- Email: [email protected]
Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments
VIROLOGY 186, 444-451
(19%)
Identification and Characterization of the Structural and Nonstructural Proteins of African Horsesickness Virus and Determination of the Genome Coding Assignments MARVIN J. GRUBMAN*,’ AND SAMUEL A. LEWIS2 *USDA, ARS, NAA, Plum island Animal Disease Center, P.O. Box 848, Greenport, Received February
13, 199 1; accepted
New York 11944
October 2 1, 199 1
Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacr-ylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were viral specific as demonstrated by immunoprecipitation. The coding assignments of all the purified genome segments were determined by in vitro translation and confirmed by immunoprecipitation.
INTRODUCTION
associated with tubular structures that are a characteristic of orbivirus-infected cells (Huismans and Els, 1979). We recently demonstrated that a second nonstructural protein, NS2, is a phosphoprotein and consists of at least four isoforms (Devaney et al., 1988). The present study was designed to obtain more complete information about the viral proteins and identify the coding assignments of the genome segments.
African horsesickness (AHS) is an arthropod-borne viral disease that causes high mortality in horses (Hess, 1988). Historically, the disease has occurred in Africa, the Middle East, and Asia and is currently enzootic in tropical Africa (Hess, 1988). AHS seasonally spreads when conditions favor the Culicoides vector (Du Toit, 1944). Summer outbreaks in Spain, since 1987, and more recently in Portugal and Morocco, have served to focus attention on AHS (Lubroth, 1987; Yubero, 1988; Mellor et al., 1990). African horsesickness virus (AHSV) is a member of the orbivirus genus, family Reoviridae (Verwoerd eta/., 1979). Nine serotypes have been identified (Howell, 1962). Like other members of this genus, the virus contains 10 segments of double-stranded RNA that are surrounded by a double-layered capsid (Oellermann et al., 1970; Verwoerd et a/., 1979). Purified AHSV serotype 3 was examined by polyacrylamide gel electrophoresis and four major and three minor proteins were identified (Bremer, 1976). Removal of the outer capsid of AHSV by different procedures demonstrated that two of the major proteins, VP2 and VP5, form the outer shell (Bremer, 1976; Van Dijk and Huismans, 1982) while surface labeling of purified virions indicated that VP2 is the major exposed protein (Lewis and Grubman, 1991). Very few studies have examined the viral proteins synthesized in AHSV-infected cells. One viral nonstructural protein, NSl (formerly P5A), was found to be ’ To whom reprint requests should be addressed. ’ Present address: ImmuCell Corp., 966 Riverside land, ME 04103. 0042.6822/92
$3.00
MATERIALS AND METHODS Cells and virus Vero cells, baby hamster kidney (BHK-21) cells, and equine dermis cells were grown as monolayers in Eagle’s minimal essential medium supplemented with 2 mPJl glutamine, 10 mM HEPES, pH 7.5, 10% tryptose phosphate, and 10% bovine serum or 10% fetal bovine serum for equine dermis cells. Equine dermis cells were provided by Drs. Carol House and James House, Foreign Animal Disease Diagnostic Laboratory, U.S. Department of Agriculture, Greenport, New York. African horsesickness virus serotype 4 was also provided by Drs. Carol House and James House and was originally obtained from South Africa as a mouse brain suspension It was passed four times in Vero cells prior to plaque purification. Virus stocks were prepared by growing plaque-purified virus in roller bottles of Vero cells until 80-100% cytopathic effects. Bottles were frozen and thawed, nuclei removed by low speed centrifugation, and the supernatants, containing both intracellular and extracellular virus, concentrated by precipitation using 8% polyethylene glycol 8000.
Antisera Convalescent horse serum against AHSV-4 was provided by Drs. Carol House and James House. To
Street, Port-
444
PROTEINS OF AFRICAN
produce antisera against inactivated AHSV-4, virus was treated with a final concentration of 3 mM binary ethylenimine (BEI) at room temperature for 48 hr (Bahnemann, 1990). Aliquots were removed prior to and at 24 and 48 hr after treatment and the infectivity was determined by plaque assay. The virus titer was reduced eight logs by 24 hr after treatment. Rabbits were inoculated, three times, with 50 pg per rabbit of 48 hr BEI-treated virus emulsified with an equal volume of Freunds complete adjuvant. Radiolabeling
of infected
cells
Vero, BHK-21, and equine dermis cell monolayers in 60-mm tissue culture dishes were infected with AHSV. Upon the appearance of virus-induced morphological alterations, cells were radiolabeled with either [35S]methionine or 14C-labeled amino acids and lysed following the procedure of Devaney et al. (1988). The effect of tunicamycin on proteins synthesized in virus-infected cells was examined by incubating infected cells for 4 hr with 1 pg/ml of tunicamycin prior to radiolabeling. Cells were then washed and incubated in media minus methionine containing tunicamycin and radiolabeled for 1 hr with [35S]methionine in the presence of 1 gg/ml of tunicamycin. A 1 mg/ml stock solution of tunicamycin was prepared in dimethyl sulfoxide. Purified, radiolabeled virus was prepared by infecting a roller bottle of Vero cells at a multiplicity of infection of 0.1. One mCi of [35S]methionine, in 0.1 ml, was added 30 hr after infection and incubation was continued for an additional 20 hr in 100 ml of media containing one-tenth the normal methionine concentration, 1OYO tryptose phosphate, and 10% dialyzed fetal calf serum. Virus was purified essentially as described for bluetongue virus (BTV) by Mecham et al. (1986).
HORSESICKNESS
In v&o translation Preparation of rabbit reticulocyte lysates and conditions for in vitro protein synthesis were as previously described (Grubman and Baxt, 1982) with the following modifications: total RNA or individual RNA segments were denatured by incubation for 5 min at room temperature in 10 mM methyl mercury hydroxide and were translated in a lysate supplemented with 0.5 mM magnesium acetate (Grubman et al., 1983; Met-tens et al., 1984). lmmunoprecipitation Radiolabeled cytoplasmic extracts from infected and mock-infected cells or in vitro translations were preincubated either with normal serum and Staphylococcus aureus bearing protein A or only with S. aureus bearing protein A to minimize nonspecific binding. Following centrifugation, supernatants were immunoprecipitated with AHSV-specific antisera as previously described (Grubman et a/., 1984). Polyacrylamide fluorography
of AHSV RNA
RNA was prepared from partially purified virus by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. The RNA was heated at 65-70” for 2-3 min and fractionated on a 10% SDS-polyacrylamide gel (SDS-PAGE). Individual segments were extracted by crushing the gel slices, diffusion eluted for 24 hr into NET buffer (0.15 M NaCI, 0.002 IVI EDTA, 0.010 R/I Tris-HCI, pH 7.5) followed by extraction with phenol:chloroform:isoamyl alcohol and chloroform: isoamyl alcohol and then ethanol precipitated. Segments 4-9 were further purified by a second round of SDS-PAGE. Samples were stored at -70”. Identity and purity of the individual segments were checked using 10% SDS-PAGE.
gel electrophoresis
(PAGE) and
SDS-PAGE was performed on 10,12.5, or 15% slab gels using a Tris-glycine buffer system (Laemmli, 1970). After electrophoresis, gels were treated with sodium salicylate (Chamberlain, 1979) dried, and fluorographed (Bonner and Laskey, 1974). Partial protease
digestion
The procedure of Cleveland et al. (1977) with modifications (Grubman et al., 1984) was used for comparison of proteins by partial protease digestion. RESULTS Identification
Isolation
445
VIRUS
of viral proteins
To identify AHSV structural and nonstructural proteins, AHSV-4 was purified from infected, radiolabeled Vero cells and compared to a radiolabeled, infected Vero cell cytoplasmic extract by SDS-PAGE (Fig. 1). One minor (VPl) and four major (VP2, VP3, VP5, and VP7) structural proteins were identified. Two minor core proteins (VP4 and VP6) that were previously identified in purified AHSV-3 (Bremer, 1976) were not visible upon analysis of purified [35S]methionine labeled virus. Infected cells contained three additional major nonstructural proteins, NSl , NS2, and NS3, as well as variable amounts of two minor nonstructural proteins, NS4 and NS4a (Figs. 2-3). It should be noted that on some gels NS3 resolved as a doublet (Fig. 3, same samples in lanes Al and B6).
