Sequence variability of the glycoprotein gene of ... - Semantic Scholar
Journal of General Virology (1993), 74, 2001 2004. Printedin Great Britain
2001
Sequence variability of the glycoprotein gene of bovine respiratory syncytial virus Sanjay K. Mailipeddi and Siba K. Samal* V A - M D Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland 20742, U.S.A.
Sequence variation in the attachment glycoprotein G of bovine respiratory syncytial virus (BRSV) was determined. The nucleotide sequences of the G mRNAs of the A51908, VC464 and FS-1 strains of BRSV were compared with the published sequence of the BRSV strain 391-2. Nucleotide sequence alignment showed that overall they are highly conserved, with 90 to 97 % identity. In addition, the coding region of strain A51908 was longer by 18 nucleotides at the 3' end. An 84
to 95% level of identity was observed among the deduced amino acid sequences of the G proteins of BRSV strains. A maximum divergence of 19% was found when the extracellular domains of the G proteins were compared. The level of diversity would be consistent, by analogy to the human respiratory syncytial viruses, with these BRSV strains forming a single subgroup.
Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory tract infections in cattle. It belongs to the genus Pneumovirus of the Paramyxoviridae, which also includes human respiratory syncytial virus (HRSV). The proteins of these two viruses are antigenically related, except for the antigenically distinct attachment (G) protein (Lerch et al., 1990). Sequence comparisons between proteins from BRSV and HRSV indicate that the G and SH proteins are least conserved (30 % and 38 % identity respectively; Lerch et al., 1990; Samal & Zamora, 1991). HRSV isolates are divided into two antigenic subgroups, A and B, on the basis of their reactions with monoclonal antibodies (MAbs) to the G protein (Anderson et al., 1985; Mufson et al., 1985; Gimenez et al., 1986). Sequencing studies have also shown that the G protein of HRSV is less conserved than other proteins between strains of the same or different subgroups (Johnson et al., 1987; Sullender et al., 1990; Cane et al., 1991). BRSV strains have not been characterized by Gspecific MAbs, nor have any sequence comparisons of the G proteins of BRSV strains been reported. Thus, BRSV is thought to be monotypic. To examine the variability of BRSV isolates, we compared the sequences of the G proteins of four strains. Strains of BRSV used in this study were A51908, 3912, FS-1, and VC464. Strain A51908 was isolated in Maryland in 1975 (Mohanty et al., 1975); strain 391-2 was isolated in North Carolina in 1985 (Fetrow et al., 1985), strain FS-1 was isolated in Iowa in 1975 (Smith et al., 1975) and strain VC464 was isolated in Missouri in
1974 (Rosenquist et al., 1974). Construction of cDNA libraries from mRNAs isolated from infected Madin Darby bovine kidney cells has been described (Samal et al., 1991). Recombinant cDNA clones containing G mRNA sequences of BRSV strain A51908 were identified by Northern blot hybridization and by comparing the sequences to the previously published G gene sequence of the BRSV strain 391-2 (Lerch et aI., 1990). cDNA clones of G genes of BRSV strains FS-1 and VC464 were identified by the colony hybridization technique with a probe made from the G gene of BRSV strain A51908. The nucleotide sequence of the mRNA encoding the G protein of each strain of BRSV was derived from at least two independent cDNA clones, using the dideoxynucleotide chain termination method (Sanger et al., 1977). The nucleotide sequence of the 5' end of the G mRNA of A51908 strain was determined by PCR amplification of the SH-G intergenic region (Zamora & Samal, 1992). The G mRNA sequences of BRSV strains FS-1 and VC464 were determined from nucleotide positions 37 and 42, respectively. The G mRNA of BRSV strain A51908 contained 835 nucleotides excluding the poly(A) tail (Fig. 1). The G mRNA sequence of strain A51908 was compared with those of strains FS-1, VC464 and 391-2 (Lerch et al., 1990). The levels of identity among the four different G mRNA sequences were 90 to 97 % (Table 1). There were three nucleotide deletions in the 3' non-coding region of all BRSV strains sequenced compared to the corresponding region of strain 391-2. In strains 391-2, FS-1 and VC464, the stop codon was at positions 787 to 789,
0001-1619 © 1993 SGM
2002
Short communication 1
A51908 391-2 FS-I VC464
G G G G C A A A T A CAAGC~--'T~TC CAACCACACC CATCATCCTA AATTCAAGAC ~ T T .................................... T .........................................
