Bovine papillomaviral gene expression in equine sarcoid tumours

Virus Research 61 (1999) 171 – 175 www.elsevier.com/locate/virusres

Short communication

Bovine papillomaviral gene expression in equine sarcoid tumours L. Nasir a,*, S.W.J. Reid a,b a

Department Of Veterinary Clinical Studies, Uni6ersity Of Glasgow Veterinary School, Bearsden Road, Glasgow G61 1QH, UK b Department Of Statistics and Modelling, Uni6ersity Of Strathclyde, Richmond Street, Glasgow G1 1XH, UK Received 30 July 1998; received in revised form 22 February 1999; accepted 15 March 1999

Abstract The sarcoid is a benign locally invasive dermal fibroblastic lesion, commonly affecting horses and donkeys. The aetiology of the equine sarcoid is equivocal. Bovine papillomaviral (BPV) DNA (type 1/2) is frequently demonstrable in equine sarcoid tumour biopsies. However, the exact role of the virus in the disease process and its contribution to the phenotypic differences in sarcoids is not known. It was sought to assess the transcriptional activity of BPV-1 found in sarcoid tissues. Of 20 tumours examined, 18 were positive for E2 expression and ten positive for L1 expression. Viral oncogenes E5, E6 and E7 transcripts were detected in 16, nine and 12 tumours, respectively. This study demonstrates BPV gene expression in equine sarcoids and provide the first evidence for a direct involvement of the virus in the pathogenesis of sarcoids. © 1999 Elsevier Science B.V. All rights reserved. Keywords: BPV; Sarcoid; Expression; Oncogene; Equine

Equine sarcoids are locally invasive, benign fibroblastic skin tumours of horses and donkeys (Jackson, 1936; Olson, 1948; Marti et al., 1993). They most commonly affect young geldings and can occur either as single or multiple tumours and are most often detected on the ventral abdomen, limbs and head (Reid et al., 1994). Lesions can occur after trauma or at sites that are predisposed to trauma, and are usually refractory to treatment. Although sarcoids do not metastasize they * Corresponding author.

exhibit variable clinical manifestations ranging from aggressive infiltrative growth to spontaneous regression (Ragland et al., 1970; McConaghy et al., 1994). The aetiology of the equine sarcoid is not clearly understood. The genomes of certain types of bovine papillomavirus (BPV types 1/2) are frequently found in equine sarcoid tumour tissues suggesting a causal relationship between infection and tumour (Amtmann et al., 1980; Angelos et al., 1991; Reid and Smith, 1992; Otten, et al., 1993; Bloch et al., 1994; Nasir et al., 1997). BPVs are small epitheliotropic DNA tumour

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viruses capable of inducing benign and malignant lesions in cattle (Lancaster and Olson, 1982). The genome of the virus is composed of early (E) and late (L) genes which can be functionally divided into several open reading frames: viral replication (E1), regulation of transcription (E2), coding for cytoplasmic proteins (E4), transforming proteins E5, E6, and E7 as the early genes and L1 and L2 coding for capsid proteins as late genes (Shaw and Howley, 1990). In productive infections, early genes are expressed throughout the epithelium, but the two late genes are expressed only in terminally differentiated keratinocytes (Spalholz and Howley, 1989; Barksdale and Baker, 1993). The presence of BPV virion particles in equine sarcoids has yet to be demonstrated, and the disease is considered to be a non-productive infection where viral DNA exists episomally (Lancaster et al., 1977, 1979; Amtmann et al., 1980; Lancaster, 1981). However, the nature of the association is unclear; whether the presence of viral DNA is coincidental or whether viral DNA actively supports the induction of the neoplastic phenotype is unknown. On this basis, the expression of specific BPV-1 viral genes was investigated in equine sarcoid tumours to understand the molecular mechanisms of BPV associated tumourigenesis in the development of the sarcoid. Total RNA was extracted from 20 sarcoid tumours using RNAzol™ B (AMS Biotechnology). All tumours had previously been characterised for the presence of BPV-1 DNA by polymerase chain reaction (PCR) and restriction enzyme analysis. Sarcoid biopsies were taken from 19 donkeys and one horse (sample 2). First strand synthesis reactions were performed using 2 mg of total RNA in a final volume of 25 ml containing 5 ml reverse transcription buffer (5× ), 200 U Moloney murine leukemia virus reverse transcriptase, 10 mM dTT, 250 mM dNTPs, 25 U RNAse inhibitor and 25 mM random primers at 37°C for 30 min followed by a 60 min incubation at 42°C and a final incubation at 95°C for 5 min. Four of the RNA preparations (samples 17 – 20) were reverse transcribed in the presence of 25 mM oligo dT primer following DNase I treatment for 10 min at 37°C. PCR reactions were performed on 2.5 ml cDNA samples in a total volume of 25 ml contain-

