J. gen. Virol. (1988), 69, 49-58. Printed in Great Britain
49
Key words: HR V-1B/nucleotide sequence/evolutionary relationships
The Nucleotide Sequence of Human Rhinovirus 1B: Molecular
Relationships within the Rhinovirus Genus
By P A M E L A J. H U G H E S , C H R I S T I N E N O R T H ,
C H R I S T O P H E R H. J E L L I S , P H I L I P D. M I N O R 1 A N D G L Y N S T A N W A Y *
Department of Biology, University of Essex, Wivenhoe Park, Colchester C04 3SQ
and 1National Institute for Biological Standards and Control, Blanche Lane, South Mimms,
Potters Bar, HertJbrdshire EN6 3QG, U.K.
(Accepted 22 September 1987)
SUMMARY
We have determined the complete nucleotide sequence of human rhinovirus 1B and
made comparisons with other rhinoviruses. Extensive homology was found with
serotypes 2 and 89 but the similarity to serotype 14 was considerably less. Rhinovirusspecific characteristics have been noted, in particular the length of the 5' non-coding
region and the pattern of codon usage, and these may be sufficient to define the
rhinoviruses as a distinct genus rather than being considered as members of the
enteroviruses as has been suggested previously.
INTRODUCTION
Human rhinoviruses (HRVs) are the major causative agents of the common cold, a disease of
considerable economic importance and one of the most frequent virus infections of man (Fox et
al., 1985). Major factors in the incidence of the disease are the existence of over 100
immunoiogically distinct HRV serotypes and the fact that several of these can co-circulate within
the community (Gwaltney, 1975). This serotype diversity probably precludes a vaccination
programme based on conventional methodologies and necessitates the development of
alternative forms of control, a process which would be facilitated by a better understanding of
the viruses.
Rhinoviruses, including HRVs, form the largest genus of the family Picornaviridae. They are
composed of an icosahedral capsid made up of 60 copies of each of four virus-coded proteins
(VPI to VP4) enclosing a single-stranded RNA genome of about 7200 nucleotides (Stanway et
al., 1984a; Callahan et al., 1985; Skern et al., 1985; Duechler et al., 1987; Rossmann et al., 1985).
As in other picornaviruses, the RNA is of positive polarity, is polyadenylated at its 3' terminus
and has a small protein, VPg, covalently attached to the 5' terminus (Ahlquist & Kaesberg,
1979). The R N A encodes one long polyprotein which is cleaved by virus proteases to give
the mature proteins. This long open reading frame is preceded by a 5' non-coding region,
approximately 600 nucleotides in length and of unknown function.
The complete nucleotide sequences of the genomes of three rhinoviruses, HRV-14, HRV-2
and more recently HRV-89 have been determined (Stanway et al., 1984a; Callahan et al., 1985;
Skern et al., 1985; Duechler et al., 1987). The availability of further HRV nucleotide sequences is
important for several reasons. Firstly, comparisons between HRV-14 and HRV-2 showed a
relatively low degree of homology, indeed a similar level to that seen between either virus and
the polioviruses, members of the enterovirus genus of the family Picornaviridae (Skern et al.,
1985). This indicated that the rhinovirus genus is relatively diverse. When the sequence of HRV89 was determined it was found that this serotype is highly homologous to HRV-2 (Duechler et
al., 1987). Further sequencing is necessary to assess properly the level of serotype diversity and to
find out whether there are groups of more closely related viruses within the genus. This could
have important implications for the design of synthetic, broadly reactive vaccines. Secondly,
0000-7965 © 1988 SGM
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50
P. J. HUGHES AND OTHERS
previous results have demonstrated a great deal of sequence conservation within the 5' noncoding region, indicating that this part of the genome has a vital function (Stanway et al., 1984a;
Skern et al., 1985). The availability of other nucleotide sequences would facilitate the
identification of important sequences within this region. Finally, rhinoviruses can be grouped
according to which cellular receptor they recognize. About 85 ~o (including HRV-14 and H R V 89) recognize one receptor and a minor group (including HRV-2) utilize a second ( A b r a h a m &
Colonno, 1984; Colonno et al., 1986). Sequence comparisons within and between receptor
groupings may give information on the molecular basis of receptor specificity. This is
particularly true in view of the availability of the three-dimensional structure of HRV-14 and the
suggestion that a striking surface feature, a deep 'canyon', may be involved in receptor binding
(Rossmann et al., 1985).
Here we present the complete nucleotide sequence of HRV-1B, a m e m b e r of the minor
receptor group, and discuss our results in terms of molecular relationships within the rhinovirus
genus, our understanding of the 5' non-coding region and the basis of receptor specificity.
METHODS
Virus. HRV-1B, obtained from the MRC Common Cold Unit, Salisbury, U.K., was propagated and purified as
already described (Stanway et al., 1984a).
Molecular cloning. The purification of RNA from HRV-1B and the cloning of c D N A - R N A hybrids was as
described previously except that different reverse transcription conditions were used and the vector was dG-tailed
pBR322 (Stanway et al., 1984b). Reverse transcription took place for 1 h at 37 °C in a 50 ~tl reaction volume
containing 50 mM-Tris-HCl pH 7-5, 75 mM-KC1, 10 mM-dithiothreitol, 3 mM-MgC12, 0-5 mM of each dNTP,
10 ~tg/ml oligo(dT), 100 ~tg/ml bovine serum albumin and 200 units of Moloney murine leukaemia virus (MMLV)
reverse transcriptase (Bethesda Research Laboratories).
Approximately 350 tetracycline-resistant, ampiciUin-sensitive transformants were obtained and these were
screened initially by hybridization using an oligo(dT)-primed cDNA probe made with limiting concentrations of
nucleotides to detect the presence of HRV-1B sequences proximal to the 3' terminus (Cann et al., 1983). Of the
cDNA inserts identified the one representing the 5'-most sequences (pOB512) was then used to probe for 5"proximal sequences. These two probing experiments enabled overlapping cDNA clones representing the complete
genome to be identified.
One cDNA insert, pOB512, was only 200 bp short of full-length and several were over 4 kb in size. This contrasts
with our previous experience with another rhinovirus, HRV-14, for which cDNA inserts did not exceed 3 kb
(Stanway et al., 1984a). We attribute this improvement to the use of MMLV reverse transcriptase in the present
experiment rather than the avian myeloblastosis virus enzyme used formerly. We have also obtained large cDNA
clones during the molecular cloning of other rhinovirus genomes using MMLV reverse transcriptase.