ATTAAAGAGG
GCTTGGAAAG
70
A51908 391-2 FS-I VC464
CCTCAAAATA
AAGTCCCTTG T C C A A A C G G C A A A
140
A51908 391-2
TTTGACCTCC T A
FS-I
T
T
A
VC464 A51908 391-2
CTTCATAGTA GGATTATCAT GTTTATATAA GTTCAATTTA T
ATAACAGCCA TTATTTACAT C
TAGTGTGGGA
210
ATCCAAACCA ACCACCCAAC AAACACAACA GCCCCAAAAC T
CACACCCCAC T T
280
TTAGCAATGA TAACCTTGAC C
ATCACTCGTC
C
A AATGCTAAAG
CCAAGCCCAC
FS-1
T
T
T
VC464
T
T
T
A51908 391-2 FS-I VC464
TACTTCCCAC C T TT C T ,'1~ C T TT
AGAGCACAAC
A51908 391-2
AAACATAGAC
ACCACTAGTG T A
FS-I
VC464
A
T
C
AACCCAAAGC TT TT TT
ACCACACTGT
CCCAACCACC T T T T AT T
350
CGGTCACCCA ATCAACAGAA T T C GA
CCCAAAACAG
AAAAATCAAA
420
CACAAATCAA CTCACACATC T T T GAACTACATA T
A
T
T
C G
GA
A
T
T
G
GA
G
A51908 391-2 FS-1 VC464
AGCCAATCTA G C C C
CTCCACTTGC T CC T CCA T CC
CACCCGAAAA CTACCAATCA ACCCACTGGA AAGCAACCCC A C T TC G T A C T TC A C TC G
CCCGAAAACC T T T
490
A51908 391-2
ACCAAGACCA T
CAACAACTCC T
CAAACACTCC
ATCCTGCCTG T T
560
FS-I
T
VC464
CTCATGTGCC CT CT
T
T
T
T
T
T
CT
T
T
T
T
T
T
A51908 391-2 FS-I VC464
TTCACCACTC C T T C T T C T T
TGCCAAATCG T T G T
GGCTGGAGAG A AC C GA C
A51908 391-2
CCAAAACCCA
AAACCACCAA T
AAAACCAACC AAGACAACAA G
FS-I
T
VC464
G
G
CTGCAGCACA TGCGAAGGCA T T T T
AGCACCAAGC
T
AGAGCTCCCA CAATCACCCT CAAAAAGGCT C T A C T A C T A
630
TCTACCACAG C
AACCAGCCCT
GAAGCCAAAC A
700
C
G
C
A51908 391-2 FS-I VC464
TGCAAACCAA CT C C
AAAAAACACG GCAACTCCAC C A C G C G
AACAAGGCAT
CCTCTCTTCA
CCAGAACACC A T A
AAACAAATCA C C C
770
A51908 391-2 FS-1 VC464
ATCTACTACA A A A
CAGATCTCAC ~ ~
CAT--AT ~
CAATTAT**G T A TAT** **
TTC*ATATGT T A * A * A
AGTTATTT
835 838 835 835
AACACACCTC
Fig. 1. Nucleotide sequences of the mRNAs of BRSV G protein genes. The sequence of strain 391-2 is taken from Lerch et al. (1990). Only the non-identical bases are shown for strains 391-2, FS-1 and VC464. The consensus gene start and stop signals are overlined. The consensus initiation codon and the stop codons are boxed. ( - ) Sequences not determined; (*) deletions in the non-coding region.
