3''-Terminal region of avian carcinoma virus MH2 shares sequence elements with avian sarcoma viruses Y73 and SR-A.

We determined the nucleotide sequence of the acute transforming avian retrovirus MH2 from an HgiAI site within the coding region of its oncogene, v-myc, to the KpnI site within the long terminal repeat. Comparison with published sequences from other retroviruses allowed us to identify all sequence elements in this region. We conclude that MH2 contains a unique assembly of 3'-terminal sequences, which includes part of the helper virus-derived SPC region of avian sarcoma virus Y73 and the complete F3 and F1 segments of Rous sarcoma virus strain SR-A.     

Molecular cloning of the avian acute transforming retrovirus MH2 reveals a novel cell-derived sequence (v-mil) in addition to the myc oncogene

Mill-Hill-2 virus (MH2) proviral DNA was cloned from a transformed non-producer cell culture (MH2QB2) through insertion of randomly cut high mol. wt. cellular DNA in the lambdoid vector L47.1. Restriction analysis of a suitable recombinant phage by Southern DNA blotting and hybridization with different probes allowed us to characterize the genetic organization of the provirus and to identify a novel MH2-specific sequence of at least 1.1kbp. Such a sequence, for which we propose the name v-mil, from MilI-Hill-2 virus, is not homologous to v-myc, the previously described oncogene of MH2, nor to avian leukaemia virus-related sequences. Evidence is presented here that v-mil has a cellular counterpart (c-mil) phylogenetically conserved in birds and mammals, including man, and expressed as a single RNA species at least in some tissues. MH2 virus might thus be regarded, like avian erythroblastosis virus or E26, as another example of retroviruses having recombined with more than one cellular gene.     

Transformed and tumorigenic phenotypes induced by avian retroviruses containing the v-mil oncogene.

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.     

myc and src oncogenes have complementary effects on cell proliferation and expression of specific extracellular matrix components in definitive chondroblasts.

The effects of the avian viral oncogenes src and myc were compared for their ability to alter the differentiated phenotype and the proliferative capacity of definitive chondroblasts. As previously demonstrated, viruses carrying the src oncogene suppressed the synthesis of the chondroblast-specific products, type II collagen and cartilage-specific sulfated proteoglycan. In contrast, infection with MC29 and HB1 viruses, which carry the myc oncogene, did not suppress the synthesis of these normal differentiated cell products, but the infected cells exhibited an increased proliferative potential. The MH2 virus, which carries both the myc and mil oncogenes, both induced the suppression of these chondroblast-specific products and increased cell proliferation. The implications of these results for cooperation between oncogenes and the multi-oncogene models for neoplastic transformation are discussed.     

Retention or loss of v-mil sequences after propagation of MH2 virus in vivo or in vitro.

During propagation of the defective avian retrovirus MH2 in the presence of replication-competent helper virus, deletion of portions of the viral genome occurred frequently. After transformation of quail cells in vitro, v-mil sequences were lost, leading to populations of MH2 viruses which were highly deficient for mil gene expression but which could transform macrophage and fibroblast cells in vitro with high efficiency. In contrast, after induction of tumors in quail with mil-deficient MH2 viral stocks, a majority of the tumor DNAs contained mil+ proviruses, suggesting that there is selection for retention of the v-mil gene in vivo and that the mil protein may play a role in the oncogenicity of MH2 virus. We also isolated MH2-transformed cell lines which contained deleted proviruses arising from packaging and subsequent integration of the subgenomic v-myc-encoding mRNA. Some of these cell lines produced viruses which encoded abnormal v-myc proteins and had altered in vitro transforming properties. These altered phenotypes may be caused by mutations within the v-myc gene.     

Cell proliferation and cooperation of v-mil and v-myc oncogenes

Retroviruses which possess the property to recombine with genetic material from the cell, have cloned and activated some oncogenes and hence are a privileged source for the study of these genes. Cellular oncogene activation can occur following two non mutually exclusive ways: (i) by over-expression of their products; (ii) by modifications of their products through mutations. Retroviruses can combine these two ways of activation leading to the over-expression of a modified product. In this paper, we present results obtained in the study of MH2, a retrovirus containing two oncogenes. We have shown that the two oncogenes of MH2 (v-mil and v-myc) cooperate in vitro to transform neuroretina cells from chicken embryos. These cells which normally do not grow in a defined medium, are induced to proliferate and become transformed upon infection by MH2. Our data enabled us to show that in MH2 v-mil was responsible for the induction of proliferation and v-myc for the transformation of the proliferating cells. Using in vitro constructs we located two regions in the protein encoded by v-mil which are important for its mitogenic property. We have also cloned the cellular counterpart of v-mil and the study of its biological activity on neuroretina cells enabled us to propose a mechanism of activation of the cellular gene by truncation of its 5' part.     