DNA sequencing. HRV-1B cDNA contains four internal PstI sites. For sequencing, the largest of the PstI
fragments (2.1 and 3.8 kb) were subcloned into M 13mp 19 in both orientations and a series of nested deletions were
produced by the method of Dale et al. (1985). The smaller PstI fragments were digested further with Sau3A and/or
HaelII and subcloned into M13. Nucleotide sequences were determined by the dideoxynucleotide method and
data were collated and analysed using published computer programs (Staden, 1980, 1982).
RESULTS AND DISCUSSION
Comparison with other rhinoviruses
The complete nucleotide sequence of HRV-1B derived from cloned c D N A is shown in Fig. 1.
The sequence of 7133 nucleotides plus a poly(A) tail is similar in length to the other rhinoviruses
studied and has a similar nucleotide composition (A, 34"1~o; C, 17.4~o; G, 19.5~o; T, 29-0~)
being somewhat A + T-rich. A comparison of the predicted amino acid sequence of the virus
proteins with those of HRV-2, HRV-14 and HRV-89 (Stanway et al., 1984a; Callahan et al.,
1985 ; Skern et al., 1985 ; Duechler et al., 1987), the other three sequenced rhinoviruses, is shown
in Table 1. All the proteins o f HRV-1B show considerable homology with those of the other
rhinoviruses, but the relationship to HRV-2 and to HRV-89 is much more m a r k e d than that to
HRV-14. The greatest degree of similarity is to HRV-2 where the homology within the
individual proteins does not fall below 7 4 ~ and in many of the proteins there is a much higher
degree of conservation. The relationship to HRV-89 is somewhat less throughout much of the
genome except that in VP4, VP2, VP3 and P3-A there is marginally greater homology than to
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Human rhinovirus 1B
51
Table 1. Protein homologies (%) between rhinoviruses
Homologous virus pair
Protein
VP4
VP2
VP3
VP1
P2-A
P2-B
P2-C
P3-A
VPg
Protease
Polymerase
HRV-1B/
HRV-2
94
77
74
74
88
88
84
74
91
83
83
HRV-1B/
HRV-89
100
78
75
67
83
68
74
75
81
76
72
HRV-IB/
HRV-14
52
61
52
36
43
39
50
52
57
52
55
HRV-2/
HRV-89
94
73
66
62
85
64
72
65
86
75
72
HRV-2/
HRV-14
51
60
49
32
34
42
44
38
58
50
55
HRV-14/
HRV-89
54
57
51
38
36
45
49
40
57
52
58
HRV-2. HRV-1B and HRV-2 are the most closely related serotypes overall since their degree of
homology also exceeds that seen in the HRV-2/HRV-89 comparison. In contrast to the
relationship to serotypes 2 and 89, the homology to HRV-14 is of a much lower level, varying
from 36~ to 61%. These are similar figures to the HRV-2/HRV-14 and HRV-89/HRV-14
comparisons although overall HRV-14 seems to be less similar to HRV-2 than to the other two
serotypes.
The distribution of amino acid differences between HRV-1B and each of the three previously
sequenced rhinoviruses is shown in Fig. 2. The polyprotein has been divided into blocks of four
amino acids and a vertical bar plotted of different height depending on whether none, one, two,
three or four of the amino acids in each block are different. Interestingly the degree of
conservation seems to be remarkably uniform throughout the genome and this is slightly
different from the pattern seen with other closely related picornaviruses which show more
homology in the non-structural proteins. Within the capsid region the homologies between the
three closely related rhinoviruses are in the range 62 to 100~ and for HRV-1B/HRV-2 are 74 to
9 4 ~ (Table 1). These figures approach those for the corresponding homologies between
poliovirus serotypes (71 to 9 2 ~ for PV-1/PV-3) (Stanway et al., 1983). In comparison to the
poliovirus situation, the homologies of some of the proteins towards the C-terminal end of the
polyprotein, particularly the polymerase (72 to 83 ~) seem relatively low. In the polioviruses this
protein is almost identical between serotypes (about 98 ~ homology) and is far more homologous
than the capsid proteins except for VP4 (Toyoda et al., 1984). This pattern of conservation of the
polymerase gene and a more flexible structure for the capsid proteins does not seem to be as
marked between HRV-1B, -2 and -89. It is therefore possible that the rhinovirus polymerase is
less stringent in its requirement for absolute maintenance of structure than its poliovirus
counterpart. On the basis of partial sequence comparisons between HRV-14 and HRV-2, we
suggested previously that the rhinoviruses are a very diverse group (Stanway et al., 1984a). The
current results on HRV-1B as well as the HRV-89 sequence indicate that this conclusion must be
modified. Clearly there is considerable diversity between some serotypes, e.g. between HRV-14
and each of HRV-2, -1B and -89, but there are also groups of more closely related serotypes.
Since HRV-1B is in the same receptor group as HRV-2 (the minor group) it would be expected
that it would share more overall homology with this serotype than with HRV-14, a member of
the major group. However, the high level of homology of HRV-1B and HRV-2 to HRV-89 (also
a member of the major group) shows that receptor grouping cannot account for overall genetic
homology. In support of this conclusion we have sequence data indicating that HRV-1B is
closely related to several other rhinoviruses in the major receptor group. The detailed picture of
the relationships within the genus will become clear with the availability of further complete or
partial sequences.