whereas in strain A51908 it was at positions 805 to 807. Correspondingly, the 3' non-coding region of the G mRNA of strain A51908 was 18 nucleotides shorter than those of strains VC464, FS-1 and 391-2. The 3' noncoding region of BRSV G mRNA is 70 nucleotides (52 in strain A51908) shorter than the corresponding sequence of HRSV. Recently, sequence analysis of the carboxyterminal third of the G protein gene of subgroup A HRSVs revealed a region prone to polymerase errors
resulting in the insertion or deletion of adenosine residues in some runs of such residues (Cane et al., 1993). Sequences of BRSV G genes also had runs of adenosine residues in the region coding for the extracellular domain, but no enhanced mutation was observed in this region among BRSV strains. The G mRNA of strain A51908 had a major open reading frame of 263 amino acids. The predicted relative molecular mass of this polypeptide is 28.9K. In vitro
Short communication
Table 1. Nucleotide and amino acid identities f o r G genes and proteins o f B R S V A51908 A51908 ~
9
0
391-2
-
3
FS-1
VC464
90.9
93.2
2 r~
14 i,2 . FS-1
84-4 (81.2)
92.0 (89-5)
~
97-1 O
VC464
87-9 (8~8)
9+0 (92-7) 95.2(942-)
Z
Amino acid identities (%) *Data in parentheses represent amino acid identity in the extracellular domain C"2TOPLASX/C DOima~V
~ ~" T R A N ~ U ~ ; M
A51908 391-2 FS-I VC464
THHPKFKTLKRAWKASKYF IVGLS6LYKFNLKSLVQTALTSLAMITLTSLVITAI i M~ L ST ....... L ST ......... T
A51908 391-2 FS-1 VC464
Y~SVGNAKAKPTSKPTTQQTQQPQNHTPLLPTE~HKSTMTSTQSTTLSQPPNIDTTSGT I ~ SPFF Y I LL R I I ~. SPFF N Y I LZ R I SPFF y I L T R
A51908
TYGHP~I~TQNRKIKSQSTPLATRKLPINPLESNPP~VHQDB~SQTLpHVp6ST~EGNp ST~E G 5P P SG I F y L
~
391-2
FS-I VC464
STDE
•
A51908 391-2 FS-I VC464
~s19o8
391-2 FS-I VC464
EXTRACE~L~R
S
LPT
P
S
60
~
,
Y
120
L
•
ACSPLCQIGLERAPSRAPT ITLKKAPKPKTTKKPTKTT IYMRTS PEAJ(LQTKKNTATPQQ LS H ET T H T P LS V PG T I H P ~ A LS p T H P A
N~
:
GILSSPEHQ STTQ ISQHTs I * T H ~ *
240
263 257 257 257
Fig. 2. Deduced amino acid sequences of O proteins of BRSV. The sequence of strain 391-2 is from Lerch et al. (1990). Only the nonidentical amino acid residues are shown for strains 391-2, FS-1 and VC464. The proposed domains are indicated above the sequences. Potential N-linked carbohydrate addition sites are shaded. The region corresponding to the conserved 13 amino acid region of HRSV (Johnson et al., 1987)is boxed. (e) Cysteineresidue; (*) end of each of the four proteins. expression of the G gene produced two unglycosylated forms of the G protein (28K and 23K). When the second methionine at position 48 was mutated to isoleucine, only one polypeptide of 28K was produced by in vitro expression, indicating that the 23K protein was produced by either leaky scanning or independent internal initiation (S. K. Mallipeddi & S. K. Samal, unpublished). The deduced G protein amino acid sequence of strain A5 ! 908 was compared with those o f strains FS- I, VC464 and 391-2 (Fig. 2). The predicted G protein of strain A51908 was longer by six amino acids at the C terminus than that of the strain 391-2 (Lerch et al., 1990). However, cross-neutralization and cross-immunoprecipitation tests using polyclonal antibodies failed to show any detectable antigenic difference between these
2003
