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Journal of Virology, December 1998, p. 10301-10304, Vol. 72, No. 12
Department of Biochemistry,
Received 13 July 1998/Accepted 1 September 1998
A new subgroup of avian leukosis virus (ALV) that includes a unique
env gene, designated J, was identified recently in England. Sequence analysis of prototype English isolate HPRS-103 revealed several other unique genetic characteristics of this strain and provided information that it arose by recombination between exogenous and endogenous virus sequences. In the past several years, ALV J type
viruses (ALV-J) have been isolated from broiler breeder flocks in the
United States. We were interested in determining the relationship
between the U.S. and English isolates of ALV-J. Based on sequence data
from two independently derived U.S. field isolates, we conclude that
the U.S. and English isolates of ALV-J derive from a common ancestor
and are not the result of independent recombination events.
Members of the leukosis/sarcoma
group of avian retroviruses are divided into subgroups based on the
identity of their envelope genes. The mature env gene
products are the gp85 surface glycoprotein (SU), which directs receptor
binding, and the gp37 transmembrane protein (TM), which is linked to SU
by disulfide bonds and which anchors the complex to the viral membrane
(reviewed in reference 19). Five major subgroups of
avian retroviruses (A to E), which differ in host range, viral
interference, and cross-neutralization, properties that are determined
by the portion of env encoding gp85 have been identified
(7, 8, 13). The envelope genes for subgroups A to D are
found in exogenous viruses, while the E subgroup is encoded by the
env gene of the ev family of endogenous proviruses (3,
6, 12, 17, 18). The gp85 proteins of subgroup A to E viruses are
approximately 85% identical to each other; subgroup-determining
regions map to discrete variable and hypervariable regions of SU
(7, 8, 14, 33).
Several years ago, a number of nonacute avian leukosis viruses were
identified in England; these viruses exhibited a novel subgroup
specificity, designated J, that differed from those of previously
characterized avian virus subgroups A to E based on patterns of viral
interference, cross-neutralization, and host range (2, 4, 5,
22-26, 35). These viruses were originally identified based on
their ability to induce myelocytic myeloid leukosis (2, 23, 26,
27). Sequence analyses of several type J avian leukosis virus
(ALV-J) isolates have demonstrated that the subgroup J gp85 genes show
only 40% overall identity to the gp85 genes of subgroup A to E viruses
(2, 23, 26, 27). In particular, the amino-terminal 43 amino
acids and a region between residues 251 and 289 (Fig. 1) of the
subgroup J gp85 protein each show a high degree of identity (84%) to
subgroup A to E gp85 proteins; the remainder of the subgroup J protein shows no significant homology to those of the other subgroups. In
addition, while the ALV-J SU proteins are over 90% identical to each
other, they exhibit localized regions of sequence alterations and also
show antigenic variation (35). Although only weakly related
to the SU proteins of the subgroup A to E viruses, the ALV-J gp85
protein does include several regions between 6 and 21 amino acids in
length that are over 90% identical to the gp85 protein of the ancient
endogenous avian proviruses (EAVs) (4, 5;
unpublished observations). Members of the EAV family of avian
endogenous viruses are distinct from the well-characterized subgroup E
endogenous viruses encoded by the ev loci (10, 11, 13); the
subgroup specificity of the EAVs is unknown since all proviruses of
this family identified to date include a defective env gene
(10, 11, 13). The finding that the HPRS-103 env gene contains sequences related to those of EAVs, together with the
genome structure of the prototype English ALV-J strain, HPRS-103 (see
below), has led to the suggestion that HPRS-103 arose by the
recombination of one or more exogenous viruses with other viruses (or a
single virus), at least one of which was related to the EAVs (4,
5).
Over the last several years, several commercial breeders in the United
States have reported the appearance of myeloid tumors similar to those
induced by ALV-J viruses identified in England (15, 16).
Biological testing (including viral interference assays and serological
screening) of viruses obtained from these tumors indicated that the
U.S. field isolates were subgroup J viruses (15, 16). Since
these viruses had not been detected previously in the United States, we
were interested in investigating the genetic content of the ALV-J U.S.
field isolates to determine whether the U.S. isolates and the English
prototype strain, HPRS-103, appeared to derive from a common ancestor
or if they more likely arose from independent events. To approach this
question, two field isolates of ALV-J, termed ADOL-R5-4 and ADOL-Hc-1
(15, 16), were subjected to partial sequence analysis. For
the ADOL-R5-4 isolate, the sequence was obtained from an infectious
molecular clone generated from infected-cell DNA, while the ADOL-Hc-1
sequence was obtained from cloned DNA.
env gene.