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52
P. J. H U G H E S A N D O T H E R S
TTAAA•CTG•GTGTG•GTT•TT•••ACTCAC•CCA•CCAATG•GTGTTGTACTCTGTTATTCCGGTAA•TTTG•ACGCC•TTTTCC•TCCCTCCCcATC•TTTTACGTA•CTTACAACTT
I0
20
30
40
50
60
70
80
90
I00
110
120
TTAAA~ACAAGACCAAT~A~G~AACTC~CA~TT~T~TAA~T~C~TCT~TTT~CG~TTG~TTGATA7G~T~TAACA~AA~A~ACT~ATAT~TT~CC~CA~
130
140
150
160
170
180
198
8~G
210
880
230
240
AGTGC•TA•ACAGAGcTTAGTAGGATTCTGAAAGATCTTTGGTTGGTCGCTcAGcTGC•TACCCAGC•GTAGAC•TTGcAGATGAGGCTGGACATTCCCcACTGGTAACAGTGGT•CAGC
250
260
270
280
290
300
310
380
930
34O
350
360
~TGCGTGGCTGCCTG~A~A~T~TTATGAGGTGTGAAG~CAAAGATTGGACAGGGTGT~AAGAG~C~CGTGTGCT~ACTTT~AGTCCTCcGGCCCCTGAATG~GGCTA~TTA~ACCTGC
370
380
390
400
410
920
430
490
450
460
470
48O
•GCCATGGCT•ATAAACCAATGAGCTTAT•GTC••AA3G•GCAATTGC•G•ATGG•AC•GAC•ACT••GGGTGTCCG•GTTTCACTTTTTCCT•TATCAATTGC••ATGGTGACAATA•A
490
500
510
520
530
540
550
560
570
580
590
)VP4
M G A Q V S R Q N V G T H S T Q N S V S N G S S L N Y F N I N
TA•ATAG•TATATATTGGCAT•ATGGGTGCCCAGGT•TCTAGGCAAAAT••TGGT•CACA•TCA••TC•AA•TTCAGT•TCAAATGGATCAAGTTTAAATTAcTTCAATATAAATTACTT
610
620
630
640
650
660
67O
68D
690
7OO
710
600
Y
F
720
~VP2
KDAASSGASRLDFSQDPSKFTOPVKDVLSKGIPTLQISPSV
T~AG~A?~C~GCTTCGAGTG~T~T~G~TTAG~C~TCTCTCAA~7~CAAGCA~TTCA~GA~CCAGTTAAAGATG~CTTAGAAA~AGGAAT~C~CACTAC~ATCACCA~CT~T
730
740
75O
/60
770
780
790
800
810
820
830
840
E A C G Y S D R I I Q I T R G D S T I T S Q D V A N A V V G Y G V W P H Y L T
TGAAGC•T•TGGCTATTcAGATAGGATTATA•AAATAA•••GAGGAGACT••ACAATCACATcCcAGGACGTGGC•AAT•CT•TGGTTGGATATGGGGTTTGGCCACA•TACTTAACCCC
850
860
870
880
890
900
910
920
930
940
950
P
Q D A T A I D K P T Q P D T S S N R F Y T L E S K H W N G D S K G W W W K
ACAA•ATG•AACCGCCATAGACAAACc••CA•AA•CCG•T•CATCATCAAA••GGTTCTA•ACACT•••AAGTAAACACTGGAATGGTGATT•TA•A•GATGGTGGTGGAA•TTACCAGA
D
970
980
990
1008
1010
1020
I030
1040
I050
1060
L
960
P
1070
1080
A L K E M G I F G E N M Y Y H F L G R S G Y T V H V Q C N A S K F H
TGCTCTTAAAGAAATGGG•ATTTTT••AGAGAATAT•TATTATCACTTCTTGGGTAGAAGTGGATATA•AGTTCACGTACAGTGTAATGCTAGCA••TTCCATCAAGG•A•CCT•TTAGT
1090
i100
IIi0
1120
1130
1140
1150
116fl
I170
1180
I190
Q
A M I P E H Q L A S A K N G S V T A G Y N L T H P G E A G R V
T•CAAT•ATACCAGAACA•CAA•TAGCAAGTGCAAA•AATGGAAGTGT•A•T•CTGGTTATAAT•TCAcACACCCA•GTG•G•CCGGTAGA•TTGTGGGTCAACAGCGT•ATGCCAATCT
1210
1230
1230
1240
1250
1260
1270
1280
1290
1300
Q
V
G
Q
1340
1350
1360
1370
1380
1390
1400
1410
1420
1460
1470
1490
1480
1500
1510
1520
1530
1540
T
L
L
V
D
A
N
L
I
V
1320
S
A
1430
T
L
P
1440
Y V N A V P M D S M L R H N N W S L V I I P I S P L R S E T T S S N I R P I
ATATGT••ATGCTGTA•CAATG•ATTcAATG•TTC•A•ACAATAATTG••GTTTAGTc•TTATACCAATCAGTCCATT••GTAGTGAAAcCA•ATCTTcTAACATAAGACcAATCACTGT
1450
R
1310
R Q P S D D S W L N F D G T L L G N L L I F P H Q F I N L R S N N
AAGGC•ACCTAGCGATGATAGCT•GCTTAATTTTGATGGCACT•TTcTT•GAAATCTGTTAATT?TC•CACATCAG•TTATAAATCTTAGAAG•AATAATTCTGCAA•TTTGATAGTACC
1330
G
1200
T
V
1560
1560
S I S P M C A E F S G A R A K N V R Q F G L ~ P S v Y I T P G S G Q F M T T O D M Q S
ATCAATTA•TCcTATGTGTGCTGAATTTTCTGGT•CAAGAGCAAA••AT•TCAGAcAAGGTTTACCTGT•TATATAACTCCAGGATCTGG•CAATTTATGACGACTGATGACATGCAGTC
1570
1580
1590
1600
1610
1620
1630
1640
1650
1660
1670
1680
P C A L P W Y H P T K E I S I P G E V K N L I E M C Q V D T L I P
ACCTTGTGCACTACCAT•GTAC•ATCCT•CTAAGGAAATATCTATTCCA••TGAAG•TAAAAACCTCAT•GAGATGTGTCAAGTGGATACCTTAATCCCAGTCAATAATGTGGGTACCAA
1690
1700
1710
1720
1730
1740
1750
1760
1770
1780
V
1820
1830
1840
1850
1860
1879
1899
1899
1900
G
1940
1950
1960
1970
1980
1990
2000
2010
2020
A T R K D A M L G T H V V W D V G L Q S T I S L V V P W V S A S
AGCT•CC••AAAA•AT•CAAT•CTG•GAACACATGTTGTGTGGGATGTCGGTTT•CAATCTACCATAT•TCTTGTTGTACCATGGGTAAGT•CCA•TCATTTTA•GTTAAC•GCAAATGA
2050
2060
2070
2080
2090
2100
2110
2120
2130
2140
G