RNA and protein encoded by MH2 virus: evidence for subgenomic expression of v-myc.

MH2 and MC29 are highly related myc-containing avian retroviruses. We found that MH2, unlike MC29, synthesizes a 2.6-kilobase subgenomic mRNA containing myc sequences as well as sequences from the 5' end of the genome. A 57-kilodalton protein containing myc, but not gag, sequences (p57myc) was detected by hybrid selection and in vitro translation of RNA from MH2-transformed cells. Gradient separation of MH2 intracellular RNAs indicated that p57myc is encoded by the subgenomic RNA. A highly oncogenic MH2 virus variant (MH2YS3) (M. Linial, Virology 119:382-391, 1982) was shown to encode only p57myc and not P100, the previously described MH2-encoded polyprotein (Hu et al., Virology, 89:162-178, 1978). Cells transformed by subclones of this virus synthesized predominantly the 2.6-kilobase RNA rather than genomic 5.4-kilobase RNA. These results suggest that only p57myc is required for maintenance of the transformed state after MH2 infection.     

Mapping by in vitro constructs of the P100gag-mil region, accounting for induction of chicken neuroretina cell proliferation.

The v-mil oncogene of the avian retrovirus MH2 is expressed as a fusion protein with viral gag determinants in infected cells. This P100gag-mil protein accounts for the proliferation of chicken embryo neuroretina cells (CNR) induced by MH2 in vitro. We constructed a series of mutants by in-frame deletions in different parts of the gag and mil domains and tested their ability to induce CNR growth. We show that gag sequences, as well as 200-base-pair 5' mil sequences, were not required to induce such a proliferation. However, gag sequences seem to contribute to a full proliferation of growing CNR. In contrast, deletions in the kinase domain abolish this induction. In particular, by deleting only 9 nucleotides localized around the unique SphI site of v-mil, we produced a totally inactive mutant (BalSp). This mutant directs the synthesis of a v-mil protein lacking the dipeptide Tyr-Leu, which is conserved in almost all the members of the large protein kinase family, and a histidine residue highly conserved in Ser-Thr protein kinase members.     

Temperature-sensitive mutants of MH2 avian leukemia virus that map in the v-mil and the v-myc oncogene respectively.

MH2 is an avian retrovirus that contains the v-mil and v-myc oncogenes. In vitro it transforms chick macrophages that are capable of proliferation in the absence of growth factor. Earlier work showed that v-myc induces macrophage transformation and that v-mil induces the production of chicken myelomonocytic growth factor (cMGF), thus generating an autocrine system. We describe the isolation of temperature-sensitive (ts) mutants of MH2 virus. As suggested by marker rescue experiments, one mutant bears a ts lesion in v-mil, whereas the other carries a mutation in v-myc. Ts v-mil MH2-transformed macrophages become factor-dependent at the non-permissive temperature (42 degrees C), while ts-v-myc MH2-transformed macrophages cease growing and acquire a more normal macrophage phenotype at 42 degrees C irrespective of the presence of cMGF. Both phenotypes can be reversed by backshift to the permissive temperature. These results suggest that the gene products of v-mil and v-myc function independently of each other and that v-mil is necessary for the maintenance of autocrine growth, whereas v-myc is required to maintain the transformed phenotype.     

Nucleotide sequence analysis of the BALB/c murine sarcoma virus transforming gene.

We determined the nucleotide sequence of the v-H-ras-related oncogene of BALB/c murine sarcoma virus. This oncogene contains an open reading frame of 189 amino acids that initiates and terminates entirely within the mouse cell-derived ras sequence. The protein encoded by this open reading frame matches the sequence predicted for the T24 human bladder carcinoma oncogene product, p21, in all but two positions. The presence of a lysine residue in position 12 of BALB/c murine sarcoma virus p21 likely accounts for its oncogenic properties.     