strains (data not shown). Our results indicate that the six amino acids from the C terminus of the BRSV G protein are probably not involved in neutralization. Similar results have been reported with HRSV, where C-terminal truncated forms o f the G protein did not show any difference in neutralization (Olmsted et al., 1989), and this region has been shown to accommodate many changes without losing its function (Garcia-Barreno et al., 1990). The levels of identity among the G proteins of the four BRSV strains were 84 to 95% (Table 1). However, between the prototype strains of subgroups A and B of HRSV, there was only 53 % amino acid identity (Johnson et al., 1987). Within these subgroups, the G protein was also highly divergent with up to 20% amino acid variability between subgroup A strains (Cane et al., 1991) and up to 9 % variability between subgroup B strains (Sullender et al., 1991). Since variability between the four BRSV strains sequenced was 5 to 16%, all appear to belong to a single subgroup. When the extracellular domains of the G proteins of BRSV strains were compared, there was a maximum divergence of 19% between A51908 and 391-2 or FS-1, and there was a minimum divergence of 6 % between FS-1 and VC464. However, the cytoplasmic and transmembrane domains were highly conserved, with less than 5 % divergence among BRSV strains. The deduced amino acid sequences of the BRSV strains showed a high level (25 %) of serine plus threonine content that are potential O-linked glycosylation sites. In addition, four or five potential N-linked glycosylation sites were found in all strains examined (Fig. 2). Only the potential N-linked glycosylation site at amino acid residue 251 was conserved in all BRSV strains and in a majority of subgroup A strains of HRSV (Cane et al., 1991); it was absent in subgroup 13 strains. This suggests that N-linked glycans at this site are probably important for structural and functional properties o f the glycoprotein. There are five cysteine residues conserved in the G proteins o f all strains, indicating similar structural features. A conserved 13-amino acid region (164 to 176; Fig. 2) in the extracellular domain of the G protein of the HRSV was proposed as a candidate for attachment to cellular receptors (Johnson et al., 1987). This region was not conserved between BRSV strain 391-2 and HRSV, further suggesting that it could relate to host specificity (Lerch et al., 1990). However, this region was not perfectly conserved among strains of BRSV. We thank Dr Sashi B. Mohanty for his support and Mr Daniel Rockemannfor excellenttechnicalassistance.This study was supported by grants from the Maryland Agricultural Experiment Station and USDA grant no. 89-34116-4855. Approved as scientific article no. A6428 and contribution no. 8622 of the Maryland Agricultural Experiment Station.
2004
Short communication
References ANDERSON,L. J., HIERHOLZER,J. C., TSOU,C., HENDRY,R. M., FERNIE, B. N., STONE,Y. & MCINTOSH,K. (1985). Antigenic characterization of respiratory syncytial virus strains with monoclonal antibodies. Journal of Infectious Diseases 151,626-633. CANE,P. A., MATTHEWS,D. A. & PRINGLE,C. R. (1991). Identification of variable domains of the attachment (G) protein of subgroup A respiratory syncytial viruses. Journal of General Virology 72, 2091-2096. CANE, P.A., MATTHEWS, D.A. & PRINGLE, C. R. (1993). Frequent polymerase errors observed in a restricted area of clones derived from the attachment (G) protein gene of respiratory syncytial virus. Journal of Virology 67, 1090-1093. FETROW, J., HENRY, E., GUY, J. & BROWN, T. (1985). North Carolina State University Agricultural Extension Service Veterinary Newsletter. GARCIA-BARRENO, B., PORTELA, A., DELGADO, T., LOPEZ, J.A. & MELERO, J. A. (1990). Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus. EMBO Journal 9, 4181-4187. GIMENEZ, H.B., HARDMAN, N., KEIR, H.M. & CASH, P. (1986). Antigenic variation between respiratory syncytial virus isolates. Journal of General Virology 67, 863 870. JOHNSON, P. R., SPRIGGS, M. K., OLMSTED, R.A. & COLLINS, P. L. (1987). The G glycoprotein of human respiratory syncytial viruses of subgroup A and B: extensive sequence divergence between antigenically related proteins. Proceedings of the National Academy of Sciencs, U.S.A. 84, 5625 5629. LERCH, R.A., ANDERSON, K. & WERTZ, G.W. (1990). Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus. Journal of Virology 64, 5559-5569. MOHANTY, S.B., INGLING, A.L. & LILLIE, M.G. (1975). Experimentally induced respiratory syncytial viral infection in calves. American Journal of Veterinary Research 36, 417-419. MUFSON, M. A., (~RVELL,C., RAFNAR,B. & NORRBY, E. (1985). TWO
distinct subtypes of human respiratory syncytial virus. Journal of General Virology 66, 21115124. OLMSTED, R.A., MURPHY, B.R., LAWRENCE, L.A., ELANGO, N., MOSS, B. & COLLINS, P. L. (1989). Processing, surface expression, and immunogenicity of carboxy-terminally truncated mutants of G protein of human respiratory syncytial virus. Journal of Virology 63, 411-420. ROSENQUIST,B. D. (1974). Isolation of respiratory syncytial virus from calves with acute respiratory disease. Journal of Infectious Diseases 130, 177-182. SAMAL,S. K. & ZAMORA,M. (1991). Nucleotide sequence analysis of a matrix and small hydrophobic protein dicistronic mRNA of bovine respiratory syncytial virus demonstrates extensive sequence divergence of the small hydrophobic protein from that of human respiratory syncytial virus. Journal of General Virology 72, 1715 1720. SAMAL, S.K., ZAMORA, M., McPHILLIPS, T.H. & MOHANTY, S.B. (1991). Molecular cloning and sequence analysis of bovine respiratory syncytial virus mRNA encoding the major nucleocapsid protein. Virology 180, 453~456. SANGER, F., NICKLEN, S. & COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences, U.S.A. 74, 5463-5467. SMITH, M.H., FREY, M.L. & DIERKS, R.E. (1975). Isolation, characterization, and pathogenicity studies of a bovine respiratory syncytial virus. Archives of Virology 47, 23%247. SULLENDER,W.M., ANDERSON,K. & WERTZ, G.W. (1990). The respiratory syncytial virus subgroup B attachment glycoprotein: analysis of sequence, expression from a recombinant vector, and evaluation as an immunogen against homologous and heterologous subgroup virus challenge. Virology 178, 195-203. SULLENDER,W. M., MUFSON,M. A., ANDERSON,L. J. & WERTZ,G. W. (1991). Genetic diversity of the attachment protein of subgroup B respiratory syncytial virus. Journal of Virology 65, 5425-5434. ZAMORA,M. & SAMAL,S. K. (1992). Gene junction sequences of bovine respiratory syncytial virus. Virus Research 24, 11M121.
(Received 9 February 1993; Accepted 30 April 1993)
2001
Sequence variability of the glycoprotein gene of bovine respiratory syncytial virus Sanjay K. Mailipeddi and Siba K. Samal* V A - M D Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland 20742, U.S.A.
Sequence variation in the attachment glycoprotein G of bovine respiratory syncytial virus (BRSV) was determined. The nucleotide sequences of the G mRNAs of the A51908, VC464 and FS-1 strains of BRSV were compared with the published sequence of the BRSV strain 391-2. Nucleotide sequence alignment showed that overall they are highly conserved, with 90 to 97 % identity. In addition, the coding region of strain A51908 was longer by 18 nucleotides at the 3' end. An 84
to 95% level of identity was observed among the deduced amino acid sequences of the G proteins of BRSV strains. A maximum divergence of 19% was found when the extracellular domains of the G proteins were compared. The level of diversity would be consistent, by analogy to the human respiratory syncytial viruses, with these BRSV strains forming a single subgroup.