T
N
I
D
K
P
T
A
N
O
F
C
L
1920
P
2030
H
V
E
19.10
I A S Y Y T H W T G S L R F S F M F C G T A N T T L K L L L A Y T P
AATTGCAA•TTATTATACCCACTGGA•TGGAAGTTTACGATTTAGTTTCATGTTTTGT•GGACTG•AAACACCACACTTAAATTATTACTTGC•TACACACCACCT•GGATTGACAAGCC
1930
N
1800
V G N I S M Y T V Q L G N Q M D M A O E V F A I K V D I T S Q P L A T T L {
TGTTGGAAACATT•GTATGTAC•CTGTG•AATTAGGAAA•C•AATGGATATGG•ACAGGAA•T•TTTGC•ATAAAAGT•GATATTA•ATCACAAc•TTTAGCTAC•AcCCTAATTGGAGA
1810
N
1790
G
2040
F
R
2150
K Y S M A G Y I T C W Y Q T N L V V P P N T P Q T A D M L C F V S A
cAAATATTC•ATGGCTGGTTATATTACATGTTGGTATCAAACTA•TCTA•TAGTGCCCCcAAACACGCCACAAACTGCTGACATGTTGTGTTTTGTTTCTGCATGTAAGGACTTTTGTCT
2170
2180
2190
2200
2210
2220
2230
2248
2250
2260
2270
)VPI
L
2160
C
K
D
2280
R M A R D T D L H I Q S G P I E Q I N P V E N Y I D E V L N E V L V V P N I K E 8
ACGGATGGCTAGGGATACAGATTTACATATACAAAGTGGTCCAATAGA•CA•A•TCCAGTG•AAAAT•ACATTGATGAA•TTTTAAATGAA•TTC•AGTA•TACCAAATATA•AAGAAAG
2290
2300
2310
2520
2330
2340
2350
2360
237O
2380
2390
2400
H H T T S N S A P L L D A A E T G H T S N V Q P E D A I E TV M
RTSQ
•CATcA•ACTACATCAAATTCTGCTCCACTCTTGGATGCTG•AGAGACAGGACACACCAGTAATGTAC•ACCAGAGGATGCTATA•AAACAAG•TATGTTATGACATCACAAACAAGAG•
2410
E
M
S
2420
I
E
S
F
2430
L
G
R
2940
S
G
C
V
2450
H
I
S
2460
R
I
K
V
2470
D
Y
N
2980
D
Y
N
G
2490
V
N
K
2500
RD
2510
2520
F T T W K
TL
TGAGATGAG~AT~AAAGTTTT~TTG~TA~ATCTGGC~GTG~GCA~ATTTCAAGAAT4~A~G~TG4TT4CAATGACTACAA~GGm~TGAA£AAA~TTTA~AA~A~GGAAAATCACA~T
2530
2540
2550
2560
2570
2580
2590
2600
2610
2620
2630
2640
Q E M A Q I R R K F E L F T Y V R F D S V T L V P C I A G R
D D I G H
GCAG•A•ATGGcACAAATTAGAAGAAAATTCGAACT•TTTACTTAT•TTAGGTTTGATTCAGAAGTAACTTTAGTACCCTGTATTGCTGGTA•AGGAGATGACATTGGTcATGT•GTAAT
2650
2660
2670
2680
2690
2700
2710
2720
2780
2740
2750
2760
VM
Q Y M Y V P P G A P I P K T R N D F S W S G T N M S I F W Q
GQPFP
GCA~TA~A~GTATGTTCC~CA~GAGCTCCAATTCCAAAAACAAGAAAT~ATTTCTCAT~CAATCAGG~ACTAATAT~TCAATATTCTGGCAACATGGACAAC~GTTC~CTA~ATTCTC
2770
2780
2790
2800
2810
2820
2830
2840
2880
2860
2878
L P F L S I A S A Y Y M F Y D G Y O G D
S S S K Y G S I V T
D M G T I
TTTA••ATTT•TTA••ATTGC•T•AGcTTATTAcATGTTTTATGATGGATATGAT••AGAT•ATTCCTcTTcC•AATATGGT•GTATAGTcACCAATGATATGGGAA•CATATGTTCAA•
2890
2900
8910
2920
2930
2940
2950
2960
2970
2980
FS
2880
SR
2990
3000
I V T E K Q E H P V V I T T H I Y R K A 8 T K A W C P R P P
A V P Y T
AATAGTTA•AGAGAAGCAGGAAcA•CCTGT••TTATT•CAAC•CACATAT•T•ACAAAGCT•AACAcACAAAAGCTTGGTG•CCTAGACCTCCTA•AGCT•TTCCTTA•ACACATAGTCG
3010
8028
3030
3040
8050
3060
3070
3080
3090
3100
3110
P2-A
V T N Y V P K T G D V T T A I V P R A S KTVG[~PSOLYV
VGNLITA~GAN
SR
8120
TGTAACTAATTATGTACCAAAAACAGGT•ATGTGACAACAGCTATAGTTCCTAGAGCTAGCATGAAAACTGTTGGACCCAGTGATTTGTATGTACATGTAGGTAACTTAATATA A
3130
3140
3150
3160
3170
3180
3190
3200
3210
3220
3230
3240
L H L F N S E M H D S I L V S Y S S D L I Y R T N T T G D D
I P S C N
TTTA•ATTTGTTTAAcTCTG•••TGcATGATTCA•TTCTGGTTTCATAC•CTTCTGATTTAATCATATACCGCACAAACACTACAGGTGATGATTATATTCCTAGTT•TAACT•cACAGA
3250
3260
3270
8280
3290
3300
3810
3320
3330
3340
TE
3350
A T Y Y C K H K N R Y Y P I K V T P H D Y E I Q E S E Y Y P
8 1 Q Y N
GGCTACCTATTATTGTAAACACAAAAATAGATATT•CC•AA•AAAGGTTACTCCACATGATTGGTATGAAAT•CAAGA•AGTGAATATTACCCTAAACATATCCAATACAATTTATTAAT
3370
3380
3390
3400
3410
3420
3430
3440
3450
3460
3470
3360
L
L
I
3480
G E G P C E P G D C G G K L L C R H G V G I I T A G G E G 8
A F T D L R Q F
TG•TGAA•GACcATGTGAACCT••TGATTGTGGTG•GAAAcTTCTTT•TAGACATGGTGTTATTGGCATAATT•CAGCAG•TGGTGAAGGc•ATGTAGCATTTA•AG•TCTTAGACAG•T
349D
3500
3510
3520
3530
3540
3550
3560
3570
3580
3590
3600
E Q I
A I N P I N S I
Q C A E E Q ~ I T )PS-B
D Y I H M L G E A F G N G F V O S V K
TCAATGTG~TGAAGAACAGG~TATAACTGATTATA~ACAcATGTTA~GAGAGGCCTTTGGTAATGGTTTTGTAGATAGTGTCAAAGAACAAATTAATGCAATAAATCCAATCAATAG~AT
