Viral Oncogenesis Group, Institute for Animal Health, Compton, Berkshire RG20 7NN, UK1
Author for correspondence: K. Venugopal. Fax +44 1635 577237. e-mail venu.gopal{at}bbsrc.ac.uk
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Abstract |
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Introduction |
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The genome structure of ALV-J resembles that of other subgroups, with the gag and pol genes showing over 96% sequence identity and the LTR showing over 90% sequence identity with the equivalents in subgroups AE (Bai et al., 1995 ; Benson et al., 1998
). However, the sequence of the ALV-J env gene shows a much greater similarity to that of a closely related group of novel chicken endogenous retroviral elements designated EAV-HP (Sacco et al., 2000
; Smith et al., 1999
), suggesting that env was acquired by recombination (Venugopal, 1999
) and raising the possibility that the myeloid lineage-specific oncogenicity of ALV-J could mainly be related to determinants within env.
The envelope glycoprotein of ALV, as in other retroviruses, functions mainly as a ligand for receptor binding for virus entry into the susceptible cell (Weiss, 1992 ). Subgroup-determining sequences in the ALV-A env gene have a significant influence on the lymphomagenic potential (Brown & Robinson, 1988
). The ALV-A receptor, encoded by the tva susceptibility allele, contains sequences related to the ligand-binding region of the low-density lipoprotein receptor (Bates et al., 1993
). The variation in the distribution of the tva susceptibility allele accounts for the differences in the susceptibility to ALV-A infection among chicken populations. The receptors for the ALV subgroups B, D and E, members of the tumour-necrosis factor receptor family (Adkins et al., 2001
), are also not expressed in all lines of chickens. However, unlike the other ALV subgroups, all lines of chickens are susceptible to infection by ALV-J, suggesting that the putative ALV-J receptor, yet to be characterized, would be distinct in terms of its distribution among chicken lines.
It is not known whether the lineage-specific oncogenicity of different ALV subgroups is associated with interactions with the receptor and virus entry into the corresponding cell types or with post-entry events. In order to examine the role of the env gene in the induction of ML by ALV-J, we have made reciprocal chimeric viral constructs by substituting the env regions between HPRS-103 and the replication-competent ALV-A vector RCAS (Hughes et al., 1987 ) to create HPRS-103(A) and RCAS(J). The oncogenicity of these two chimeric viruses was evaluated in line 0 chickens, which are highly susceptible to ML induction (Payne et al., 1992a
) and line 15I chickens, which are selected for susceptibility to LL induction (reviewed by Bacon et al., 2000
). Examination of the oncogenicity of HPRS-103(A) is particularly significant because of a recent report of the isolation of three such chimeric ALV-J viruses from alv6 transgenic chicken cells (Crittenden & Salter, 1992
) naturally infected with ALV-J strains (Lupiani et al., 2000
).
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Methods |
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Cell culture, virus propagation and virological assays.
The propagation of the chimeric viruses was initiated by transfection of 12 µg of plasmid DNA into primary chicken embryo fibroblasts (CEF) (Payne et al., 1992b ) derived from 10-day-old, line 0 (C/E) embryos (Astrin et al., 1979
). Production of virus in the transfected cells was monitored by assaying culture supernatants for ALV p27 using ELISA (Smith et al., 1979
). Microneutralization tests (Fadly & Witter, 1998
) for typing the virus stocks and detecting specific antibodies in the serum were carried out using a single dilution of 10 µl of heat-inactivated serum sample and 100 tissue culture infectious units (TCIU) of the virus.
Infection of chickens.