Bovine respiratory syncytial virus (BRSV) is an important cause of respiratory tract infections in cattle. It belongs to the genus Pneumovirus of the Paramyxoviridae, which also includes human respiratory syncytial virus (HRSV). The proteins of these two viruses are antigenically related, except for the antigenically distinct attachment (G) protein (Lerch et al., 1990). Sequence comparisons between proteins from BRSV and HRSV indicate that the G and SH proteins are least conserved (30 % and 38 % identity respectively; Lerch et al., 1990; Samal & Zamora, 1991). HRSV isolates are divided into two antigenic subgroups, A and B, on the basis of their reactions with monoclonal antibodies (MAbs) to the G protein (Anderson et al., 1985; Mufson et al., 1985; Gimenez et al., 1986). Sequencing studies have also shown that the G protein of HRSV is less conserved than other proteins between strains of the same or different subgroups (Johnson et al., 1987; Sullender et al., 1990; Cane et al., 1991). BRSV strains have not been characterized by Gspecific MAbs, nor have any sequence comparisons of the G proteins of BRSV strains been reported. Thus, BRSV is thought to be monotypic. To examine the variability of BRSV isolates, we compared the sequences of the G proteins of four strains. Strains of BRSV used in this study were A51908, 3912, FS-1, and VC464. Strain A51908 was isolated in Maryland in 1975 (Mohanty et al., 1975); strain 391-2 was isolated in North Carolina in 1985 (Fetrow et al., 1985), strain FS-1 was isolated in Iowa in 1975 (Smith et al., 1975) and strain VC464 was isolated in Missouri in
1974 (Rosenquist et al., 1974). Construction of cDNA libraries from mRNAs isolated from infected Madin Darby bovine kidney cells has been described (Samal et al., 1991). Recombinant cDNA clones containing G mRNA sequences of BRSV strain A51908 were identified by Northern blot hybridization and by comparing the sequences to the previously published G gene sequence of the BRSV strain 391-2 (Lerch et aI., 1990). cDNA clones of G genes of BRSV strains FS-1 and VC464 were identified by the colony hybridization technique with a probe made from the G gene of BRSV strain A51908. The nucleotide sequence of the mRNA encoding the G protein of each strain of BRSV was derived from at least two independent cDNA clones, using the dideoxynucleotide chain termination method (Sanger et al., 1977). The nucleotide sequence of the 5' end of the G mRNA of A51908 strain was determined by PCR amplification of the SH-G intergenic region (Zamora & Samal, 1992). The G mRNA sequences of BRSV strains FS-1 and VC464 were determined from nucleotide positions 37 and 42, respectively. The G mRNA of BRSV strain A51908 contained 835 nucleotides excluding the poly(A) tail (Fig. 1). The G mRNA sequence of strain A51908 was compared with those of strains FS-1, VC464 and 391-2 (Lerch et al., 1990). The levels of identity among the four different G mRNA sequences were 90 to 97 % (Table 1). There were three nucleotide deletions in the 3' non-coding region of all BRSV strains sequenced compared to the corresponding region of strain 391-2. In strains 391-2, FS-1 and VC464, the stop codon was at positions 787 to 789,
0001-1619 © 1993 SGM
2002
Short communication 1
A51908 391-2 FS-I VC464
G G G G C A A A T A CAAGC~--'T~TC CAACCACACC CATCATCCTA AATTCAAGAC ~ T T .................................... T .........................................
ATTAAAGAGG
GCTTGGAAAG
70
A51908 391-2 FS-I VC464
CCTCAAAATA
AAGTCCCTTG T C C A A A C G G C A A A
140
A51908 391-2
TTTGACCTCC T A
FS-I
T
T
A
VC464 A51908 391-2
CTTCATAGTA GGATTATCAT GTTTATATAA GTTCAATTTA T
ATAACAGCCA TTATTTACAT C
TAGTGTGGGA
210
ATCCAAACCA ACCACCCAAC AAACACAACA GCCCCAAAAC T
CACACCCCAC T T
280
TTAGCAATGA TAACCTTGAC C
ATCACTCGTC
C
A AATGCTAAAG
CCAAGCCCAC
FS-1
T
T
T
VC464
T
T
T
A51908 391-2 FS-I VC464
TACTTCCCAC C T TT C T ,'1~ C T TT
AGAGCACAAC
A51908 391-2
AAACATAGAC