Isolation of an MH2 retrovirus mutant temperature sensitive for macrophage but not fibroblast transformation.

A conditional mutant of the MH2 avian retrovirus, termed ts41MH2, was isolated. Unlike wtMH2, ts41MH2 permitted transformed macrophages to differentiate during a 5- to 7-day temperature shift from 37 to 42 degrees C. Mutant-infected cells incubated at 42 degrees C exhibited a flattened morphology and then fused to form giant multinucleated cells that closely resembled normal macrophage maturation in vitro. These differentiated cells reacted strongly with a myeloid-macrophage-specific monoclonal antibody. The process of differentiation was inhibited when ts41MH2-transformed nonproducer clones were superinfected before the temperature shift with the myc gene-containing MC29 or OK10 viruses. By contrast, no inhibition was observed in clones superinfected with the MH2-PA200 virus that contains only the mil gene. The mutant also demonstrated a reduced oncogenic potential relative to that of wtMH2 when it was inoculated intravenously into young birds. However, in contrast to the results obtained with hematopoietic cells, none of the five fibroblast transformation parameters tested for ts41MH2 were altered from those induced by wtMH2. These results suggest that the mutation in ts41MH2 is located in a region of myc required for macrophage transformation, but not required for fibroblast transformation.     

The transforming protein of the MC29-related virus CMII is a nuclear DNA-binding protein whereas MH2 codes for a cytoplasmic RNA-DNA binding polyprotein.

The acute avian leukemia viruses MH2 and CMII belong to the group of avian myelocytomatosis viruses, the prototype virus of which is MC29. This group of viruses is characterized by myc-specific oncogenes which are presumably expressed as gag-myc polyproteins. These polyproteins are synthesized in non-producer cells transformed by MH2 and CMII and have mol. wts. of 100 000 (p100) and 90 000 (p90), respectively. Monoclonal antibodies against the N terminus of gag, p19, were used to localize the protein in MH2- and CMII-transformed non-producer fibroblasts. Immunofluorescence and cell fractionation indicated that greater than 90% of p100 from MH2 was located in the cytoplasm, whereas greater than 70% of p90 from CMII resided in the nucleus. Isolation of p100 and p90 by immunoaffinity chromatography resulted in an approximately 2000-fold purification of the two polyproteins. Both of them, as well as p110 of MC29, bound to double-stranded DNA of chick fibroblasts in vitro. However, only the MH2-specific polyprotein p100 bound to RNA in vitro. Such a binding was not observed for p90 or p110, or for the purified gag precursor Pr76. Another polyprotein, gag-erbA, from avian erythroblastosis virus, which is also located in the cytoplasm, did not bind to RNA. Our results indicate that the CMII-specific polyprotein p90 behaved indistinguishably from the p110 of MC29. However, the MH2-specific polyprotein p100 exhibited unique and novel properties which were distinct from a gag-myc-type protein.     

一种1型单纯庖疹病毒载体系统的建立

1型单纯疱疹病毒（HSV-1）作为溶瘤病毒和病毒载体的研究已有很长的历史. 本研究利用细菌人工染色体技术建立了一种HSV-1载体系统. 首先,将HSV-1内部反向重复序列（internal inverted repeat sequences, IR）两侧的片段克隆入pKO5获得穿梭质粒pKO5/BN,其电转含pHSV-BAC的大肠杆菌后筛选获得删除IR区重组DNA的 pHSVΔIR-BAC. pHSVΔIR-BAC转染Vero细胞获得删除IR区的重组病毒HSVΔIR（MH1001）.上述pKO5/BN和含pHSVΔIR BAC的大肠杆菌构成了HSV-1载体系统. 利用该系统获得了表达绿色荧光蛋白EGFP的重组病毒HSVΔIR/EGFP（MH1002）.MH1001和MH1002在感染的Vero细胞中增殖水平略低于野生型HSV-1,但无显著差异|Western印迹检测表明,重组病毒早期蛋白质ICP0、ICP4、ICP8、ICP22、ICP27在感染细胞中的表达水平下降|免疫荧光及激光共聚焦检测表明,重组病毒与野生型病毒均存在于细胞质中.以上结果表明,删除IR区的重组HSV-1保留了复制能力,能够携载并表达外源基因,建立的HSV-1载体系统可用于构建携载外源基因的复制型重组HSV-1.     