Specific-pathogen-free (SPF) line 0 and line 15I chickens were infected as 11-day-old embryos by intravenous inoculation via a chorioallantoic vein with 50 µl of virus stock containing 5x103 TCIU of HPRS-103(A) or 5 TCIU of RCAS(J) viruses. Lower doses of RCAS(J) virus were used, since higher-titre virus stocks could not be generated, in spite of repeated transfection attempts with the DNA constructs. Control birds belonging to the uninfected group were injected with tissue culture medium from CEF. As the oncogenic characteristics of both subgroup A and subgroup J ALV, in terms of their lymphoid and myeloid leukaemogenicity, respectively, are well documented through several previous studies (Payne et al., 1991 ; 1992a
; Purchase et al., 1977
), we did not include control groups of birds infected with these viruses to reduce the unnecessary usage of animals. Virus-infected and control chicks of each line were incubated, hatched and reared in separate incubators and rooms in the experimental animal house. In addition, 1-day-old chicks from both lines were also infected by intraperitoneal inoculation of 200 µl of the above two virus stocks, and blood samples were collected 12 weeks later to obtain specific antiserum against the virus. All experiments were carried out in accordance with UK Home Office Guidelines, and birds that showed signs of disease were euthanized, autopsied and examined for gross and microscopic lesions. Experiments were terminated at 150 days post-infection (p.i.) for birds infected with HPRS-103(A) and 220 days p.i. for birds infected with RCAS(J). All birds from the infected and control groups were examined for lesions.
DNA extraction, Southern blotting and hybridization.
Frozen tumour tissue was collected during post-mortem examination from five birds from the HPRS-103(A) virus-infected group, four (nos 1261, 1262, 1285 and 1302) diagnosed as LL and one (no. 1303) diagnosed as EB, and from two birds from the RCAS(J) virus-infected group (nos 1335 and 1346) diagnosed as ML by histopathological examination. Birds 1261, 1262, 1335 and 1346 belonged to line 0 and birds 1285, 1302 and 1303 belonged to line 15I. High molecular mass DNA samples extracted from these tumours were used for Southern blot hybridization and PCR, using methods described previously (Chesters et al., 2001 ). For Southern blotting, 10 µg samples digested with EcoRI and BamHI were separated by agarose gel electrophoresis, transferred to nylon membranes and hybridized with a 32P-labelled probe derived from c-myc (kindly provided by Dr Don Ewert, Wistar Institute, Philadelphia, USA) or c-myb (Klempnauer et al., 1982
) DNA.
PCR amplification.
Two types of PCR tests were carried out on tumour DNA samples (2 µg) using a long template PCR kit (Roche Molecular Biochemicals). The first test, using sense primers that annealed to the ALV-A env sequences at positions between nt 5420 and 5776 of RCAS and antisense primers that annealed to HPRS-103 sequences at positions between nt 7386 and 7438, specifically detected the chimeric HPRS-103(A) DNA. The second PCR test was designed to distinguish between the two chimeric and parent viruses based on the ClaI restriction profile of the PCR products, which gives bands of sizes 2 kb and 0·4 kb for RCAS, 2 kb and 0·5 kb for HPRS-103(A), 1·4 kb and 1·1 kb for HPRS-103 and 1·4 kb, 0·6 kb and 0·4 kb for RCAS(J). In this PCR, the upstream primer annealed at nt 52585277 or 50335052, and the downstream primer annealed at nt 77227749 or 73747401 in HPRS-103 and RCAS, respectively. The amplifications were performed as follows: one cycle at 94 °C for 5 min, 14 cycles consisting of 94 °C for 10 s, 60 °C (-1 °C/cycle) for 30 s and 68 °C for 4 min, followed by 30 cycles of 94 °C for 10 s, 48 °C for 30 s and 68 °C for 4 min, and one cycle of 68 °C for 6 min. The second-round PCR was performed under the same conditions using 10 µl of a 1:500 dilution of the first-round product. Amplification of the proviral/c-myc or c-erbB junction sequences from the tumour DNA was carried out using a nested PCR with primers that annealed to the LTR U5 region and exon 2 of c-myc or exon 15 of c-erbB, as described previously (Gong et al., 1998 ).
DNA sequencing.
PCR products representing the c-myc and c-erbB integration junctions were agarose gel-purified and sequenced, either directly or after cloning into the pGEM-T vector (Promega), using insert-or vector-specific primers. The sequences were analysed using the Genetics Computer Group version 10 software.