446
GRUBMAN V
I
M
AND LEWIS
3A). However, in vitro, the synthesis of NS3 was significantly reduced as compared to the level of this protein found in infected cells. Denaturation of AHSV-4 RNA by dimethyl sulfoxide treatment or by boiling followed by in vitro translation did not result in increased synthesis of NS3, nor did addition to the in vitro reaction of lysates from uninfected or infected Vero cells (data not shown). Identical results were found when the level of in vitro and in vivo protein synthesis was compared for serotypes 3, 6, and 9 (data not shown). By in vitro translation, two minor proteins, designated VP4 and VP6, were identified (Fig. 3A). These proteins were synthesized in variable amounts in infected cells. To attempt to enhance viral-specific protein synthesis, AHSV-4-infected Vero cells were radiolabeled after hypertonic salt treatment, a procedure which has been used to selectively block the initiation of host protein synthesis (Saborio et a/., 1974; Nuss et a/., 1975). In viral-infected cells the background of cellular proteins synthesized at 230 mM NaCl was reduced compared to normal media, while the synthesis
kna
FIG. 1. Comparison of AHSV-4 structural and nonstructural proteins, [?S]Methionine-labeled proteins from purified AHSV-4 (lane V), from infected Vero cell cytoplasm (lane I) and from mock-infected Vero cell cytoplasm (lane M) are shown. Molecular weight standards are indicated on the right. Samples were resolved using SDS-PAGE on a 12.5% gel.
The viral-specific proteins do not contain N-linked carbohydrates based on radiolabeling with [3H]mannose (data not shown) or with [35S]methionine in the presence of tunicamycin (Fig. 2). As previously shown NS2 is a phosphoprotein (Devaney er a/., 1988). Estimated molecular weights of the viral proteins are listed in Table 1. To determine whether the viral-specific proteins are similar in different cells, three susceptible cell lines were infected with AHSV-4 and radiolabeled with [35S]methionine (Fig. 2). The patterns of viral-specific proteins synthesized in infected Vero, equine dermis, and BHK cells appear qualitatively similar. In a second experiment, infected BHK cells were radiolabeled with 14C-amino acids. Comparison of [35S]methionine and 14C-amino acid-labeled cytoplasmic extracts showed that the quantitative patterns of viral proteins are very similar (Fig. 2, lanes 8 and 9). Identification
of minor viral-specific
proteins
Total AHSV-4 RNA, isolated from partially purified virus, was translated in a reticulocyte cell-free system. The quantitative pattern of most of the proteins synthesized in vitro and in infected Vero cells was similar (Fig.
NS3NS4 NS4a
1
FIG. 2. Comparison of AHSV-4 proteins synthesized in different cell lines. Vero (V), equine dermis (ED), and BHK cells were infected with AHSV-4 and radiolabeled with [?S]methionine (lanes 2-4, and 8). Infected Vero cells were also radiolabeled in the presence of tunicamycin (lane 5, VT), while infected BHK cells were radiolabeled with ?.-labeled amino acids (lane 9). Mock-infected equine dermis (MED), Vero (MV), and BHK (MB) cells, radiolabeled with [?S]methionine, are also shown (lanes 1, 6, and 7, respectively). Samples were resolved using SDS-PAGE on a 12.5% gel.
PROTEINS OF AFRICAN
I
M
-7
1
2
3
190 MI
4
230 ii-l
123456
FIG. 3. Identification of minor viral-specific proteins. [35S]Methionine-labeled proteins were synthesized either in a reticulocyte cellfree system programmed with AHSV4 RNA or in mock-infected or AHSV-Cinfected Vero cells incubated for 30 min in normal media, media containing 190 or 230 mM NaCI. (A) Lane 1, cytoplasmic extract from AHSV-4-infected Vero cells (I) incubated in media containing 230 mM NaCI; lane 2, cytoplasmic extract from mock-infected Vero cells (M) incubated in media containing 230 mM NaCI; lane 3, in vitro translation in the absence of exogenous RNA(-): lane 4, in v&o translation in the presence of total AHSV RNA(T). (B) Lanes 1, 3, 5, cytoplasmic extracts from mock-infected (M) Vero cells incubated with normal media, or media containing 190 or 230 mM NaCI; lanes 2, 4, 6, cytoplasmic extracts from AHSV4infected Vero cells (I) incubated with normal media, 190 or 230 mM NaCI. Samples were resolved using SDS-PAGE on a 12.59/o gel.
of viral proteins, including VP4 and VP6, was not significantly affected (Fig. 3B, lanes 2 and 6). All viral proteins synthesized in in vitro translation reactions or in infected cells were immunoprecipitated with convalescent horse serum (Fig. 4), although VP4 was not efficiently immunoprecipitated from infected cells. Identification
of genome coding assignments
Individual genome segments were isolated by either one or two rounds of SDS-PAGE. By this procedure, the segments were found to be relatively pure (Fig. 5). Translation of segment 1 resulted in synthesis of VP1 and a slightly lower molecular weight protein, while segments 2 and 3 coded for VP2 and VP3, respectively (Fig. 6). Segment 4 coded for VP4, segment 5 coded for NSl , and segment 6 coded for VP5 and VP6. Segment 7 coded for VP7, segment 8 coded for NS2, and
HORSESICKNESS
447
VIRUS
segment 9 coded for NS3 while segment 10 coded for NS4 and NS4a. Coding assignments of the individual segments are summarized in Table 1. To confirm that the proteins synthesized by the individual genome segments are viral specific, convalescent horse serum was used to immunoprecipitate the translation products. As shown in Fig. 7 all the in vitro synthesized proteins are specifically precipitated. To aid in the identification of the minor viral structural proteins, rabbit antisera against inactivated virus was produced and used to immunoprecipitate the translation products of total virion RNA and individual genome segments l-7 (Fig. 8). Proteins VP2, VP3, VP4, VP5, VP6, and VP7 were efficiently precipitated with both inactivated virus and convalescent serum (compare Figs. 7 and 8). VP1 was weakly reactive with both sera. In contrast, the presumed nonstructural proteins NSl , NS2, NS3, NS4, and NS4a, which were efficiently precipitated with convalescent serum, were weakly reactive or unreactive with inactivated virus serum. The two proteins coded for by segment 6 (VP5 and VP6) were compared to each other by partial protease analysis as were the two proteins coded for by segment 10 (NS4 and NS4a). As demonstrated in Fig. 9 VP5 and VP6 contain similar peptide profiles as do NS4 and NS4a. Pulse-chase experiments suggest that there is no precursor-product relationship between the proteins (data not shown). DISCUSSION In this study we have determined the coding assignments of the 10 genome segments of AHSV-4 and have identified the viral structural and nonstructural
TABLE 1 AHSV-4 PROTEINSAND GENOME CODING ASSIGNMENTS
Viral protein
Molecular weight’
VP1 VP2 VP3 VP4 VP5 NSl VP6 NS2 VP7 NS3 NS4 NS4a
128,500 107,000 88,500 69,000 65,000 62,500 60,500 54,000 42,000 27,500 26,000 25,000
a Molecular weights tein standards.