3610
3620
3630
3640
3650
3660
3670
3680
3690
3700
3710
S K K V I K W L L R I I S A M V I I I R N S S D g Q T I I A T L T L
TAGCAAAAAAGTTATCAAGTGGCTACTTAGAATAATTTCAGCTATGGTCATTATAATTAGAAATTCTTCTGACcCTCAAAC•ATCATAGCAACTCTAA•TTTAATTG•CTGCAATGGTTC
3730
3740
3750
3760
3770
3780
3790
3800
3810
3820
3830
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3720
I
G
C
3840
N
G
S
53
Human rhinovirus 1B
~P2-C
PWR
LKE
F C K W T Q
T Y I H K E I S D S W L K K F
E M C N A A R G L E
GCCATG•AGGTTT•TCAAGGAAA•GTTT•GTAAATGGACCCA•TTAACTTA•ATCCACAAAGAATCTGATT•ATGGCTCAAGAAATTTACTGAAATGTGTAATGcCG•G••TGGTCTTGA
3880
3860
3870
3880
3890
3900
3910
3920
3930
394D
3950
3960
WIG
KIS
F I D W M
M
L
P
Q
A
Q
L
K
V
K
Y
L
N
E
K
K
L
S
L
L
E
K
Q
I
GTGGATTGGcAATAAAATTT•AAAGT•C•TAG•T•G•AT•AAAT•AAT•CTACCTCA••CTCAGTTGAAAGT•AAATATCTGAATGAGATAAAGAAACTTA•CTTGCTT•AAAAACAAAT
3970
3980
3990
4000
4010
4020
4030
4040
4050
4060
4070
4080
ENL
AAO
A T Q E K
C E I O T
H D L S C K F L
L Y A H E A K
TGA•AATCTA•GCGCAGCAGATAATGCTAC•CAAGAAAAGAT•AAATGTGAAATTGA•AC•TTGCATGACTTATCATG•AAATTT•TC••TTTATACGCACATGAAGCAAAAAGGATTAA
4090
4100
4110
4120
4130
4140
4150
4160
4170
4180
4190
VLY
KCS
I I K Q R
S E P V A
M I H G P P G T
K S I T
AGTG•TCTA•AATAAATGTTC•AATATAATCAAACAAAGAAAGA•AAGTGAGCCAGTGGCA••AATGATA•AT•GACC•C•TGGTACT••TA•GT•TATAACAACCAATTTCCTAG•CAG
4210
428Q
4230
4240
4250
4260
4270
4280
4290
4300
T
N
R
I
K
4200
F
L
A
4310
R
4380
MI
ESD
YS
PP
KYF
G
D N Q S V V I M
D [ M Q N P D G E D
AATGATAACAAATGAAAGTGATGTGTATTCATTAcCCCCAGATCCTAAATATTTTGAT•GATATGACAATCAGAGTGTTGTGATCATGGATGATATCATGCAGAAcCcTGATGGA•AAGA
4330
4340
4350
4360
4370
4380
4990
4400
4410
4420
4430
4440
MT
CQM
SS
TF
PMA
L
D K G K P F D S
F V L C S T N H S L
CATGACACTATTTTGTCAAATG•TTTcAAGTGTTACATTCATAcCACCTATGG•TGACCTGCCTGATAAGGGTAAGCCGTTTGACTCAA•ATTTGTTTTGTGTAGTACTAATcACTCTCT
4450
4460
4470
4480
4490
4500
4510
4520
4530
4540
4550
4560
LA
T [ 5 S L P
MN
FFF
L
I V V H D N Y K
A Q G K L N V S K A
CCTA~CTCCACCCAC~ATATCT~CATTACC~GCAATGAA~A~AAG~T~TTC~TT~AC~AGA~A~T~AG~TCAT~ATAATTA~GGATGCACAAGGGAAATTAAAT~IATCTAAGGC
4570
4580
4590
4600
4610
4620
4630
4640
4650
4660
4670
4680
FQ
N V N T K I
NAK
CPF
C
K A V S F K D R
T C S T Y T L A Q V
TTT••AACCTTGTAATGTcAAT•CTAAAATTGGCAATGCAAAATGTTGTCCATTTGTGTGTG•TAAGGCAGTGTCAT•TAAGGATCGTAGCACTTGTTCAACATATACCTTGGCTCAAGT
4690
4700
4713
4720
4730
4740
4750
4760
4770
4780
4790
48D0
)03-A
YN
L E E D K R
R Q V V D V M
A
F Q I G P I S L D
p p P P A I A D L L
T•ACAATCACATTTT••AAGAAGATAAGAGAAGGAGACAGGTGGTAGATGTAATGTCTGCAATTTTCCA•GGGCCAATTTCTCTAGATGCTCCGCCGC•ACcAG•C•TAGCAGATCTGTT
4810
4820
4830
4840
4850
4860
4870
4880
9890
4900
4910
4980
QS
T P E V I K
C Q D N K W I
P A E C Q I E R D L
I A N S [ ] T I I A
ACAATCAGTTAG•ACACCTGAAGTAATTAAATATTGTCAGGACAACAAATGGATAGTCCCAGCAGAATGCCAAATAGAGAGAGACTTAAG•ATAGCCAATA•CATAATAACTAT•ATAGC
4930
4940
4950
4960
4970
4980
4990
5000
5010
5020
5030
5040
VP9
V
NI
S I A G I I F
IYKLFCTLQG~-~;~PYSGEPKP
T K M P E ~ R V A T A
AAATATAATAAGTATAG•TG•TATTATATTTGTAATTTACAAATTGTTTTGCACA•TA•AAGGACCATA•TCAGGTGAG•CTAAACCCAA•ACCAAGATGCCTGAAAGGAGAGT
G TGC
5050
8060
5070
5080
5090
5100
5110
5120
5130
5140
5150
5160
~PROTEASE
Q [ G P E E E F G R S I L K N N T C V I T T D N G K F T G L G I Y D R T L I
CCAAGGTCCAGAAGA#`GAATTTGGAAGATCAATCTTAAAGAACAACACTTGTGTGA~TACTACAGACAA~GGAAAATTTACAGGTCTTGGTATCTATGACAGAACTTTGATCATTCCAAC
5170
5180
5190
5200
5210
5220
523O
5240
5250
5260
5270
5280
HAOP
R E V Q V N G [ H T K V L D S Y D L Y R R D G V K L E I T
ACATGCTGATC•AGGTAGAGAG•TTCAAGTCAA•GGCATTCACACTAAGGTCTTAGATTCATATGACCTTTATAATAGGGAT•GAGTTAAACTTGAAATAACAGTTATACA•TTA•A•AG
R
NEKF
D I R K Y I P E T E D D Y P E C N L A L S A N Q V E P T I I K V G
AAAT~AAAA~TT~A~GATATTA~GAAGTACATA~CT~AAACAG~GGATGATTATC~AGAATGTAATTTAG~ACTTTCAGCTAATCAAGTTGAACCAACTATAATTAAAGTAGG~GATGT