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Results |
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Oncogenicity of HPRS-103(A) and RCAS(J) viruses
Chimeric HPRS-103(A) virus was highly oncogenic for both lines of birds, as shown by its ability to induce tumours in a large proportion of birds during the 150-day experimental period. Line 0 birds were more susceptible to the induction of tumours by the HPRS-103(A) virus than line 15I birds, as 24/32 (75%) of the former developed tumours detected by gross or histological examination. In comparison, only 13/32 (40·6%) line 15I chickens infected in a similar manner developed tumours during the same period. Chimeric RCAS(J) virus was not highly oncogenic, and even after 220 days, only 3/31 (9·7%) line 0 chickens were shown to have any type of tumour by gross or histological examination. None of the line 15I chickens infected with RCAS(J) virus and none of the birds from the control group developed tumours detectable by gross or histological examination.
The tumours were classified into LL, EB or ML, based on the typical gross characteristics of the tumour, tumour tissue distribution and the histological phenotype of the transformed cells. Most of the LL tumours included involvement of the bursa of Fabricius, where the tumour appeared to be localized to single [Fig. 2a(1)] or multiple lymphoid follicles. Histologically, the tumour cells appeared to be lymphoblastic and mainly showed extravascular infiltration [Fig. 2a
(2)] in the affected tissues. The two RCAS(J) virus-induced ML tumours showed massive infiltration of the characteristic myelocytes [Fig. 2a
(3)] in the various organs. The EB tumours could be differentiated by the morphology of the erythroblasts and the typical intravascular accumulation in the sinusoidal spaces of the liver [Fig. 2a
(4)]. In line 0 birds infected with HPRS-103(A) virus, 87·5% of the tumours were LL, while the remaining 12·5% were EB. In a single case in line 0 birds, the tumours consisted of both lymphoid and myeloid cells. In line 15I chickens, 53·8% of the tumours were diagnosed as LL, while 46·2% consisted of EB tumours. The time of detection varied between the types of tumours and the lines of birds (Fig. 2b
). The shortest time to detection of LL in line 0 was 71 days with a mean period of 101·2 days. In line 15I birds, the first case of LL was seen 117 days after infection, with a mean period of 141·6 days. The onset of EB in both lines of birds was faster with a mean latency period of 82·7 days in line 0 and 84·7 days in line 15I. RCAS(J) virus induced tumours of slow onset, both ML tumours being detected 200 days after infection and the single case of EB 126 days after infection (Fig. 2b
).
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Discussion |
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The reasons for the lower oncogenicity of RCAS(J) are thought to be directly related to the poor replication, since the virus titres obtained from CEF transfected with the RCAS(J) construct were at least 103-fold less than those of the parent viruses and the HPRS-103(A) chimeric virus. This was further demonstrated by the negative antibody response in all the birds infected with the virus after hatching, as well as the small proportion of birds developing viraemia after embryonic infection. However, it has been shown that viruses of subgroup A are the most consistently pathogenic, even at relatively low doses (Purchase et al., 1977 ). Furthermore, a virus dose response has been demonstrated, with high virus dosage inducing death by haemorrhages, erythroblastosis and sarcomas, and low virus doses inducing lymphoid leukosis (Payne et al., 1968
). Thus, the shift to a myeloid tropism by RCAS(J) most probably cannot be ascribed to the low infectious dose. The induction of ML in two line 0 birds and the demonstration of virus integration in the c-myc locus confirmed that the ALV-J env region is the major determinant for the myeloid lineage-specific oncogenicity.
Cell lineage-specific oncogenicity of ALV is also dependent on host genetic factors. We have previously demonstrated that while strain HPRS-103 can infect several inbred and commercial lines of chickens, a greatly variable incidence of ML was shown among chicken lines. Commercial meat-type lines of chickens and, among the inbred lines, line 0 chickens were the most susceptible to the development of ML by HPRS-103, with the incidence in line 0 birds often close to that seen in some of the meat-type lines (Arshad et al., 1997 ; Payne et al., 1992a
). On the other hand, line 15I, selected for increased susceptibility to the induction of LL (reviewed by Bacon et al., 2000
), showed increased resistance to the induction of ML tumours (Payne et al., 1992a
). Line 0 birds were highly susceptible to the induction of LL by chimeric virus (similar to the high incidence of ML induced by HPRS-103 virus) with 75% of the infected birds developing tumours, compared with only 40·6% in line 15I birds. The lower incidence of LL induced by the chimeric virus in line 15I also correlates with their increased resistance to the induction of ML by HPRS-103 (Payne et al., 1992a
). These differences in tumour susceptibility between inbred lines are thought to be related to the differences in the LTR between ALV-A and ALV-J viruses, which could affect the interaction with various enhancer binding factors (Ruddell et al., 1989
). The results from this present study indicate that the two major determinants of oncogenicity, the env gene and the LTR (Brown et al., 1988), act in concert as the factors associated with lineage specificity and tumour induction, respectively.