were estimated
Genome segment 1 2 3 4 6 5 6 8 7 9 10 10
Location Core Outer capsid Core Core Outer capsid Nonstructural Core Nonstructural Core Nonstructural Nonstructural Nonstructural
from SDS-PAGE
using pro-
446
GRUBMAN invitro ?iFGT-i-
I
in vim aA aN
VP1 VP2 VP3
VP4 VP5 NSl VP6 NS2
VP7
NS3 NS4 NS4a
FIG. 4. lmmunoprecipitation of AHSV4 proteins synthesized in vitro and in viva. [35S]Methionine-labeled cell-free extract programmed with AHSV-4 RNA or a cytoplasmic extract from infected Vero cells radiolabeled in media containing 230 rnn/l NaCl were immunoprecipitated with either normal horse serum (aN) or AHSV-4 convalescent horse serum (aA). Lanes labeled I contain either a clarified cell-free extract (in vitro) or a cytoplasmic extract from infected cells (in viva). Samples were resolved using SDS-PAGE on a 12.5% gel.
proteins. ln vitro translation of double-stranded genomic RNA in a rabbit reticulocyte cell-free system resuited in the synthesis of viral proteins which werequalitatively and quantitatively very similar to the pattern of proteins synthesized in infected cells. Translation of the individual genome segments clearly revealed the coding assignments and immunoprecipitation confirmed that all the in virro synthesized proteins are viral specific. Most of the genome segments coded for only one protein. Segment 1, however, coded for VP1 and a slightly lower molecular weight protein. This protein was not usually observed in infected cells or after translation of total RNA and has not yet been examined further. Segment 6 coded for VP5 and VP6. Comparison of these two proteins by partial protease digestion suggests that VP6 is a truncated form of VP5, which may arise by in-frame initiation at a downstream methionine codon since pulse-chase analysis did not reveal a precursor-product relationship. Segment 10
AND LEWIS
codes for NS4 and NS4a, two low molecular weight proteins which are related as demonstrated by partial protease digestion. The nucleic acid sequence of segment 10 from AHSV-3 and -9 has been determined and this segment contains two potential initiation sites in the same open reading frame (Van Staden and Huismans, 1991). Furthermore, segment 10 from serotype 4 has been translated in vitro into two proteins termed NS3 and NS3a (Van Staden and Huismans, 1991). Thus, it is possible that NS4a may be a truncated form of NS4, similar to the relationship of BTV NS3 and NS3a (Mertens et a/., 1984; Lee and Roy, 1986; Gould, 1988; French et a/., 1989). Purified, radiolabeled AHSV-4 contains four major proteins, VP2, VP3, VP5, and VP7, and one minor protein, VPl. By examination of radiolabeled virus, we were not able to identify two additional minor structural proteins, VP4 and VP6, that are present in purified AHSV-3 (Bremer, 1976) and also in the prototype orbivirus bluetongue (Verwoerd eta/., 1979). However, two minor proteins, designated VP4 and VP6, were clearly visible after radiolabeling AHSV-infected cells in hypertonic salt conditions and were also synthesized after in vitro translation of total genomic RNA or genome segments 4 and 6, respectively. These proteins are viral specific as demonstrated by reactivity with convalescent horse serum. Silver staining of purified AHSV-4 revealed, in addition to VP1 , VP2, VP3, VP5, and VP7, a few minor proteins, two of which comigrated with VP4
B
FIG. 5. Purity of AHSV-4 individual genome segments. AHSV-4 RNA was separated into individual segments by 10% SDS-PAGE. RNA was extracted from gel slices and segments 4-9 were further purified by a second round of SDS-PAGE. (A) Lanes l-4; Segments l-3 and 10. (B) Lanes 1-6; Segments 4-9; Lane T, total AHSV-4 RNA.
PROTEINS OF AFRICAN D-1
2345679
9
10
T
IE
IV
MV
VPlVP2VP3-
VWVPS“,g NS2-
VP7-
NS3-
HORSESICKNESS
VIRUS
449
NS2, and NS3) and variable amounts of two minor (NS4 and NS4a) nonstructural proteins are synthesized, while only two major and two minor nonstructural proteins are found in BlV- and EHDV-infected cells (Roy, 1989; Huismans er al., 1979; Mecham and Dean, 1988). This information has been confirmed in Vero, BHK, and equine dermis cells infected with other AHSV serotypes (data not shown). Our results thus suggest that AHSV codes for an additional nonstructural protein as compared to BTV and EHDV and that two apparently related structural proteins, VP5 and VP6, are coded for by the same genome segment. This later observation has some similarities with reovirus where segment M2 codes for structural protein ~1 which is proteolytically processed, during virion assembly, to PIG (Schiff and Fields, 1990). The smaller reovirus product, &, is a major structural protein, while only a few molecules of /*I are found in virus. In contrast, with AHSV the larger protein, VP5, is a major component of the virus, while VP6 is a minor constituent. Pulse-
FIG. 6. Identification of proteins coded for by the individual genome segments of AHSV4 RNA. Genome segments were isolated from 10% SDS-PAGE (the preparations of ds RNA analyzed in Fig. 5) and were translated in a reticulocyte cell-free system. In vitro translation products in the absence of exogenous RNA (-), with individual genome segments (l-10, respectively), or with total RNA (T) are shown. Lanes marked IE and IV contain [35S]methionine-labeled infected equine dermis and Vero cell cytoplasmic extracts, respectively, while the lane marked MV represents a [35S]methioninelabeled, mock-infected Vero cell cytoplasmic extract. Samples were resolved using SDS-PAGE on a 15% gel.
and VP6 (data not shown). Most importantly, serum produced against inactivated AHSV-4 clearly did not react with nonstructural proteins NSl, NS2, NS3, NS4, and NS4a, but was as efficient in precipitation of structural proteins VP1 , VP2, VP3, VP5, and VP7 and minor proteins VP4 and VP6 as convalescent serum. We believe that the sum of our data suggests that the minor viral specific proteins VP4 and VP6 are equivalent to the minor structural proteins found in AHSV-3 (Bremer, 1976). Previously, we demonstrated that AHSV NS2 is a phosphoprotein that is phosphorylated at serine residues (Devaney et al., 1988). In the present study, infected cells were examined for N-linked glycoproteins by radiolabeling with [3H]mannose or with [35S]methionine in the presence of tunicamycin. No viral proteins were found to contain N-linked carbohydrates. The protein pattern in AHSV-infected cells differs from that found in other orbivirus-infected cells examined such as BTV or epizootic hemorrhagic disease virus (EHDV). In AHSV-infected cells three major (NSl,
FIG. 7. lmmunoprecipitation of proteins encoded by the individual genome segments of AHSV4 with convalescent serum. Translation products of the individual genome segments (lanes l-l 0. respectively) and total RNA (lane 11) were each immunoprecipitated with AHSV-4 convalescent horse serum. Lanes marked IV and IE contain [35S]methionine-labeled infected Vero and equine dermis cell cytoplasmic extracts, respectively. Samples were resolved using SDSPAGE on a 15% gel.
450
GRUBMAN
AND LEWIS
chase analysis also reveals that there is no apparent precursor-product relationship between AHSV VP5 and VP6. More definitive information about the relationship of VP5 and VP6 will be obtained when the nucleic acid sequence of segment 6 is determined. AHSV genome segment 2 codes for the 107,000 MW capsid protein VP2. Based on hybridization studies with AHSV-3 cDNA clones, segment 2 is serotypespecific, although under low stringency conditions, there was a slight cross-hybridization with segment 2 of serotype 4 (Bremer et al., 1990). In addition, VP2 is the major surface-exposed viral capsid protein based on radioiodination of purified virus with a number of reagents (Lewis and Grubman, 1991). Expression of cDNA from this segment alone or in combination with segments coding for other viral structural proteins may be useful as a potential subunit vaccine. In this regard, vaccine trials have been reported that successfully used BTV proteins derived from baculovirus expression vectors to protect sheep against challenge (Roy et al., 1990). FIG. 9. Partial protease analysis of proteins coded for by RNA segments 6 and 10. ln vitro synthesized proteins VP5, VP6, NS4, and NS4a were separated on a 12.5% gel. The lndlvidual proteins were excised, partially digested with S. aureus V8 protease, and resolved using SDS-PAGE on a 17.5% gel. Lanes 1 and 2, VP5 and VP6; Lanes 3 and 4, NS4 and NS4a. .VPl .VP2 WP3
-VP4 -VP5 -NSl ‘VP6
The information obtained from this study will now allow us to rationally select genome segments that, either directly or through expression of their protein products, may be of use in both development of improved diagnostic reagents and disease prevention procedures.