ing 0.4 mM of both 3% and 5% oligonucleotides (Table 1), 250 mM dATP, dCTP, dTTP and dGTP, 10 mM Tris–HCl, 1.5 mmol/l MgCl2, 50 mM/l KCl, 0.1 mg/ml gelatin and 2.5 U Dynazyme (Flowgen, UK). Samples were subjected to an initial denaturation at 95°C for 5 min followed by 35 cycles at 96°C for 1 min, 55°C for 1 min and 72°C for 1 min, with a final extension at 72°C for 5 min. Products of amplification were fractionated by gel electrophoresis and visualised by ethidium bromide staining (Fig. 1). The housekeeping gene Abl (abelson tyrosine kinase) was used to check for DNA contamination and successful first strand synthesis. The sensitivity of the PCRs were determined using serial dilutions of purified PCR product. Twenty sarcoid tumours containing BPV-1 DNA were examined for the presence of viral specific E2, E5, E6, E7 and L1 transcripts using reverse transcription coupled PCR. The results Table 1 Oligonucleotide primer sequences Region in BPV-1a

Primer sequence (5%–3% orientation)

L1

tat cgc tat ata gag let cct g

E2

E5

E6

E7

Abl gene (control)

(nts 6853– 6874) (nts 7166– 7187) (nts 2723– 2741) (nts 2953– 2971) (nts 3841– 3858) (nts 3972– 3991) (nts 313– 334) (nts 483– 504) (nts 483– 500) (nts 740– 757)

gac ace gac cat tgc tat tea g age ace cac tgc ttt atg c caa act cca cct cta cca c cac tgc cat tgc ctt ttc gga gca ctc aaa tga tcc c act gaa tta ttg cat ggc aaa a cta tag gta ttg gga cct tga a ttc aag gtc caa ata ccc cac agc aaa agt cag ctc cag cgg cca gta gca let gac tt tat get tat agc cta age ccc gga g

a Nucleotide positions (nts) correspond to the Bovine papillomaviral (BPV)-1 sequence in accession no. X02346.

L. Nasir, S.W.J. Reid / Virus Research 61 (1999) 171–175

Fig. 1. Detection of reverse transcriptase-polymerase chain reaction (RT-PCR) products in tumour sample 18. Lane 1, E2 (248 bp); lane 2, E5 (150 bp); lane 3, E6 (191 bp); lane 4, E7 (274 bp); lane 5, L1 (344 bp); lane 6, Abl (200 bp); lane 7, pBR322/HaeIII marker.

are summarised in Table 2 and an example of the amplification product profile shown in Fig. 1. BPV-E2 transcript amplification products were identified in 18 out of 20 tumours. The BPV-L1 amplification product was identified in ten tumours. The BPV oncogene transcripts E5, E6 and E7 were detected in 16, nine and 12 tumours, respectively. One tumour (sample 15) showed no detectable viral transcripts. The absence of detectable transcripts in some samples may be attributed to low levels of expression undetectable by the RT-PCR assay. The sensitivity of the E2, E6, E7 and L1 PCRs revealed a limit of detection of 0.01 fg equivalent to approximately 40, 52, 37 and 30 viral molecules, respectively. The data showed that BPV viral genes including oncogenes are expressed in the majority of tumours. These data provide evidence for an aetiogical role of the virus in the disease. The transforming potential of BPV has been studied widely in a range of tissue culture systems and transgenic models (Dimaio et al., 1986; Lacey et al., 1986; Neary and Dimaio, 1989). Studies from BPV-1 transgenic mice have demonstrated that oncogene expression is sufficient for the development of benign precancerous lesions, and malignant trans-