5410
5420
5430
5440
5450
5460
54•0
5480
5490
5500
5510
6520
D
V
5310
5320
5330
5340
5350
5360
5370
5980
5390
VSYG
I L L S G N Q T A R M
K Y N y P T K S G Y C G G V L Y K I
AG•GTC•TATGGTAACATTTTACTT•GTGG•AACC•AACA•CTAGAATGCTAAAATATAATTACCCTACAAAATCAGGATATTGTGGAGGTGTATTATATAAAATTG•TCAGATTCTAGG
5530
5540
5550
5560
5570
5580
5590
5600
5610
562D
Q
T
O
5300
I
P
L
5290
V
I
5400
G
Q
5630
I
L
G
5640
)POLYMERASE
IHVG
N G R D G F S A M L L
SYFTDTQtGQIKISKHANECGLPT
TATTCATGTAGGTGGAAATGGAAGAGATGGATTTTCAGCC•TGTTACTT•GGTCATACTTTACAGACACTCAGGGTCAGATTAAAATCTCCAAACATGCTAATGAATGTGGTCTTCCAAC
5650
5660
567O
5680
5690
5700
5710
5720
5730
5740
5750
5760
I H T P
K T K L Q R S V F Y D
F P G S K E P A V S R D N D P R L K V N F K
CATACATACCCCTAGTAAAACTAAACTTCAA~TAGTG~GTICTACGAT~TCTTCCCAGGTT~TAA~GAACCAGCT~TCTCAC~AGAT~A~GACCC~A~ACTA~AA~TTAATTTT~AA~A
5770
5780
5790
5800
5810
5820
5830
5840
5850
5850
5870
5880
E
ALFS
Y K G N T E C S L N Q
M E I A I A H Y S A Q L I T L D I D
AG•T•TATTCTCTAAATATAAAGGTAATACAGAATGTA••TTAAATCAACATATGGAAATTGCCA•C•CTCACTATTC•GCACAATTAATAACATTA•ATAT•GATT•TAAACCAATAGC
I
A
T
K
5890
5900
5910
5920
5930
5940
5990
5960
5970
5980
L£OS
F 6 1 5 G L E A L D L
T S A G F P Y V T M G I K K R D L
ATTAGAGGACA•TGTGTTCGG•ATAGAG•GGCTCGAA•CTTTAGACTTA•ACACTAGTGCTGGTTTTCCTTAT•TTA•A•T••G•ATC8••AAGA•AGACCTAATAAATAATAAAA•AAA
6010
6020
6030
6040
6050
6060
6070
6080
6090
S
K
5990
6100
I
P
6000
S
N
6110
K
8120
D I S R L K E A L D K Y G V O L
M I T F L K O £ L R K K E K I
A G K T R V I
AGACATAT•TAGACTTAAAGAGGCTTTAGA•AAATATG•TGTTGACTTGCCTATGATTACTTTC•TAAAGGATGAACTTAGAAAGAAGGAGAAAATCTCAGCAGGTAAAACTAGAGTTAT
6130
6140
6150
6160
6170
6180
6190
6200
6210
6220
6830
6240
E A S S I N O T I L F R T T F G
L F S K F H L N P G V V T G S A
AGAAGCAAGTAGCATAAATGA•ACAATAC•ATTTAGAACTACTTTTGGT•A•TTATT•T••AA•TTT•ACTTGAAT••AGGTGTTGTTA•TGGTTCT••AGTAGGGTGTGATCCTGAG••
6250
6260
6270
6280
629O
6300
6310
6320
6330
6340
V
G
6350
C
D
P
5380
F W S K I P V M L D G D C I M A F D Y T N Y D G S I H P V W F LQK K V L E N
TTTCTG•TCCAAAATC•CTGTTATGCTTGATGGAGACTGCATAATGGCCTTTGATTATA•AAAC•ATGATGGTAGCATACACCCTGTTTGGTTTCAAGCTTTAAAGAAAGTTCTTGAAAA
6370
6380
6390
6400
6410
6420
6430
6440
6450
6460
6470
6480
L S F Q S N L I D R L C Y S K H L F K S T Y Y E V A G G V P S S GG T S I F N
CTT•TCCTTTCAATcTAATTTAATTGATAGGTT•TGTTA•TCTAAGCATTTGTTTAAGT•AACATACTATGAAGTGGCAGGTGGAGT•CCT•CTGGATGTTCTGGGACTAGCATATTTAA
6490
6500
6510
6520
6530
6540
6550
6560
6570
6580
6590
6600
T M I N N I I I R T L V L D A Y K N I D L D K L K ] I A Y G DI D
F S Y K Y T
TACTATGATTAATAACATTATAATAAGAACATTAGT~TTAGA~CATATAAGAATATT~ATTTGGACAAGTTGAA~ATAATCGCATAT~GTGAT~ATGT~ATTTTTTC~T~C~AGT~TAC
6610
6620
6630
6640
6650
6660
6670
6680
6690
6700
6710
6720
L D M E A I A N E G K K Y G L T I T P A D K S T E F K K L D Y V NT F L K R G
TTTAGATAT•GAAGCCA•T•CTAATGAAGGA•AGAAATATGGACTCACAATAACACCAG•AGATAAAT•TACTGAATT•AAGAAACTT•ATTA•AACA•TGTGACTTTTCTTAAACGTGG
6730
6740
6780
6760
6770
6780
6790
6800
6810
6880
6830
6840
F K Q D E K H T F L I H P T F P V E E ~ Y E S I R W T K K P S Q QE H V L S L
TTTTAAACAAGATGAGAAACACACATTTC•TATTCACCCCACATTTCCAGTAGAAGAAATATATGAATCAATTAGATGGACTAAGAAG•CTTCAcAAATGCAAGAACATGTACTAT•ATT
6850
6860
6870
6880
6890
6900
6910
6920
6930
6940
6950
6960
C H L M W O N G R K V Y E O F S S K I R S V S A G R A L Y I P OP L t K H E W
•TG•CATTTGATGTGGCAcAA•GGACGTAAGGTGTATGAG•ATTTTTCCAGTAAGATA•GCAGTGTCAGCGCT•GTCGT•CACTGTATATCcCACC•TATGATCTATTAAAG•ATGAATG
6970
6980
6990
7000
7010
7020
7030
7040
7050
7060
7070
7080
Y E K F *
GTATGAAAAATTTTAGATATAGAAATAATGAATGAATGATICTTTAATTCTAT-poly
7090
7100
7110
7120
7130
A
Fig. 1. The nucleotide and predicted amino acid sequences of cDNA representing the entire genome of
HRV-IB. Protein boundaries were predicted by alignment with other rhinoviruses.