ACKNOWLEDGMENTS -VP7
We thank Marla Zellner, Douglas Soroka, Brenda Rodd, and Elizabeth Sibbet for technical assistance and Adriene Ciupryk for typing the manuscript.
REFERENCES
FIG. 8. lmmunoprecipitation of proteins encoded by individual genome segments with antisera against inactivated virus. Translation products of individual genome segments l-7 (lanes l-7, respectively) and total RNA (lane 8) were immunoprecipitated with rabbit antisera agatnst inactivated virus. Lane 9 represents an in vitro translation of total RNA. Samples were resolved using SDS-PAGE on a 15% gel.
BAHNEMANN, H. G. (1990). InactIvation of viral antigens for vaccine preparation with particular reference to the applications of binary ethylenimine. Vaccine 8, 299-303. BONNER,W. M., and LASKEY, R. A. (1974). A film detection method for tritium labeled proteins and nucleic acids in polyacrylamide gels. Eur. 1. Biochem. 46, 83-88. BREMER,C. W. (1976). A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus, OnderstepoortJ. Vet. Res. 43, 1933199. BREMER,C. W., HUISMANS, H., and VAN DIJK, A. A. (1990). Characterization and cloning of the African homesickness virus genome. J. Gen. Viral. 71, 793-799. CHAMBERLAIN,1. P. (1979). Fluorographic detection of radioactivity In
PROTEINS OF AFRICAN polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal. Biochem. 98, 132-l 35. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER,W. M., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. 1. Biol. Chem. 252, 1102-l 106. DEVANEY, M. A., KENDALL, I., and GRUBMAN, M. J. (1988). Characterization of a nonstructural phosphoprotein of two orbiviruses. Virus Res. 11, 151-164. Du TOIT, R. M. (1944). The transmission of bluetongue and horsesickness by Culicoides. Onderstepoort /. Vet. Sci. Aim. Ind. 19, 7-16. FRENCH, T. J., INUMARA, S., and ROY, P. (1989). Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTVinfected BHK cells. J. Viral. 63, 3270-3278. GOULD, A. R. (1988). Nucleotide sequence of the Australian bluetongue virus serotype 1 RNA segment 10. /. Gen. Viol. 69, 945949. GRUBMAN, M. J., APPLETON,J. A., and LETCHWORTH,G. J. (1983). Identification of bluetongue virus type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology 131, 355-366. GRUBMAN, M. J., and BAXT, B. (1982). Translation of foot-and-mouth disease virion RNA and processing of the primary cleavage products in a rabbit reticulocyte lysate. Virology 116, 19-30. GRUBMAN, M. I.. ROBERTSON.B. J., MORGAN, D. O., MOORE, D. M., and DOWBENKO,D. (1984). Biochemical map of polypeptides specified by foot-and-mouth disease virus. /. Viral. 50, 579-586. HESS, W. R. (1988). The Arboviruses: Epidemiology and Ecology. In ’ ‘Arboviruses-Epidemiology and Ecology” (T. P. Monath, Ed.), Vol. 2, pp. 1-18. CRC Press, Boca Raton, FI. HOWELL, P. G. (1962). The isolation and identification of further antigenie types of African horsesickness virus. Onderstepoort J. Vet. Res. 29, 139-149. HUISMANS, H., BREMER,C. W., and BARBER,T. L. (1979). The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoolt J. Vet. Res. 46, 95-l 04. HUISMANS, H., and ELS, H. J. (1979). Characterization of the tubules associated with the replication of three different orbiviruses. L&o/ogy 92,397-406. HUISMANS. H., VAN DIJK, A. A., and BAUSKIN, A. R. (1987). In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA. J. Vkol. 61, 3589-3595. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
HORSESICKNESS
VIRUS
451
LEE, 1. W., and ROY, P. (1986). Complete sequence of a cDNA clone of RNA segment 10 of bluetongue virus (serotype 10). J. Gen. Viral. 67,2833-2837. LEWIS, S. A., and GRUBMAN, M. J. (1991). VP2 is the major exposed protein on orbiviruses. Arch. i&o/., in press. LUBROTH,J. (1988). African horsesickness and the epizootic in Spain in 1987. Equine Practice 10, 26-33. MECHAM, 1. O., and DEAN, V. C. (1988). Protein coding assignment for the genome of epizootic haemorrhagic disease virus. J. Gen. Viral. 69, 1255-l 262. MECHAM. J. O., DEAN, V. C., and JOCHIM, M. M. (1986). Correlation of serotype specificity and protein structure of the five U.S. serotypes of bluetongue virus. J. Gen. viral. 67, 2617-2624. MELLOR, P. S., BONED, J., HAMBLIN, C., and GRAHAM, S. (1990). Isolations of African horsesickness virus from vector insects made during the 1988 epizootic in Spain. Epidemiol. Infect. 105, 447-454. MERTENS, P. P. C., BROWN, F., and SANGAR, D. V. (1984). Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135, 207-217. Nuss, D. L., OPPERMANN,H., and KOCH, G. (1975). Selective blockage of initiation of host protein synthesis in RNA-virus-infected cells. Proc. Natl. Acad. SC;. USA 72, 1258-l 262. OELLERMANN. R. A., ELS, H. J., and ERASMUS, B. J. (1970). Characterization of African horsesickness virus. Archiv. fur die gesamte Virusforschung 29, 163-l 74. ROY, P. (1989). Bluetongue virus genetics and genome structure. virus Res. 13, 179-206. ROY, P., URAKAWA, T., VAN DIJK, A. A., and ERASMUS, B. J. (1990). Recombinant virus vaccine for bluetongue disease in sheep. j. Viral. 64, 1998-2003. SABORIO,J. L., PONG, S.-S., and KOCH, G. (1974). Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. Mol. Biol. 85, 195-21 1, SCHIFF, L. A., and FIELDS, B. N. (1990). Reoviruses and their replication. In “Virology, Second Edition” (B. N. Fields and D. M. Knipe et al., Eds.), pp. 1275-l 306. Raven Press, New York. VAN DIJK, A. A., and HUISMANS. H. (1982). The effect of temperature on the in vitro transcriptase reaction of bluetongue virus, epizootic haemorrhagic disease virus and African horsesickness virus. Onderstepoort J. Vet. Res. 49, 227-232. VAN STADEN, V., and HUISMANS, H. (1991). A comparison of the genes which encode nonstructural protein NS3 of different orbiviruses. J. Gen. Hrol. 72, 1073-l 079. VERWOERD,D. W., HUISMANS, H., and ERASMUS, B. J. (1979). Orbiviruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, Eds.), Vol. 14, pp. 285-345. Plenum, New York. YUBERO, M. A. D. (1988). African horsesickness in Spain. O/E Disease Information 1, 37-38.
(19%)
Identification and Characterization of the Structural and Nonstructural Proteins of African Horsesickness Virus and Determination of the Genome Coding Assignments MARVIN J. GRUBMAN*,’ AND SAMUEL A. LEWIS2 *USDA, ARS, NAA, Plum island Animal Disease Center, P.O. Box 848, Greenport, Received February
13, 199 1; accepted
New York 11944
October 2 1, 199 1
Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacr-ylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were viral specific as demonstrated by immunoprecipitation. The coding assignments of all the purified genome segments were determined by in vitro translation and confirmed by immunoprecipitation.