ACCACTAGTG T A
FS-I
VC464
A
T
C
AACCCAAAGC TT TT TT
ACCACACTGT
CCCAACCACC T T T T AT T
350
CGGTCACCCA ATCAACAGAA T T C GA
CCCAAAACAG
AAAAATCAAA
420
CACAAATCAA CTCACACATC T T T GAACTACATA T
A
T
T
C G
GA
A
T
T
G
GA
G
A51908 391-2 FS-1 VC464
AGCCAATCTA G C C C
CTCCACTTGC T CC T CCA T CC
CACCCGAAAA CTACCAATCA ACCCACTGGA AAGCAACCCC A C T TC G T A C T TC A C TC G
CCCGAAAACC T T T
490
A51908 391-2
ACCAAGACCA T
CAACAACTCC T
CAAACACTCC
ATCCTGCCTG T T
560
FS-I
T
VC464
CTCATGTGCC CT CT
T
T
T
T
T
T
CT
T
T
T
T
T
T
A51908 391-2 FS-I VC464
TTCACCACTC C T T C T T C T T
TGCCAAATCG T T G T
GGCTGGAGAG A AC C GA C
A51908 391-2
CCAAAACCCA
AAACCACCAA T
AAAACCAACC AAGACAACAA G
FS-I
T
VC464
G
G
CTGCAGCACA TGCGAAGGCA T T T T
AGCACCAAGC
T
AGAGCTCCCA CAATCACCCT CAAAAAGGCT C T A C T A C T A
630
TCTACCACAG C
AACCAGCCCT
GAAGCCAAAC A
700
C
G
C
A51908 391-2 FS-I VC464
TGCAAACCAA CT C C
AAAAAACACG GCAACTCCAC C A C G C G
AACAAGGCAT
CCTCTCTTCA
CCAGAACACC A T A
AAACAAATCA C C C
770
A51908 391-2 FS-1 VC464
ATCTACTACA A A A
CAGATCTCAC ~ ~
CAT--AT ~
CAATTAT**G T A TAT** **
TTC*ATATGT T A * A * A
AGTTATTT
835 838 835 835
AACACACCTC
Fig. 1. Nucleotide sequences of the mRNAs of BRSV G protein genes. The sequence of strain 391-2 is taken from Lerch et al. (1990). Only the non-identical bases are shown for strains 391-2, FS-1 and VC464. The consensus gene start and stop signals are overlined. The consensus initiation codon and the stop codons are boxed. ( - ) Sequences not determined; (*) deletions in the non-coding region.
whereas in strain A51908 it was at positions 805 to 807. Correspondingly, the 3' non-coding region of the G mRNA of strain A51908 was 18 nucleotides shorter than those of strains VC464, FS-1 and 391-2. The 3' noncoding region of BRSV G mRNA is 70 nucleotides (52 in strain A51908) shorter than the corresponding sequence of HRSV. Recently, sequence analysis of the carboxyterminal third of the G protein gene of subgroup A HRSVs revealed a region prone to polymerase errors
resulting in the insertion or deletion of adenosine residues in some runs of such residues (Cane et al., 1993). Sequences of BRSV G genes also had runs of adenosine residues in the region coding for the extracellular domain, but no enhanced mutation was observed in this region among BRSV strains. The G mRNA of strain A51908 had a major open reading frame of 263 amino acids. The predicted relative molecular mass of this polypeptide is 28.9K. In vitro
Short communication
Table 1. Nucleotide and amino acid identities f o r G genes and proteins o f B R S V A51908 A51908 ~
9
0
391-2
-
3
FS-1
VC464
90.9
93.2
2 r~
14 i,2 . FS-1
84-4 (81.2)
92.0 (89-5)
~
97-1 O
VC464
87-9 (8~8)
9+0 (92-7) 95.2(942-)
Z
Amino acid identities (%) *Data in parentheses represent amino acid identity in the extracellular domain C"2TOPLASX/C DOima~V
~ ~" T R A N ~ U ~ ; M
A51908 391-2 FS-I VC464
THHPKFKTLKRAWKASKYF IVGLS6LYKFNLKSLVQTALTSLAMITLTSLVITAI i M~ L ST ....... L ST ......... T
A51908 391-2 FS-1 VC464
Y~SVGNAKAKPTSKPTTQQTQQPQNHTPLLPTE~HKSTMTSTQSTTLSQPPNIDTTSGT I ~ SPFF Y I LL R I I ~. SPFF N Y I LZ R I SPFF y I L T R
A51908
TYGHP~I~TQNRKIKSQSTPLATRKLPINPLESNPP~VHQDB~SQTLpHVp6ST~EGNp ST~E G 5P P SG I F y L
~
391-2
FS-I VC464
STDE
•
A51908 391-2 FS-I VC464
~s19o8
391-2 FS-I VC464
EXTRACE~L~R
S
LPT
P
S
60
~
,
Y
120
L
•
ACSPLCQIGLERAPSRAPT ITLKKAPKPKTTKKPTKTT IYMRTS PEAJ(LQTKKNTATPQQ LS H ET T H T P LS V PG T I H P ~ A LS p T H P A
N~
:
GILSSPEHQ STTQ ISQHTs I * T H ~ *
240
263 257 257 257
