rotavirus and adult diarrhea. Adv Virus Res 35:193-218 10. Fang ZY, Ye Y, Ho M-S, Dons H, Qing S, Penaranda M, Hung T, Wen L, Glass RI

(1989) Investigation of an outbreak of adult diarrhea rotavirus in China. J Infect Dis 160:948-953

11. Penaranda ME, Ho M-S, Fang ZY, Dong H, Bai XS, Duan SC, Ye WW, Estes MK, Echeverria P, Hung T, Glass RI (1989) Seroepidemiology of adult diarrhea rotavims in China. J Clin Microbiol 27:2180-2183

12. Fang ZY, Glass RI, Penaranda ME, Dong H, Monroe SS, Wen L, Estes MK, Eiden J, Yolken RH, Saif L, Gouvea V, Hung T (1989) Purification and characterization of adult diarrhea rotavirus: identification of viral structural proteins. J Virol 63: 2191- 2197

13. Chen G-M, Hung T, Mackow ER (1990) eDNA cloning of each genomic segment of the Group B rotavirus ADRV: molecular characterization of the 1 lth RNA segment. Virology 175:605-609

14. Chen G-M, Hung T, Mackow ER (1990) Identification of the gene encoding and group

ADRV gene-coding assignments 69

B rotavirus VP7 equivalent: primary characterization of the ADRV segment 9 RNA. Virology 178:311-315

15. Chen G-M, Werner-Eckert R, Tao H, Mackow ER (1991) Expression of the major inner capsid protein of the group B rotavirus ADRV: primary characterization of genome segment 5. Virology 182:620-629

16. Eiden JJ, Allen JR (1992) Identification of cognate genes among heterologous strains of group B rotavirus. J Virol 66:1232-1235

17. Mason BB, Graham DY, Estes MK (1980) In vitro transcription and translation of simian rotavirus SA11 gene products. J Virol 33:1111-1112

18. Fraenkel-Conrat H, Singer HB, Tsugita A (1961) Purification of viral RNA by means of bentonite. Virology 14:54-58

19. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685

20. Smith ML, Lazdins I, Holmes IH (1980) Coding assignments of double-stranded RNA segments of SA11 rotavirus established by in vitro translation. J Virol 33:976-982

21. Tanaka TN, Conner ME, Graham DY, Estes MK (1988) Molecular characterization of three rabbit rotavirus strains. Arch Virol 98:253-265

22. Coffin JM, Billeter MA (1976) A physical map of the Rous Sarcoma virus genome. J Mol Biol 100:293-318

23. Flores J, Green KY, Garcia D, Sears J, Perez-Schaet I, Avendano LF, Rodriguez WB, Taniguchi K, Urasawa S, Kapikian AZ (1989) Dot hybridization assay for detection of rotavirus serotypes. J Clin Microbiol 27:29-34

24. Mason BB, Graham DY, Estes MK (1983) Biochemical mapping of the simian rotavirus SA11 genome. J Virol 46:413-423

25. McCrae MA, McCorquodale JG (1982) Molecular biology of rotavirus. II. Identifi- cation of the protein-coding assignments of calf rotavirus genome RNA species. Vi- rology 117:435-443

26. Shatkin AJ, Kozak M (1983) Biochemical aspects of reovirus transcription and trans- lation. In: Joklik WK (ed) The Reoviridae. Plenum, New York, pp 79-106

27. Both GW, Lavi S, Shatkin AJ (1975) Synthesis of all gene products of the reovirus genome in vivo and in vitro. Cell 4:173-180

28. Dyall-Smith ML, Holmes IH (1981) Gene-coding assignments of rotavirus double- stranded RNA segments 10 and 11. J Virol 38:1099-1103

29. Liu M, Offit PA, Estes MK (1988) Identification of the simian rotavirus SAI 1 genome segment 3 product. Virology 163:26-32

30. Welch S-KW, Crawford SE, Estes MK (1989) The rotavirus SA11 genome segment 11 protein is a nonstructural phosphoprotein. J Virol 63:3874-3982

31. Estes MK, Cohen J (1989) Rotavirus gene structure and function. Microbiol Rev 53: 410-419

Authors' address: Dr. Z.-Y. Fang, Viral Gastroenteritis Unit (G04), Centers for Disease Control, Atlanta, GA 30333, U.S.A.

Received September 27, 1991