INTRODUCTION
associated with tubular structures that are a characteristic of orbivirus-infected cells (Huismans and Els, 1979). We recently demonstrated that a second nonstructural protein, NS2, is a phosphoprotein and consists of at least four isoforms (Devaney et al., 1988). The present study was designed to obtain more complete information about the viral proteins and identify the coding assignments of the genome segments.
African horsesickness (AHS) is an arthropod-borne viral disease that causes high mortality in horses (Hess, 1988). Historically, the disease has occurred in Africa, the Middle East, and Asia and is currently enzootic in tropical Africa (Hess, 1988). AHS seasonally spreads when conditions favor the Culicoides vector (Du Toit, 1944). Summer outbreaks in Spain, since 1987, and more recently in Portugal and Morocco, have served to focus attention on AHS (Lubroth, 1987; Yubero, 1988; Mellor et al., 1990). African horsesickness virus (AHSV) is a member of the orbivirus genus, family Reoviridae (Verwoerd eta/., 1979). Nine serotypes have been identified (Howell, 1962). Like other members of this genus, the virus contains 10 segments of double-stranded RNA that are surrounded by a double-layered capsid (Oellermann et al., 1970; Verwoerd et a/., 1979). Purified AHSV serotype 3 was examined by polyacrylamide gel electrophoresis and four major and three minor proteins were identified (Bremer, 1976). Removal of the outer capsid of AHSV by different procedures demonstrated that two of the major proteins, VP2 and VP5, form the outer shell (Bremer, 1976; Van Dijk and Huismans, 1982) while surface labeling of purified virions indicated that VP2 is the major exposed protein (Lewis and Grubman, 1991). Very few studies have examined the viral proteins synthesized in AHSV-infected cells. One viral nonstructural protein, NSl (formerly P5A), was found to be ’ To whom reprint requests should be addressed. ’ Present address: ImmuCell Corp., 966 Riverside land, ME 04103. 0042.6822/92
$3.00
MATERIALS AND METHODS Cells and virus Vero cells, baby hamster kidney (BHK-21) cells, and equine dermis cells were grown as monolayers in Eagle’s minimal essential medium supplemented with 2 mPJl glutamine, 10 mM HEPES, pH 7.5, 10% tryptose phosphate, and 10% bovine serum or 10% fetal bovine serum for equine dermis cells. Equine dermis cells were provided by Drs. Carol House and James House, Foreign Animal Disease Diagnostic Laboratory, U.S. Department of Agriculture, Greenport, New York. African horsesickness virus serotype 4 was also provided by Drs. Carol House and James House and was originally obtained from South Africa as a mouse brain suspension It was passed four times in Vero cells prior to plaque purification. Virus stocks were prepared by growing plaque-purified virus in roller bottles of Vero cells until 80-100% cytopathic effects. Bottles were frozen and thawed, nuclei removed by low speed centrifugation, and the supernatants, containing both intracellular and extracellular virus, concentrated by precipitation using 8% polyethylene glycol 8000.
Antisera Convalescent horse serum against AHSV-4 was provided by Drs. Carol House and James House. To
Street, Port-
444
PROTEINS OF AFRICAN
produce antisera against inactivated AHSV-4, virus was treated with a final concentration of 3 mM binary ethylenimine (BEI) at room temperature for 48 hr (Bahnemann, 1990). Aliquots were removed prior to and at 24 and 48 hr after treatment and the infectivity was determined by plaque assay. The virus titer was reduced eight logs by 24 hr after treatment. Rabbits were inoculated, three times, with 50 pg per rabbit of 48 hr BEI-treated virus emulsified with an equal volume of Freunds complete adjuvant. Radiolabeling
of infected
cells
Vero, BHK-21, and equine dermis cell monolayers in 60-mm tissue culture dishes were infected with AHSV. Upon the appearance of virus-induced morphological alterations, cells were radiolabeled with either [35S]methionine or 14C-labeled amino acids and lysed following the procedure of Devaney et al. (1988). The effect of tunicamycin on proteins synthesized in virus-infected cells was examined by incubating infected cells for 4 hr with 1 pg/ml of tunicamycin prior to radiolabeling. Cells were then washed and incubated in media minus methionine containing tunicamycin and radiolabeled for 1 hr with [35S]methionine in the presence of 1 gg/ml of tunicamycin. A 1 mg/ml stock solution of tunicamycin was prepared in dimethyl sulfoxide. Purified, radiolabeled virus was prepared by infecting a roller bottle of Vero cells at a multiplicity of infection of 0.1. One mCi of [35S]methionine, in 0.1 ml, was added 30 hr after infection and incubation was continued for an additional 20 hr in 100 ml of media containing one-tenth the normal methionine concentration, 1OYO tryptose phosphate, and 10% dialyzed fetal calf serum. Virus was purified essentially as described for bluetongue virus (BTV) by Mecham et al. (1986).
HORSESICKNESS
In v&o translation Preparation of rabbit reticulocyte lysates and conditions for in vitro protein synthesis were as previously described (Grubman and Baxt, 1982) with the following modifications: total RNA or individual RNA segments were denatured by incubation for 5 min at room temperature in 10 mM methyl mercury hydroxide and were translated in a lysate supplemented with 0.5 mM magnesium acetate (Grubman et al., 1983; Met-tens et al., 1984). lmmunoprecipitation Radiolabeled cytoplasmic extracts from infected and mock-infected cells or in vitro translations were preincubated either with normal serum and Staphylococcus aureus bearing protein A or only with S. aureus bearing protein A to minimize nonspecific binding. Following centrifugation, supernatants were immunoprecipitated with AHSV-specific antisera as previously described (Grubman et a/., 1984). Polyacrylamide fluorography
of AHSV RNA
RNA was prepared from partially purified virus by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. The RNA was heated at 65-70” for 2-3 min and fractionated on a 10% SDS-polyacrylamide gel (SDS-PAGE). Individual segments were extracted by crushing the gel slices, diffusion eluted for 24 hr into NET buffer (0.15 M NaCI, 0.002 IVI EDTA, 0.010 R/I Tris-HCI, pH 7.5) followed by extraction with phenol:chloroform:isoamyl alcohol and chloroform: isoamyl alcohol and then ethanol precipitated. Segments 4-9 were further purified by a second round of SDS-PAGE. Samples were stored at -70”. Identity and purity of the individual segments were checked using 10% SDS-PAGE.
gel electrophoresis
(PAGE) and
SDS-PAGE was performed on 10,12.5, or 15% slab gels using a Tris-glycine buffer system (Laemmli, 1970). After electrophoresis, gels were treated with sodium salicylate (Chamberlain, 1979) dried, and fluorographed (Bonner and Laskey, 1974). Partial protease
digestion
The procedure of Cleveland et al. (1977) with modifications (Grubman et al., 1984) was used for comparison of proteins by partial protease digestion. RESULTS Identification
Isolation
445
VIRUS
of viral proteins
To identify AHSV structural and nonstructural proteins, AHSV-4 was purified from infected, radiolabeled Vero cells and compared to a radiolabeled, infected Vero cell cytoplasmic extract by SDS-PAGE (Fig. 1). One minor (VPl) and four major (VP2, VP3, VP5, and VP7) structural proteins were identified. Two minor core proteins (VP4 and VP6) that were previously identified in purified AHSV-3 (Bremer, 1976) were not visible upon analysis of purified [35S]methionine labeled virus. Infected cells contained three additional major nonstructural proteins, NSl , NS2, and NS3, as well as variable amounts of two minor nonstructural proteins, NS4 and NS4a (Figs. 2-3). It should be noted that on some gels NS3 resolved as a doublet (Fig. 3, same samples in lanes Al and B6).