Fig. 2. Deduced amino acid sequences of O proteins of BRSV. The sequence of strain 391-2 is from Lerch et al. (1990). Only the nonidentical amino acid residues are shown for strains 391-2, FS-1 and VC464. The proposed domains are indicated above the sequences. Potential N-linked carbohydrate addition sites are shaded. The region corresponding to the conserved 13 amino acid region of HRSV (Johnson et al., 1987)is boxed. (e) Cysteineresidue; (*) end of each of the four proteins. expression of the G gene produced two unglycosylated forms of the G protein (28K and 23K). When the second methionine at position 48 was mutated to isoleucine, only one polypeptide of 28K was produced by in vitro expression, indicating that the 23K protein was produced by either leaky scanning or independent internal initiation (S. K. Mallipeddi & S. K. Samal, unpublished). The deduced G protein amino acid sequence of strain A5 ! 908 was compared with those o f strains FS- I, VC464 and 391-2 (Fig. 2). The predicted G protein of strain A51908 was longer by six amino acids at the C terminus than that of the strain 391-2 (Lerch et al., 1990). However, cross-neutralization and cross-immunoprecipitation tests using polyclonal antibodies failed to show any detectable antigenic difference between these
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strains (data not shown). Our results indicate that the six amino acids from the C terminus of the BRSV G protein are probably not involved in neutralization. Similar results have been reported with HRSV, where C-terminal truncated forms o f the G protein did not show any difference in neutralization (Olmsted et al., 1989), and this region has been shown to accommodate many changes without losing its function (Garcia-Barreno et al., 1990). The levels of identity among the G proteins of the four BRSV strains were 84 to 95% (Table 1). However, between the prototype strains of subgroups A and B of HRSV, there was only 53 % amino acid identity (Johnson et al., 1987). Within these subgroups, the G protein was also highly divergent with up to 20% amino acid variability between subgroup A strains (Cane et al., 1991) and up to 9 % variability between subgroup B strains (Sullender et al., 1991). Since variability between the four BRSV strains sequenced was 5 to 16%, all appear to belong to a single subgroup. When the extracellular domains of the G proteins of BRSV strains were compared, there was a maximum divergence of 19% between A51908 and 391-2 or FS-1, and there was a minimum divergence of 6 % between FS-1 and VC464. However, the cytoplasmic and transmembrane domains were highly conserved, with less than 5 % divergence among BRSV strains. The deduced amino acid sequences of the BRSV strains showed a high level (25 %) of serine plus threonine content that are potential O-linked glycosylation sites. In addition, four or five potential N-linked glycosylation sites were found in all strains examined (Fig. 2). Only the potential N-linked glycosylation site at amino acid residue 251 was conserved in all BRSV strains and in a majority of subgroup A strains of HRSV (Cane et al., 1991); it was absent in subgroup 13 strains. This suggests that N-linked glycans at this site are probably important for structural and functional properties o f the glycoprotein. There are five cysteine residues conserved in the G proteins o f all strains, indicating similar structural features. A conserved 13-amino acid region (164 to 176; Fig. 2) in the extracellular domain of the G protein of the HRSV was proposed as a candidate for attachment to cellular receptors (Johnson et al., 1987). This region was not conserved between BRSV strain 391-2 and HRSV, further suggesting that it could relate to host specificity (Lerch et al., 1990). However, this region was not perfectly conserved among strains of BRSV. We thank Dr Sashi B. Mohanty for his support and Mr Daniel Rockemannfor excellenttechnicalassistance.This study was supported by grants from the Maryland Agricultural Experiment Station and USDA grant no. 89-34116-4855. Approved as scientific article no. A6428 and contribution no. 8622 of the Maryland Agricultural Experiment Station.
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Short communication
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(Received 9 February 1993; Accepted 30 April 1993)