The incidence of LL and EB induced by HPRS-103(A) virus varied between the two lines. In line 0 birds, the incidence of these tumours was 87·5% and 12·5%, respectively, compared with 53·8% and 46·2% in line 15I. Line 0 birds also appeared to be more susceptible than line 15I, based on the shorter time for the detection of LL (Fig. 2b). The slower progression of LL tumours in line 15I could be a reflection of genetic resistance to the induction of tumours by the chimeric virus. Genetic resistance to tumour development is manifested as a failure of the cells with c-myc integration to hyperproliferate in the bursal environment (Bird et al., 1999
). The slow expansion of cells with proviral c-myc genes has also been attributed to the delay of onset of tumours in line 6, another line genetically resistant to the development of ALV tumours (Bacon et al., 2000
). However, as line 15I is highly susceptible to the induction of LL by RAV-1 (Bacon et al., 2000
), the degree of resistance to the induction of LL by the chimeric virus is likely to be related to determinants in the HPRS-103 LTR and the interaction with cell type-specific transcription factors (Curristin et al., 1997
).
Induction of LL or EB tumours by ALV-A strains such as RAV-1 occurs by integration of the virus in the c-myc (Hayward et al., 1981 ) or c-erbB (Fung et al., 1983
) loci, respectively. Induction of ML by ALV-J strains also occurs through the involvement of the c-myc oncogene (Chesters et al., 2001
). Using a PCR assay (Gong et al., 1998
), we showed here that LL and ML induced by the chimeric HPRS-103(A) and RCAS(J) viruses, respectively, also resulted from proviral integration within the c-myc locus. Similarly, we also showed that the c-erbB locus was involved in the induction of EB by the chimeric HPRS-103(A) virus. These results indicate that activation of oncogenes is a common pathway of induction of tumours, regardless of cell type or virus subgroup. However, one of the major differences between LL tumours induced by ALV-A or ALV-B and ML tumours induced by ALV-J is the high frequency of generation by ALV-J of acutely transforming viruses with transduction of v-myc (Chesters et al., 2001
; Payne et al., 1993
). High levels of transduction of erbB sequences have also been reported in EB tumours induced by activation of c-erbB (Miles & Robinson, 1985
). We were not able to isolate any acutely transforming viruses from any of the tumours induced by the chimeric viruses. However, sequencing PCR products from the EB tumour (1303) and one of the ML tumours (1335) showed that the 5' LTR and part of the gag gene had fused with the c-erbB and c-myc sequences, respectively. These structures resemble those of acutely transforming viruses with transduced oncogene sequences. Compared with this, sequencing PCR products from all the four LL tumours showed 3' LTR integration in c-myc intron 1, a feature of insertional activation (Kung & Liu, 1997
). The reasons for the changes in frequency of transduction of c-myc between LL and ML tumours are not clear. However, it is possible that the interaction between the LTR and cell-type specific factors in myeloid and lymphoid cells could influence the frequency of the events involved in the transduction of v-myc and the generation of acutely transforming viruses.
Although the chimeric viruses described here were derived by molecular manipulation of the HPRS-103 and RCAS proviral clones, three chimeric viruses that probably arose naturally by recombination between exogenous subgroup J virus and a recombinant-defective endogenous virus with subgroup A env gene have recently been described (Lupiani et al., 2000 ). Although the tropism and oncogenicity of these viruses have yet to be described, the data from our studies suggest that they are likely to cause LL or EB rather than ML tumours.
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Acknowledgments |
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Footnotes |
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References |
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Received 3 December 2001;
accepted 4 June 2002.