446
GRUBMAN V
I
M
AND LEWIS
3A). However, in vitro, the synthesis of NS3 was significantly reduced as compared to the level of this protein found in infected cells. Denaturation of AHSV-4 RNA by dimethyl sulfoxide treatment or by boiling followed by in vitro translation did not result in increased synthesis of NS3, nor did addition to the in vitro reaction of lysates from uninfected or infected Vero cells (data not shown). Identical results were found when the level of in vitro and in vivo protein synthesis was compared for serotypes 3, 6, and 9 (data not shown). By in vitro translation, two minor proteins, designated VP4 and VP6, were identified (Fig. 3A). These proteins were synthesized in variable amounts in infected cells. To attempt to enhance viral-specific protein synthesis, AHSV-4-infected Vero cells were radiolabeled after hypertonic salt treatment, a procedure which has been used to selectively block the initiation of host protein synthesis (Saborio et a/., 1974; Nuss et a/., 1975). In viral-infected cells the background of cellular proteins synthesized at 230 mM NaCl was reduced compared to normal media, while the synthesis
kna
FIG. 1. Comparison of AHSV-4 structural and nonstructural proteins, [?S]Methionine-labeled proteins from purified AHSV-4 (lane V), from infected Vero cell cytoplasm (lane I) and from mock-infected Vero cell cytoplasm (lane M) are shown. Molecular weight standards are indicated on the right. Samples were resolved using SDS-PAGE on a 12.5% gel.
The viral-specific proteins do not contain N-linked carbohydrates based on radiolabeling with [3H]mannose (data not shown) or with [35S]methionine in the presence of tunicamycin (Fig. 2). As previously shown NS2 is a phosphoprotein (Devaney er a/., 1988). Estimated molecular weights of the viral proteins are listed in Table 1. To determine whether the viral-specific proteins are similar in different cells, three susceptible cell lines were infected with AHSV-4 and radiolabeled with [35S]methionine (Fig. 2). The patterns of viral-specific proteins synthesized in infected Vero, equine dermis, and BHK cells appear qualitatively similar. In a second experiment, infected BHK cells were radiolabeled with 14C-amino acids. Comparison of [35S]methionine and 14C-amino acid-labeled cytoplasmic extracts showed that the quantitative patterns of viral proteins are very similar (Fig. 2, lanes 8 and 9). Identification
of minor viral-specific
proteins
Total AHSV-4 RNA, isolated from partially purified virus, was translated in a reticulocyte cell-free system. The quantitative pattern of most of the proteins synthesized in vitro and in infected Vero cells was similar (Fig.
NS3NS4 NS4a
1
FIG. 2. Comparison of AHSV-4 proteins synthesized in different cell lines. Vero (V), equine dermis (ED), and BHK cells were infected with AHSV-4 and radiolabeled with [?S]methionine (lanes 2-4, and 8). Infected Vero cells were also radiolabeled in the presence of tunicamycin (lane 5, VT), while infected BHK cells were radiolabeled with ?.-labeled amino acids (lane 9). Mock-infected equine dermis (MED), Vero (MV), and BHK (MB) cells, radiolabeled with [?S]methionine, are also shown (lanes 1, 6, and 7, respectively). Samples were resolved using SDS-PAGE on a 12.5% gel.
PROTEINS OF AFRICAN
I
M
-7
1
2
3
190 MI
4
230 ii-l
123456
FIG. 3. Identification of minor viral-specific proteins. [35S]Methionine-labeled proteins were synthesized either in a reticulocyte cellfree system programmed with AHSV4 RNA or in mock-infected or AHSV-Cinfected Vero cells incubated for 30 min in normal media, media containing 190 or 230 mM NaCI. (A) Lane 1, cytoplasmic extract from AHSV-4-infected Vero cells (I) incubated in media containing 230 mM NaCI; lane 2, cytoplasmic extract from mock-infected Vero cells (M) incubated in media containing 230 mM NaCI; lane 3, in vitro translation in the absence of exogenous RNA(-): lane 4, in v&o translation in the presence of total AHSV RNA(T). (B) Lanes 1, 3, 5, cytoplasmic extracts from mock-infected (M) Vero cells incubated with normal media, or media containing 190 or 230 mM NaCI; lanes 2, 4, 6, cytoplasmic extracts from AHSV4infected Vero cells (I) incubated with normal media, 190 or 230 mM NaCI. Samples were resolved using SDS-PAGE on a 12.59/o gel.
of viral proteins, including VP4 and VP6, was not significantly affected (Fig. 3B, lanes 2 and 6). All viral proteins synthesized in in vitro translation reactions or in infected cells were immunoprecipitated with convalescent horse serum (Fig. 4), although VP4 was not efficiently immunoprecipitated from infected cells. Identification
of genome coding assignments
Individual genome segments were isolated by either one or two rounds of SDS-PAGE. By this procedure, the segments were found to be relatively pure (Fig. 5). Translation of segment 1 resulted in synthesis of VP1 and a slightly lower molecular weight protein, while segments 2 and 3 coded for VP2 and VP3, respectively (Fig. 6). Segment 4 coded for VP4, segment 5 coded for NSl , and segment 6 coded for VP5 and VP6. Segment 7 coded for VP7, segment 8 coded for NS2, and
HORSESICKNESS
447
VIRUS
segment 9 coded for NS3 while segment 10 coded for NS4 and NS4a. Coding assignments of the individual segments are summarized in Table 1. To confirm that the proteins synthesized by the individual genome segments are viral specific, convalescent horse serum was used to immunoprecipitate the translation products. As shown in Fig. 7 all the in vitro synthesized proteins are specifically precipitated. To aid in the identification of the minor viral structural proteins, rabbit antisera against inactivated virus was produced and used to immunoprecipitate the translation products of total virion RNA and individual genome segments l-7 (Fig. 8). Proteins VP2, VP3, VP4, VP5, VP6, and VP7 were efficiently precipitated with both inactivated virus and convalescent serum (compare Figs. 7 and 8). VP1 was weakly reactive with both sera. In contrast, the presumed nonstructural proteins NSl , NS2, NS3, NS4, and NS4a, which were efficiently precipitated with convalescent serum, were weakly reactive or unreactive with inactivated virus serum. The two proteins coded for by segment 6 (VP5 and VP6) were compared to each other by partial protease analysis as were the two proteins coded for by segment 10 (NS4 and NS4a). As demonstrated in Fig. 9 VP5 and VP6 contain similar peptide profiles as do NS4 and NS4a. Pulse-chase experiments suggest that there is no precursor-product relationship between the proteins (data not shown). DISCUSSION In this study we have determined the coding assignments of the 10 genome segments of AHSV-4 and have identified the viral structural and nonstructural
TABLE 1 AHSV-4 PROTEINSAND GENOME CODING ASSIGNMENTS
Viral protein
Molecular weight’
VP1 VP2 VP3 VP4 VP5 NSl VP6 NS2 VP7 NS3 NS4 NS4a
128,500 107,000 88,500 69,000 65,000 62,500 60,500 54,000 42,000 27,500 26,000 25,000
a Molecular weights tein standards.
were estimated
Genome segment 1 2 3 4 6 5 6 8 7 9 10 10
Location Core Outer capsid Core Core Outer capsid Nonstructural Core Nonstructural Core Nonstructural Nonstructural Nonstructural
from SDS-PAGE
using pro-
446
GRUBMAN invitro ?iFGT-i-
I
in vim aA aN
VP1 VP2 VP3
VP4 VP5 NSl VP6 NS2
VP7
NS3 NS4 NS4a
FIG. 4. lmmunoprecipitation of AHSV4 proteins synthesized in vitro and in viva. [35S]Methionine-labeled cell-free extract programmed with AHSV-4 RNA or a cytoplasmic extract from infected Vero cells radiolabeled in media containing 230 rnn/l NaCl were immunoprecipitated with either normal horse serum (aN) or AHSV-4 convalescent horse serum (aA). Lanes labeled I contain either a clarified cell-free extract (in vitro) or a cytoplasmic extract from infected cells (in viva). Samples were resolved using SDS-PAGE on a 12.5% gel.