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E
T
54
P. J, HUGHES AND OTHERS
NIm-IB
Nlm-II,Nlm-llI NIm-IA I
HRV-2
2
: I ,m,l, ,ILL,l,m, i i,IL UlIili,LllllUl , L,I ,Ill,, l,hll
HRV-89 "~ 2
<~ 4
3
HRV-14
2
1
0
I
l
VP4
l
VP2
I
VP3
l
I
I
I
II
I
I
VP1
P2-A P2-B P2-C P3-A]Protease
Polymerase
VPg
Virus genome
Fig. 2. The amino acid homologiesof HRV-1B to HRVs 2, 89 and 14. The genome has been divided
into blocks of four amino acids and a vertical line of height 0 to 4 plotted to represent the number of
amino acid mismatches within the block.
Table 2. Nucleotide frequencies in rhinoviruses and enteroviruses
Position in codon
First
U
A
C
G
Second
U
A
C
G
Third
U
A
C
G
HRV-1B
HRV-2
HRB-89
HRV-14
PV-3*
CB-4t
20.5
35.0
16.3
28,2
20.5
35-2
16.6
27-7
21.1
31.9
17-6
29.4
19-9
33.6
17.7
28.8
19.3
31.8
18-0
30.9
19.9
30.7
17-9
31.5
28.2
32.9
22-5
16.4
28-2
33.0
22.5
16.3
28-6
31.2
23.7
16.5
28.9
31-9
23.8
15.4
27-4
31.8
24.9
15.9
28-3
31-5
22.9
17-3
38.6
34.7
13.7
13.0
36.0
32-8
15.9
15-3
39-0
33,4
15.2
12,4
32.4
33.6
17-8
16.2
25-2
25.8
26-2
22,8
22.8
24.4
27.2
25-6
* Poliovirus type 3 (Stanway et al., 1983).
t Coxsackievirus B4 (Jenkins et al., 1987).
Comparisons with viruses sequenced previously enable the prediction of the cleavage sites in
the polyprotein which give rise to the mature virus proteins. These have been well established
for the polioviruses and the capsid cleavage sites have been determined for HRV-2 and HRV-89
by protein sequencing and for HRV-14 from the three-dimensional structure (Kitamura eta[.,
1981 ; Skern et al., 1985; Duechler et al., 1987; Rossmann et aL, 1985). In all these viruses the
sequence Q G predominates as the major recognition site for the virus protease protein 3C,
which performs most of the cleavages. At all junctions in H RV-1 B, predicted by alignment with
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Human rhinovirus 1B
55
the sequences of other viruses, there is a corresponding QG except for the VP3/VP1 (QN as in
HRV-2) and P2-B/P2-C (probably ES) boundaries. Thus QG is probably the preferred cleavage
site for the HRV- 1B 3C protease. The site for the second protease, P2-A, is less clear due to poor
homology between the rhinoviruses in the region of the boundary (VP1/P2-A). Assuming that
the HRV-1B VP 1 protein is of similar length to that of HRV-14 (289 amino acids) this cleavage
should occur somewhere around position 3380 in the primary structure. The sequence YG, the
recognition sequence for the P2-A protease of the polioviruses and HRV-14, is absent from this
region and thus a different signal must be utilized. We suggest that the VG sequence marked on
Fig. 1 is used but this needs to be confirmed by direct protein sequence analysis.
Recently, the nucleotide sequence of the P3-C protease region of HRV-1A has been published
and this gives the possibility of a preliminary comparison (Werner et al., 1986). The two viruses
are remarkably homologous in this region, 89.6~ and 96.7~ at the nucleotide and amino acid
levels respectively. This is much greater homology than that seen between any other pair of
rhinoviruses and it may suggest that HRV-1A and HRV-1B are correctly regarded as subtypes
rather than distinct serotypes.
Comparison with enteroviruses
Our previous results on HRV-14 indicated that the rhinovirus and enterovirus genera are
closely related; indeed, throughout much of the genome, HRV-14 was found to be more
homologous to the polioviruses (enteroviruses) than to HRV-2 (Stanway et al., 1984a; Skern et
al., 1985). This prompted us to suggest that rhinoviruses and enteroviruses can be considered as
one genus of the family Picornaviridae rather than being divided as at present (Stanway et al.,
1984a). The present finding of tighter grouping within the rhinovirus genus tends to weaken our
argtiment about the amalgamation of the enteroviruses and rhinoviruses. It is also interesting
that as more information becomes available, distinct rhinovirus characteristics are emerging.
For instance, if codon usage is analysed, it can be seen that in the first two positions of the triplet,
the occurrence of each nucleotide follows more or less the same pattern between the
enteroviruses and rhinoviruses (Table 2). However, there is a marked difference in the third
base position, where A, G, C and T occur about equally in the enteroviruses but where there is a
preponderance of A + T in the rhinoviruses. This effect is particularly striking in HRV-1B and
HRV-89 where 73.3~ and 72-4~ respectively of the codons have an A or T in the third position.
Considering only the codons where the amino acid is specified by the first two nucleotides (i.e.
any nucleotide can be in the third position without changing the amino acid) the figure for A + T
in this position rises to over 80~. We currently believe that this A and T preference is a result of
the low optimum temperature for growth of rhinoviruses (33 °C) and that it may be related to the
weakness of A - T base pairs compared with G-C. Base pairing is marginally more stable at
33 °C than at the optimum temperature for enteroviruses (37 °C) and if this adversely affects
some stage of viral replication a drift to a higher A + T content would be advantageous. It may
be envisaged that this could be important during at least two stages of the replicative cycle.
Firstly, during RNA replication weaker interactions between the nascent and template strands
may facilitate strand displacement and lead to enhanced replication. Secondly, during
translation of the long open reading frame the high A + T content could lead to a reduction of
fortuitous secondary structure which may otherwise inhibit translation.
The non-coding regions
As with other picornaviruses, the 5' non-coding region is one of the most interesting regions of
the HRV-1B genome. This region is highly conserved between related viruses and is the most
homologous part of the genome when rhinoviruses and enteroviruses are compared (Stanway
et al., 1984a; Skern et al., 1985). One important observation which has implications for
classification is the fact that this region is of very similar length (about 620 nucleotides) in the
three rhinoviruses sequenced to date but is much shorter than that of the enteroviruses (about
740 nucleotides; Toyoda et al., 1984; Jenkins et al., 1987). This difference in length is due to a
relative deletion or insertion immediately prior to the A U G that initiates the long open reading
frame. The HRV-1B 5' non-coding region is 622 nucleotides in length, a length similar to that of
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56
P. J. H U G H E S AND O T H E R S
HRV-IB
TTATGGTGACAATATATAcata-gaTATATATTGGCATC ATG
HRV-2
TTATGGTGACAATATATAc--aataTATATATTGGCATC ATG
HRV-85
TTATGGTGACAATATATAtacaacaTATATATTGGCAcC
HRV-89
TTATGGTGACAATtTATA . . . . gtgTATAgATTGtCATC ATG
HRV-14
TTATGGTcACAgcATATAtatac---ATATAcTGtgATC ATG
ATG
Fig. 3. The nucleotide sequence preceding the initiation codon in several rhinoviruses. Conserved
nucleotides are shown in upper case,
the other rhinoviruses. This suggests that the length of this region of the genome may be
approximately the same in all rhinoviruses but it is not clear how this could contribute to the
characteristic properties of the genus.
