Identification Of The Oncogene In The Genome Of Defective Avian Leukosis Virus Subgroup J Causing Acute Fibrosarcomas

Avian Leukosis/Sarcoma Viruses (AL/SVs) had been divided into non-acute transformingvirus and acute transforming virus in accordance with different oncogenesis mechanisms.Non-acute transforming virus caused tumors after a long latent period of over90days byinsertional activation of cellular proto-oncogene, while acute transforming virus could inducea fast tumorigenesis within several weeks through direct transcription of viral oncogene(Fadly and Nair,2008).The HPRS-103strain, prototype of ALV-J, has a genome without any viral oncogene,neither cause tumors rapidly in vivo and nor transform myeloid cell in vitro (Payne et al.,1992). However, acutely transforming viruses with v-myc oncogene had been isolated fromcases of myelocytomatosis induced experimentally by HPRS-103(Payne et al.,1993), mostof them can transform cultured chicken bone marrow cells in vitro and induce a variety ofacute tumors including myelocytomatosis, nephroblastoma, renal adenomas/adenocarcinomasin chickens. Lately, acutely transforming ALV-J strains with v-myc have also been isolatedfrom naturally infected parent flocks (Venugopal et al.,2000), although they mainly inducederythroblastosis as well as myelocytomatosis.Fujinami Sarcoma Virus (FSV) was discovered in Japan (Fujinami and Inamoto,1914).Previous studies have shown that it has a genome with v-fps oncogene which is different fromv-src in Rous Sarcoma Virus (RSV), although both of them can cause acute fibrosarcomas inchickens (Shibuya and Hanafusa,1982). PRCII is a naturally obtained5′-fps deletion of1020nucleotides mutant, showed low efficiency of transforming activity on cells and decreasedcarcinogenicity in chickens (Carlberg et al.,1984; Huang et al.,1984).Over the past few years, we have observed the occurrence of lesions indicative offibrosarcoma in tissues from parent layers and hybrid chickens (cross-breeding between commercial female layers and white meat-type male) in China. Researchers in our lab havemade a definite diagnosis of acute fibrosarcoma in these chickens, which is characterized byrapid growth and infiltration into surrounding tissues and metastasis, and demonstrated that itis associated with the infection of ALV-J (Liu et al.,2011; Li et al.,2012). The same acutefibrosarcoma occurred at the9thday after the filtered supernatant of sarcomas and the1stpassage DF1infected by sarcoma supernatant. So we suspect that there exists some certaindefective virus with viral oncogene in the fibrosarcoma.This study preliminarily explored the biological activity of defective virus in vitro CEFcell culture; extracted the viral RNA and proviral DNA, then usd the PCR method to screenthe relevant v-oncogene succesfully; prepared artificial protein encoded by v-fps and specificserum against Fps protein, established the indirect immunofluorescence assay for detection ofviral antigens in infected cells and demonstrated the existence of oncoprotein; constructed4recombinant plasmid carrying v-fps, then injected them into SPF chicken, the similar tumorwas observed. We provided the evidence to prove that the defective viruses Fu-Js were thereal reason to cause acute fibrosarcomas from two perspects of gene and protein.In order to observe the productivity of defective virus cultured in vitro CEF, tumorsupernatant and the1st,3rdpassage of CEF cell infected by it were inoculated respectively into10SPF chickens. The results indicated that the1stpassage of CEF had the same strongtumorigenicity as the tumor supernatant, while the3rdpassage of CEF had a remarkabledecreased tumorigenic ability. It suggested that progeny virus particles may gradually reducedin the process of vitro culture. Combined with the absence of CPE, we inferred that this typeof defective virus is likely not suited to grow in CEFs.PCR amplified the whole genome sequence of the replication-competent ALV-J (SD1005),alignment of the complete sequence demonstrated that it has no remarkable mutation in thegenome and appears not to be the real pathogen inducing the acute tumorigensis. Additionally,we obtained several genome sequences associated with ALV-J (Fu-J1~5), which is defective,with parts of gag, pol and env genes replaced by a sequence of fps gene. The sequences ofFu-Js revealed that their genomes are closely related to that of SD1005(Wu et al.,2010), and probably derived from it in accordance with the high homology (＞99%) in env gene betweenthem. Notably, compared with homologous region of Fujinami and PRCII, the obtained v-fpsgenes in our study have two deletion mutations at both ends, with the major diversity at the3′-fps end especially.Anti-Fps polyclonal antiserum was prepared by standard methods by references. Aprokaryotic expression vector containing v-fps oncogene was constructed and inverted intoEscherichia coli (E. coli) expression system. Balb/c mouse were immunized with purifiedproteins expressed from Escherichia coli (E. coli) and antiserum was monitored by ELISAmethod. These antisera was shown to be special, sensitive and useful in detection tests (datasin press). CEF cells inoculated with tumor supernatant were detected by the indirectfluorescence assay (IFA) with anti-Fps polyclonal antisera. The result was positive, implyingthat natural Fps oncoprotein indeed existed in the infected cells.Plasmid pTZ57-Fu-J1and pTZ57-Fu-J3were obtaind successfully by SOE-PCR andrestriction endonuclease technology. Reference to Fujinami genome, pTZ57-Fu-J1E andpTZ57-Fu-J1M were acquired by the restruction of pTZ57-Fu-J1by the addition of219nt at3′-fps end. These four plasmids were transfected into CEFs, and detected by IFA with theanti-Fps serum as the first antibody. The positive cell can be observed, suggesting thattransient transfection was successful. However, the infectious molecular clone of virus can notbe saved. In the experiment of direct inoculation into SPF chickens of plasmid DNA, sarcomacan be detected in one chicken at the19thdays after inoculation, despite of the low incidenceratio, but it had already demonstrated that the constructed plasmid has the ability to inducetumors to some extent.So we infer that it seems to be unstable for junction site in the process of recombinationbetween c-fps oncogene and retrovirus ALV, a majority of variable individuals at gene level(quasispecies) will generate, one strain of which has biological activity at least. Obviously, theinduction of acute fibrosarcomas in our experiment occurs through mechanisms involving thedirect activation of viral oncogene v-fps in Fu-Js rather than insertional activation of cellularoncogene c-fps, revealing that the tumorigenesis induced by ALV-J is probable to involve fps oncogene besides for the myc oncogene (Chesters et al.,2000). This is the first report on acutefibrosarcomas caused by ALV-J containing v-fps oncogene since the identification of thisvirus in1988.