Figure 1
shows the predicted amino acid sequences of the SU portions of the
ADOL-R5-4 and ADOL-Hc-1 env gene products aligned with that
of HPRS-103 (4). Also included is the consensus sequence derived from the 13 sequenced English isolates of ALV-J
(35). These data revealed that the ADOL-R5-4 SU protein
showed 90.9% identity with the SU protein of HPRS-103, differing at
only 28 residues. A comparison with the consensus sequence obtained
from all sequenced English isolates revealed an even higher level of identity (95.1%). Similarly, the ADOL-Hc-1 strain SU proteins showed a
92.2 and 90.3% identity with the SU proteins of HPRS-103 and the
consensus sequence, respectively. The level of variation between the
two U.S. isolates was 11%. These results demonstrate that the
HPRS-103, ADOL-R5-4, and ADOL-Hc-1 SU proteins are highly related and
that the degrees of variation between the two U.S. isolates and between
these isolates and the English isolates are similar to those seen among
the group of English isolates (35). Interestingly, most of
the amino acid substitutions seen in the U.S. field isolates were also
seen in at least one of the English ALV-J isolates sequenced
(35). There were in fact only 4 substitutions in the
ADOL-R5-4 sequence and 11 substitutions in the ADOL-Hc-1 sequence that
were not seen in any of the English isolates; none of these were shared
between the two U.S. isolates. Of note was the finding that the
ADOL-Hc-1 sequence showed a one-amino-acid deletion within
hypervariable region 1 (hr1), a finding also reported for one of the
English isolates (X12) (35). ADOL-Hc-1 was unique among all
isolates sequenced in having a three-amino-acid insertion within the
hr2 region; the significance of these findings are currently being
investigated. Together, these data demonstrate that, while each SU gene
is unique, they are all very highly related and are likely to have
arisen from a common source.
pol gene.
As mentioned above, the HPRS-103 genome
exhibits several features that have not been reported previously for
any other avian retrovirus (Fig. 2A). One
such feature is the presence of a premature stop codon near the end of
the pol gene (4). This mutation is unlikely to
affect either the mature Pol proteins or virus growth. This conclusion
is based on the fact that the codon resides in a region of
pol that in other avian leukosis/sarcoma viruses (ALSVs)
encodes the carboxy-terminal portion of the unprocessed Pol protein;
this portion of Pol is removed during maturation to generate the mature
integrase protein and is therefore not required for Pol or IN function.
It has in fact been demonstrated that this carboxy-terminal tail is not
essential for virus growth, at least in vitro (20). In our
search of the GenBank database (which includes 16 different avian
retrovirus sequences that span this region), we have not found another
example of this mutation, suggesting that it might serve as a
diagnostic for ALV-J and/or the virus(es) that might have given rise to
ALV-J. We therefore sequenced this region from the two U.S. isolates;
the data obtained are shown in Fig. 2B. As shown, the mutation that
gives rise to the premature stop codon is found in both U.S. field
isolates of ALV-J. These data indicate that the presence of the
premature stop codon is not unique to the HPRS-103 strain but instead
is a common feature of all three of the ALV-J strains. This finding, therefore, supports the hypothesis that either each of the ALV-J isolates analyzed traces to a common ALV-J ancestor or that one of the
virus parents that gave rise to all of these isolates contained the
pol gene mutation.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Independent Isolates of the Emerging Subgroup J
Avian Leukosis Virus Derive from a Common Ancestor
ABSTRACT
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FIG. 1.
Comparison of the predicted amino acid sequences
of ALV-J gp85 proteins. Shown are the predicted amino acid sequence of
gp85 from HPRS-103 (4), the consensus sequence of the 13 English ALV-J strains (35), and the sequences of the two
U.S. field isolates ADOL-R5-4 and ADOL-Hc-1. The consensus sequence is
defined as the sequence found in seven or more of the sequenced English
isolates. Dashes indicate identical residues, while letters indicate
amino acid substitutions. The locations of the variable (vr) and
hypervariable (hr) regions are based on alignments with gp85 proteins
of subgroups A to E and do not necessarily reflect variation among the
subgroup J proteins. Underlined residues in the U.S. field isolate
sequences identify amino acids in the U.S. field isolates that,
although not identical to those of the consensus sequence,
are also found in one or more of the English isolates and
therefore do not represent substitutions unique to U.S. strains. Amino
acid substitutions in italics are unique to ADOL-R5-4 (residues 101, 212, 239, and 307) or ADOL-Hc-1 (residues 48, 51, 63, 110, 115, 117, 119, 211, and a three-amino-acid substitution after residue 194).