Two unrelated cell-derived sequences in the genome of avian leukemia and carcinoma inducing retrovirus MH2.

Molecularly cloned proviral DNA of avian replication-defective retrovirus Mill Hill No. 2 (MH2) was analyzed. The MH2 provirus measures 5.5 kb including two long terminal repeats (LTR), and contains a partial complement of the structural gene gag, 1.5 kb in size, near the 5' terminus, and a 1.3-kb segment of the v-myc transforming gene near the 3' terminus. These v-myc sequences are closely related to the v-myc transforming gene of avian acute leukemia virus MC29, and to the cellular chicken gene c-myc. The gag and myc domains on the MH2 provirus are separated by unique sequences, 1.3 kb in size and termed v-mil, which are unrelated to v-myc, or to other oncogenes or structural genes of the avian leukemia-sarcoma group of retroviruses. Normal chicken DNA contains sequences closely related to v-mil, termed c-mil. Analyses of chicken c-mil clones isolated from a recombinant DNA library of the chicken genome reveal that c-mil is a single genetic locus with a complex split gene structure. In the MH2 genome, v-mil is expressed via genome-sized mRNA as a gag-related hybrid protein, p100gag-mil, while v-myc is apparently expressed via subgenomic mRNA independently from major coding regions of structural genes. The presence in the MH2 genome of two unrelated cell-derived sequences and their independent expression may be significant for the oncogenic specificities of this virus.     

env-encoded residues are not required for transformation by p48v-myb.

The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus.     

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deletion of the gag region from FBR murine osteosarcoma virus does not affect its enhanced transforming activity.

Both FBJ murine osteosarcoma virus (FBJ-MSV) and FBR-MSV induce transformation in tissue culture and osteogenic sarcomas in mice. In tissue culture, however, FBR-MSV induces larger foci with a shorter latency than those induced by FBJ-MSV. Transformation is dependent on expression of the fos oncogene in FBJ-MSV and of a gag-fos fusion protein in FBR-MSV. We have determined that the gag sequences can be deleted from FBR-MSV without affecting the high transforming activity of this virus in comparison to FBJ-MSV. The resultant virus, designated FBJ/R-MSV, has a protein coding region that is half the size of that of FBR-MSV and about one-third smaller than that of FBJ-MSV. Thus, FBJ/R-MSV will provide a useful tool for studying the transforming activity of the fos oncogene.     

Antitumor activity of eosinophils activated by IL-5 and eotaxin against hepatocellular carcinoma

We examined the antitumor effects of eosinophils to explore the potential of eosinophils as effector cells in tumor cytotoxicity. We expressed eotaxin in hepatocellular carcinoma cells, MH134, and injected them into either normal or IL-5 TG mice intradermally and monitored cell growth. In normal mice, growth of MH134 cells containing the expression plasmid pCXN2-eotaxin was similar to that of vector-transfected MH134 cells for a period of 2 weeks, suggesting that expression of eotaxin does not change the growth rate of tumor cells. In IL-5 TG mice, however, the growth of eotaxin expressing MH134 cells was significantly suppressed. LPS induced eosinophils to produce TNF-alpha to kill MH134 cells in vitro. Intratumor injection of LPS is effective to kill MH134-pCXN2 and MH134-pCXN2-eotaxin only in normal mice. Administration of anti-CD4 or anti-CD8 antibodies suppressed growth of MH134-pCXN2-eotaxin cells compared with control antibodies, suggesting that T cells may interfere with immunity against MH134. Administration of anti-IL-5Ralpha and anti-asialo GM1 antibodies enhanced growth of MH134-pCXN2-eotaxin cells, suggesting involvement of eosinophils and NK cells in suppression of tumor cell growth. Although we cannot exclude the possibility that NK cells participate in tumor cell killing in vivo, the presence of NK markers such as DX5, asialo GM1, Ly49, and CD94, and NKG2D on large numbers of eosinophils activated by eotaxin suggests that eosinophils function in such suppression of tumor cell growth. Furthermore, we showed that anti-NKG2D antibodies could significantly inhibit the LPS-induced cytotoxicity against MH134 by highly enriched fraction of eosinophils.