proteins. ln vitro translation of double-stranded genomic RNA in a rabbit reticulocyte cell-free system resuited in the synthesis of viral proteins which werequalitatively and quantitatively very similar to the pattern of proteins synthesized in infected cells. Translation of the individual genome segments clearly revealed the coding assignments and immunoprecipitation confirmed that all the in virro synthesized proteins are viral specific. Most of the genome segments coded for only one protein. Segment 1, however, coded for VP1 and a slightly lower molecular weight protein. This protein was not usually observed in infected cells or after translation of total RNA and has not yet been examined further. Segment 6 coded for VP5 and VP6. Comparison of these two proteins by partial protease digestion suggests that VP6 is a truncated form of VP5, which may arise by in-frame initiation at a downstream methionine codon since pulse-chase analysis did not reveal a precursor-product relationship. Segment 10
AND LEWIS
codes for NS4 and NS4a, two low molecular weight proteins which are related as demonstrated by partial protease digestion. The nucleic acid sequence of segment 10 from AHSV-3 and -9 has been determined and this segment contains two potential initiation sites in the same open reading frame (Van Staden and Huismans, 1991). Furthermore, segment 10 from serotype 4 has been translated in vitro into two proteins termed NS3 and NS3a (Van Staden and Huismans, 1991). Thus, it is possible that NS4a may be a truncated form of NS4, similar to the relationship of BTV NS3 and NS3a (Mertens et a/., 1984; Lee and Roy, 1986; Gould, 1988; French et a/., 1989). Purified, radiolabeled AHSV-4 contains four major proteins, VP2, VP3, VP5, and VP7, and one minor protein, VPl. By examination of radiolabeled virus, we were not able to identify two additional minor structural proteins, VP4 and VP6, that are present in purified AHSV-3 (Bremer, 1976) and also in the prototype orbivirus bluetongue (Verwoerd eta/., 1979). However, two minor proteins, designated VP4 and VP6, were clearly visible after radiolabeling AHSV-infected cells in hypertonic salt conditions and were also synthesized after in vitro translation of total genomic RNA or genome segments 4 and 6, respectively. These proteins are viral specific as demonstrated by reactivity with convalescent horse serum. Silver staining of purified AHSV-4 revealed, in addition to VP1 , VP2, VP3, VP5, and VP7, a few minor proteins, two of which comigrated with VP4
B
FIG. 5. Purity of AHSV-4 individual genome segments. AHSV-4 RNA was separated into individual segments by 10% SDS-PAGE. RNA was extracted from gel slices and segments 4-9 were further purified by a second round of SDS-PAGE. (A) Lanes l-4; Segments l-3 and 10. (B) Lanes 1-6; Segments 4-9; Lane T, total AHSV-4 RNA.
PROTEINS OF AFRICAN D-1
2345679
9
10
T
IE
IV
MV
VPlVP2VP3-
VWVPS“,g NS2-
VP7-
NS3-
HORSESICKNESS
VIRUS
449
NS2, and NS3) and variable amounts of two minor (NS4 and NS4a) nonstructural proteins are synthesized, while only two major and two minor nonstructural proteins are found in BlV- and EHDV-infected cells (Roy, 1989; Huismans er al., 1979; Mecham and Dean, 1988). This information has been confirmed in Vero, BHK, and equine dermis cells infected with other AHSV serotypes (data not shown). Our results thus suggest that AHSV codes for an additional nonstructural protein as compared to BTV and EHDV and that two apparently related structural proteins, VP5 and VP6, are coded for by the same genome segment. This later observation has some similarities with reovirus where segment M2 codes for structural protein ~1 which is proteolytically processed, during virion assembly, to PIG (Schiff and Fields, 1990). The smaller reovirus product, &, is a major structural protein, while only a few molecules of /*I are found in virus. In contrast, with AHSV the larger protein, VP5, is a major component of the virus, while VP6 is a minor constituent. Pulse-
FIG. 6. Identification of proteins coded for by the individual genome segments of AHSV4 RNA. Genome segments were isolated from 10% SDS-PAGE (the preparations of ds RNA analyzed in Fig. 5) and were translated in a reticulocyte cell-free system. In vitro translation products in the absence of exogenous RNA (-), with individual genome segments (l-10, respectively), or with total RNA (T) are shown. Lanes marked IE and IV contain [35S]methionine-labeled infected equine dermis and Vero cell cytoplasmic extracts, respectively, while the lane marked MV represents a [35S]methioninelabeled, mock-infected Vero cell cytoplasmic extract. Samples were resolved using SDS-PAGE on a 15% gel.
and VP6 (data not shown). Most importantly, serum produced against inactivated AHSV-4 clearly did not react with nonstructural proteins NSl, NS2, NS3, NS4, and NS4a, but was as efficient in precipitation of structural proteins VP1 , VP2, VP3, VP5, and VP7 and minor proteins VP4 and VP6 as convalescent serum. We believe that the sum of our data suggests that the minor viral specific proteins VP4 and VP6 are equivalent to the minor structural proteins found in AHSV-3 (Bremer, 1976). Previously, we demonstrated that AHSV NS2 is a phosphoprotein that is phosphorylated at serine residues (Devaney et al., 1988). In the present study, infected cells were examined for N-linked glycoproteins by radiolabeling with [3H]mannose or with [35S]methionine in the presence of tunicamycin. No viral proteins were found to contain N-linked carbohydrates. The protein pattern in AHSV-infected cells differs from that found in other orbivirus-infected cells examined such as BTV or epizootic hemorrhagic disease virus (EHDV). In AHSV-infected cells three major (NSl,
FIG. 7. lmmunoprecipitation of proteins encoded by the individual genome segments of AHSV4 with convalescent serum. Translation products of the individual genome segments (lanes l-l 0. respectively) and total RNA (lane 11) were each immunoprecipitated with AHSV-4 convalescent horse serum. Lanes marked IV and IE contain [35S]methionine-labeled infected Vero and equine dermis cell cytoplasmic extracts, respectively. Samples were resolved using SDSPAGE on a 15% gel.
450
GRUBMAN
AND LEWIS
chase analysis also reveals that there is no apparent precursor-product relationship between AHSV VP5 and VP6. More definitive information about the relationship of VP5 and VP6 will be obtained when the nucleic acid sequence of segment 6 is determined. AHSV genome segment 2 codes for the 107,000 MW capsid protein VP2. Based on hybridization studies with AHSV-3 cDNA clones, segment 2 is serotypespecific, although under low stringency conditions, there was a slight cross-hybridization with segment 2 of serotype 4 (Bremer et al., 1990). In addition, VP2 is the major surface-exposed viral capsid protein based on radioiodination of purified virus with a number of reagents (Lewis and Grubman, 1991). Expression of cDNA from this segment alone or in combination with segments coding for other viral structural proteins may be useful as a potential subunit vaccine. In this regard, vaccine trials have been reported that successfully used BTV proteins derived from baculovirus expression vectors to protect sheep against challenge (Roy et al., 1990). FIG. 9. Partial protease analysis of proteins coded for by RNA segments 6 and 10. ln vitro synthesized proteins VP5, VP6, NS4, and NS4a were separated on a 12.5% gel. The lndlvidual proteins were excised, partially digested with S. aureus V8 protease, and resolved using SDS-PAGE on a 17.5% gel. Lanes 1 and 2, VP5 and VP6; Lanes 3 and 4, NS4 and NS4a. .VPl .VP2 WP3
-VP4 -VP5 -NSl ‘VP6
The information obtained from this study will now allow us to rationally select genome segments that, either directly or through expression of their protein products, may be of use in both development of improved diagnostic reagents and disease prevention procedures.