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FIG. 2.
Sequence comparison of selected regions of the HPRS-103
and U.S. field isolates. (A) Line drawing of the genome of HPRS-103
(see text for a description of each region). An asterisk indicates a
premature stop codon near the end of the pol gene. (B)
Sequence comparison of the 3' ends of the pol genes from the
indicated viruses. The IN processing site is the site in the Pol
polyprotein that is subject to proteolytic processing to generate the
carboxy-terminal end of the mature IN protein. The boxed TAA sequence
is the location of the premature stop codon found in HPRS-103 and the
two U.S. ALV-J field isolates. This sequence has not been found in
other avian retrovirus sequences in the GenBank database (unpublished
observations). The coding sequence for the normal Pol processing site
in Rous sarcoma virus (RSV) is 66 nucleotides downstream from the
premature stop codon shown in this figure. (C) Shown is the junction
between the portion of the ALV-J env gene that encodes the
J-specific gp37 protein and that which encodes the partially duplicated
copy of TM (rTM) that is related to the gp37 from subgroup A to E
viruses.
rTM. A third unusual feature of the HPRS-103 isolate is the presence of a partially duplicated copy of the transmembrane (TM)-encoding portion of the env gene (the redundant TM or rTM). As originally noted by Bai et al. (5), the rTM is over 90% identical to that of the TM-encoding portions of the env genes of ALSVs of the A to E subgroups but shows only approximately 60% identity to that of the HPRS-103 TM-encoding portion of the env gene. It is therefore presumed to have arisen from the exogenous virus parent of HPRS-103 after an illegitimate recombination event with one or more other exogenous or endogenous viruses. As with the pol mutation, the partial duplication of the TM-encoding portion of the env gene found in HPRS-103 has not been described previously in other avian viruses of any subgroup. The retention of the TM-rTM junction, therefore, would be highly suggestive of a common recombinant having given rise to the U.S. and English isolates of ALV-J. Figure 2C shows a sequence alignment of this region from ADOL-R5-4, ADOL-Hc-1, and HPRS-103. As shown, each of the U.S. field isolates shows the same junction sequence as HPRS-103 between the end of the subgroup J gp37/TM-encoding portion of the env gene and the rTM.
In addition to the sequence data shown here, we also have sequenced the 3'-untranslated regions (3'-UTRs) and the long terminal repeats (LTRs) of the two U.S. field isolates and find that they are virtually identical to those of the HPRS-103 strain (data not shown). In particular, both the ADOL-R5-4 and the ADOL-Hc-1 viruses contain 150-bp sequences within their 3'-UTRs, called E elements, that are also found in several strains of Rous sarcoma virus (21, 28, 34) but that have not been reported previously in other strains of ALV. All isolates also contain a direct repeat (dr) sequence in the 3'-UTR. In addition, the two U.S. field isolates, like HPRS-103, contain only one copy of an enhancer motif, called EFII, that is found in two copies in other avian retroviral LTRs (9, 29-32). Together, these data support the hypothesis that all ALV-J viruses analyzed to date arose from a common ancestor that was generated by one or more rare recombination events between exogenous and endogenous virus sequences. We are currently extending these analyses to identify the origin of the ALV-J env gene and have identified novel endogenous virus sequences as the likely source.Nucleotide sequence accession numbers. The env sequences used to generate the predicted amino acid sequences presented here have been submitted to GenBank. The ADOL-R5-4 sequence has been assigned accession no. AF076887, and the ADOL-Hc-1 sequence has been assigned accession no. AF097731.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grant R01-GM41571 from the National Institute of General Medical Sciences and by a grant from the Leukemia Task Force. S.J.B. was supported by training grant CA09138 from the National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Human Genetics, University of Minnesota Medical School, Box 206 FUMC, 515 Delaware St. S.E., Minneapolis, MN 55455. Phone: (612) 626-0445. Fax: (612) 626-7031. E-mail: kathleen{at}lenti.med.umn.edu.
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Journal of Virology, December 1998, p. 10301-10304, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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