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formation requires specific gene alterations within the host genome (Spalholz and Howley, 1989; Lindgren et al., 1989). The oncogenes of BPV-1 are encoded by E5, E6 and E7 open reading frames (ORFs). For BPV-1, E5 has generally been regarded as the major transforming gene relative to E6 and E7. In the present study 90% of tumours examined showed detectable levels of oncogenic transcripts and 80% were positive for E5 expression. The recognised transforming abilities of these genes and the high frequency of transcripts in sarcoid tumours suggest that these viral sequences play an important role in the induction and/or maintenance of cellular proliferation and the neoplastic phenotype associated with sarcoids. The proteins encoded by E6 and E7 of the human papillomaviruses (HPV) have been shown to complex with tumour suppressors p53 and retinoblastoma protein (Rb), respectively (Werness and Levine, 1990; Munger et al., 1989). These proteins play an important role in regulating normal cell growth and division and binding to papillomaviral proteins is thought to result in the loss of these Table 2 Detection of Bovine papillomaviral (BPV)-1 early gene (E2, E5, E6, E7) and late gene (L1) transcripts in sarcoid tumoursa Tumour

E2

E5

E6

E7

L1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

+ + + + + + + + + + + + + + − − + + + +

+ + + + + + + − + + + + − + − − + + + +

− − + + − − + − + + + + − + − − − − − +

+ + − + + + − − − + + + + + − + − − − +

+ − − − − + + + + + − − + + − + − + − −

a

+, RNA transcript detected; −, transcript undetected.

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functions. Although BPV-E6 does not bind human p53 in vitro (Werness and Levine, 1990), BPV-E6 transformed human cells have markedly reduced levels of p53 (Band et al., 1993). This suggests augmented p53 degradation is required for immortalisation and may be involved in the pathogenesis of equine sarcoids. The investigation of p53 in sarcoids has been limited to gene mutational analysis (Bucher et al., 1996; Nasir et al., 1997) and further examination of p53 protein levels following BPV-E6 transfection of equine fibroblasts and epithelial cells is required to test this hypothesis. In the present study, tumours were also examined for BPV-E2. BPV-E2 is recognised as playing an important role in tumourigenesis and encodes proteins that transactivate or repress transcription of other BPV early genes (McBride et al., 1991). Indeed, E5, E6 and E7 transcripts were mainly detected in tumours exhibiting E2 transcripts. Tumour 16, however, showed E7 expression in the absence of E2. This may be due to low levels of E2 expression that were not detected by the PCR assay. Detailed investigation of viral sequences in sarcoid tumours have demonstrated the presence of DNA sequences that differ from BPV-1 published sequences (Trenfield et al., 1985; Otten et al., 1993; Reid et al., 1994). This may also explain the absence of detectable RT-PCR amplification products, since oligonucleotides are based on the BPV-1 sequence. The high percentage of tumour displaying BPV-E2 transcripts may be associated with episomal viral DNA in equine sarcoids, since BPV-E2 is also required for stable extrachromosomal maintenance (Piirsoo et al., 1996). The BPV-1 late gene (L1) encodes the viral capsid protein. Late region mRNAs are expressed only after vegetative viral DNA replication and are restricted to the most differentiated layers (Spalholz and Howley, 1989). Sarcoid cells are thought to be nonpermissive for BPV virion replication and propagation. Thus the detection of BPV-1 L1 transcripts in approximately 50% of sarcoid tumours implicates L1 expression, but this does not confirm the production of L1 protein in equine sarcoids. The presence of viral proteins requires further investigation using viral antibodies.

In summary, the present communication describes the detection of papillomaviral gene transcripts in equine sarcoid tumours and provides strong evidence for a direct role of BPV in the pathogenesis of the sarcoid.

Acknowledgements We thank Professors S. Campo and Max Murray for advice and comments and S.T. McFarlane for technical assistance. This work was supported by The Donkey Sanctuary Registered Charity No. 264818, Sidmouth, Devon.

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