ACKNOWLEDGMENTS -VP7
We thank Marla Zellner, Douglas Soroka, Brenda Rodd, and Elizabeth Sibbet for technical assistance and Adriene Ciupryk for typing the manuscript.
REFERENCES
FIG. 8. lmmunoprecipitation of proteins encoded by individual genome segments with antisera against inactivated virus. Translation products of individual genome segments l-7 (lanes l-7, respectively) and total RNA (lane 8) were immunoprecipitated with rabbit antisera agatnst inactivated virus. Lane 9 represents an in vitro translation of total RNA. Samples were resolved using SDS-PAGE on a 15% gel.
BAHNEMANN, H. G. (1990). InactIvation of viral antigens for vaccine preparation with particular reference to the applications of binary ethylenimine. Vaccine 8, 299-303. BONNER,W. M., and LASKEY, R. A. (1974). A film detection method for tritium labeled proteins and nucleic acids in polyacrylamide gels. Eur. 1. Biochem. 46, 83-88. BREMER,C. W. (1976). A gel electrophoretic study of the protein and nucleic acid components of African horsesickness virus, OnderstepoortJ. Vet. Res. 43, 1933199. BREMER,C. W., HUISMANS, H., and VAN DIJK, A. A. (1990). Characterization and cloning of the African homesickness virus genome. J. Gen. Viral. 71, 793-799. CHAMBERLAIN,1. P. (1979). Fluorographic detection of radioactivity In
PROTEINS OF AFRICAN polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal. Biochem. 98, 132-l 35. CLEVELAND, D. W., FISCHER, S. G., KIRSCHNER,W. M., and LAEMMLI, U. K. (1977). Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. 1. Biol. Chem. 252, 1102-l 106. DEVANEY, M. A., KENDALL, I., and GRUBMAN, M. J. (1988). Characterization of a nonstructural phosphoprotein of two orbiviruses. Virus Res. 11, 151-164. Du TOIT, R. M. (1944). The transmission of bluetongue and horsesickness by Culicoides. Onderstepoort /. Vet. Sci. Aim. Ind. 19, 7-16. FRENCH, T. J., INUMARA, S., and ROY, P. (1989). Expression of two related nonstructural proteins of bluetongue virus (BTV) type 10 in insect cells by a recombinant baculovirus: production of polyclonal ascitic fluid and characterization of the gene product in BTVinfected BHK cells. J. Viral. 63, 3270-3278. GOULD, A. R. (1988). Nucleotide sequence of the Australian bluetongue virus serotype 1 RNA segment 10. /. Gen. Viol. 69, 945949. GRUBMAN, M. J., APPLETON,J. A., and LETCHWORTH,G. J. (1983). Identification of bluetongue virus type 17 genome segments coding for polypeptides associated with virus neutralization and intergroup reactivity. Virology 131, 355-366. GRUBMAN, M. J., and BAXT, B. (1982). Translation of foot-and-mouth disease virion RNA and processing of the primary cleavage products in a rabbit reticulocyte lysate. Virology 116, 19-30. GRUBMAN, M. I.. ROBERTSON.B. J., MORGAN, D. O., MOORE, D. M., and DOWBENKO,D. (1984). Biochemical map of polypeptides specified by foot-and-mouth disease virus. /. Viral. 50, 579-586. HESS, W. R. (1988). The Arboviruses: Epidemiology and Ecology. In ’ ‘Arboviruses-Epidemiology and Ecology” (T. P. Monath, Ed.), Vol. 2, pp. 1-18. CRC Press, Boca Raton, FI. HOWELL, P. G. (1962). The isolation and identification of further antigenie types of African horsesickness virus. Onderstepoort J. Vet. Res. 29, 139-149. HUISMANS, H., BREMER,C. W., and BARBER,T. L. (1979). The nucleic acid and proteins of epizootic haemorrhagic disease virus. Onderstepoolt J. Vet. Res. 46, 95-l 04. HUISMANS, H., and ELS, H. J. (1979). Characterization of the tubules associated with the replication of three different orbiviruses. L&o/ogy 92,397-406. HUISMANS. H., VAN DIJK, A. A., and BAUSKIN, A. R. (1987). In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA. J. Vkol. 61, 3589-3595. LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227, 680-685.
HORSESICKNESS
VIRUS
451
LEE, 1. W., and ROY, P. (1986). Complete sequence of a cDNA clone of RNA segment 10 of bluetongue virus (serotype 10). J. Gen. Viral. 67,2833-2837. LEWIS, S. A., and GRUBMAN, M. J. (1991). VP2 is the major exposed protein on orbiviruses. Arch. i&o/., in press. LUBROTH,J. (1988). African horsesickness and the epizootic in Spain in 1987. Equine Practice 10, 26-33. MECHAM, 1. O., and DEAN, V. C. (1988). Protein coding assignment for the genome of epizootic haemorrhagic disease virus. J. Gen. Viral. 69, 1255-l 262. MECHAM. J. O., DEAN, V. C., and JOCHIM, M. M. (1986). Correlation of serotype specificity and protein structure of the five U.S. serotypes of bluetongue virus. J. Gen. viral. 67, 2617-2624. MELLOR, P. S., BONED, J., HAMBLIN, C., and GRAHAM, S. (1990). Isolations of African horsesickness virus from vector insects made during the 1988 epizootic in Spain. Epidemiol. Infect. 105, 447-454. MERTENS, P. P. C., BROWN, F., and SANGAR, D. V. (1984). Assignment of the genome segments of bluetongue virus type 1 to the proteins which they encode. Virology 135, 207-217. Nuss, D. L., OPPERMANN,H., and KOCH, G. (1975). Selective blockage of initiation of host protein synthesis in RNA-virus-infected cells. Proc. Natl. Acad. SC;. USA 72, 1258-l 262. OELLERMANN. R. A., ELS, H. J., and ERASMUS, B. J. (1970). Characterization of African horsesickness virus. Archiv. fur die gesamte Virusforschung 29, 163-l 74. ROY, P. (1989). Bluetongue virus genetics and genome structure. virus Res. 13, 179-206. ROY, P., URAKAWA, T., VAN DIJK, A. A., and ERASMUS, B. J. (1990). Recombinant virus vaccine for bluetongue disease in sheep. j. Viral. 64, 1998-2003. SABORIO,J. L., PONG, S.-S., and KOCH, G. (1974). Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. J. Mol. Biol. 85, 195-21 1, SCHIFF, L. A., and FIELDS, B. N. (1990). Reoviruses and their replication. In “Virology, Second Edition” (B. N. Fields and D. M. Knipe et al., Eds.), pp. 1275-l 306. Raven Press, New York. VAN DIJK, A. A., and HUISMANS. H. (1982). The effect of temperature on the in vitro transcriptase reaction of bluetongue virus, epizootic haemorrhagic disease virus and African horsesickness virus. Onderstepoort J. Vet. Res. 49, 227-232. VAN STADEN, V., and HUISMANS, H. (1991). A comparison of the genes which encode nonstructural protein NS3 of different orbiviruses. J. Gen. Hrol. 72, 1073-l 079. VERWOERD,D. W., HUISMANS, H., and ERASMUS, B. J. (1979). Orbiviruses. In “Comprehensive Virology” (H. Fraenkel-Conrat and R. R. Wagner, Eds.), Vol. 14, pp. 285-345. Plenum, New York. YUBERO, M. A. D. (1988). African horsesickness in Spain. O/E Disease Information 1, 37-38.