A further interesting feature is that prior to the initiation codon there is a characteristic
A + T-rich region in HRV-14, HRV-2 and HRV-89 which although not exactly conserved
between the serotypes, nevertheless shows great similarity (Fig. 3). At the corresponding point
of HRV-1B there is a sequence very similar to that found in HRV-2 and such a sequence is also
found in HRV-85 (unpublished observations). In view of its location this sequence may play
some role in initiation of translation of the rhinovirus long open reading frame.
An analysis of the relationship between HRV-1B and HRV-2, HRV-14 and HRV-89
throughout the 5' non-coding region demonstrates that in this region, as in the rest of the
genome, HRV-1B is most closely related to HRV-2 and to HRV-89. The overall homology is
83-1~ and 80.5~ respectively. Homology is found throughout the 5' non-coding region but
certain blocks of conservation are particularly evident. Although the overall homology to HRV14 is less (69.1 ~ ) there is striking conservation of two of these blocks (found around positions
180 and 440 to 560). These sequences, which are also seen in the enteroviruses, must play some
central role in the virus replicative cycle but this role, and indeed the function of the whole
region, has not yet been defined. Interestingly, in poliovirus type 3 a single mutation in the
vicinity of one of the blocks (position 472) has been shown to exert a marked effect on the
virulence of different strains (Evans et al., 1985). Most of the differences between the four
rhinovirus serotypes in the 5' non-coding region are base substitutions but at position 98 in
HRV-1B there is some degree of length variability since HRV-14 has an insertion of 13 to 17
nucleotides relative to the other three serotypes. This again suggests that HRV-14 is more
distantly related to the other serotypes.
In confirmation of the importance of the non-coding regions, the 3' non-coding region of
HRV-1B also exhibits rhinovirus-specific characteristics. Whereas in enteroviruses the region is
about 70 to 120 nucleotides long, the four sequenced rhinoviruses have much shorter non-coding
regions, about 40 nucleotides long. There is also some nucleotide homology between all the
rhinoviruses and this is considerable between HRV-IB, HRV-2 and HRV-89. For instance the
last 17 nucleotides before the poly(A) tract are identical between HRV-2 and HRV-89 and there
is a stretch of 13 identical nucleotides towards the start of the 3' non-coding region between
HRV-1B and HRV-2. There is essentially no homology with the enteroviruses.
The virion surface
The homology to previously sequenced viruses enables the location of features such as sites
involved in virus neutralization. For HRV-14 several such sites have been identified and termed
NIm-IA, NIm-IB, N I m - I I and N I m - I I I (Sherry et al., 1986). When HRV-1B is compared with
the other rhinoviruses it is found that these regions are highly diverse (Fig. 2). This is true even
when the comparison is with HRV-2 and HRV-89 where there is otherwise a high degree of
homology. By aligning the sequence with that of HRV-14 it is clear that the diverse regions are in
the exterior loops of the capsid proteins and outside the fl-barrel core structures (data not
shown). Most of the diversity is due to amino acid substitution but there are also several small
deletions and insertions. In view of these characteristics it is likely that these areas of HRV-I B
are recognized by neutralizing antibodies.
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Human rhinovirus 1B
57
Another interesting aspect of the capsid sequence is the conservation of the N terminus of
VP1 between HRV-1B, HRV-2 and HRV-89. This part of VP1 has not been shown to induce
neutralizing antibodies, indeed it is believed to be buried within the capsid. However, between
the three serotypes of poliovirus there is considerable diversity and such diversity is also seen
between HRV-14 and HRV-2. In contrast only one amino acid of the first 20 is different between
HRV-1B, HRV-2 and HRV-89. The reason for this pattern of homology is not clear.
The determination of the three-dimensional structures of HRVol4 and poliovirus type 1
(Rossmann et al., 1985; Hogle et al., 1985) have important implications for an understanding of
virus assembly, uncoating and interaction with the host cell, which are attractive targets for
novel anti-rhinoviral agents. One obvious target is the initial interaction between the cell
receptor and the receptor-binding site-on the virus surface. The identification of the receptorbinding domain could lead to the development of agents which block this interaction and thus
act therapeutically against rhinovirus infections. Deep 'canyon' structures on the surface of
HRV- 14 have been postulated as the receptor-binding sites of this virus (Rossmann et al., 1985;
Smith et al., 1986). It might be expected that members of the same receptor group would show
some similarity of primary structure in the areas which make up the receptor-binding domain.
We have compared the predicted amino acid sequences of HRV-1B, HRV-2, HRV-14 and
HRV-89 in the regions of the genome which make up the 'canyon' but the results are somewhat
equivocal (data not shown). Even between HRV-1B and HRV-2, members of the same group,
there are considerable differences in these regions, although these are not as great as when each
serotype is compared with HRV-14. This may be just a reflection of the close overall similarity
between HRV-IB and HRV-2. An exception is the C terminus of VP3, which partially lines the
'canyon'. Here HRV-1B and HRV-2 are closely related and are very diverse from HRV-14.
However, the homology to HRV-89 is also quite high in this region of VP3. The definitive
assessment of the role of this surface feature in receptor binding, however, must await further
sequence comparisons and the direct manipulation of the region via an infectious cDNA copy of
the genome. These experiments are currently in progress.
In conclusion, the complete nucleotide sequence of HRV-1B presented here is important for
several reasons. It gives a further indication of the relationship between rhinovirus serotypes
and confirms that some share more extensive homology than previously thought. It emphasizes
the importance of the 5' non-coding region and it strengthens the conclusion that there are
certain rhinovirus-specific features such as the length of the 5' and 3' non-coding regions and
codon usage. We are currently exploring the role of the 5' non-coding region and whether these
rhinovirus-specific features determine the characteristic properties of the viruses.
This work was supported by the Medical Research Council, grant numbers G8324256CB and G8608957CA.
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