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Epstein-Barr virus (EBV) is a B cell transforming virus that causes B cell malignancies under conditions of immune suppression. EBV orchestrates B cell transformation through its latent membrane proteins (LMPs) and Epstein-Barr nuclear antigens (EBNAs). We here identify secondary mutations in mouse B cell lymphomas induced by LMP1, to predict and identify key functions of other EBV genes during transformation. We find aberrant activation of early B cell factor 1 (EBF1) to promote transformation of LMP1-expressing B cells by inhibiting their differentiation to plasma cells. EBV EBNA3A phenocopies EBF1 activities in LMP1-expressing B cells, promoting transformation while inhibiting differentiation. In cells expressing LMP1 together with LMP2A, EBNA3A only promotes lymphomagenesis when the EBNA2 target Myc is also overexpressed. Collectively, our data support a model where proproliferative activities of LMP1, LMP2A, and EBNA2 in combination with EBNA3A-mediated inhibition of terminal plasma cell differentiation critically control EBV-mediated B cell lymphomagenesis.

Although viruses comprise the most abundant genetic material in the biosphere, to date only several thousand virus species have been formally defined. Such a limited perspective on virus diversity has in part arisen because viruses were traditionally considered only as etiologic agents of overt disease in humans or economically important species and were often difficult to identify using cell culture. This view has dramatically changed with the rise of metagenomics, which is transforming virus discovery and revealing a remarkable diversity of viruses sampled from diverse cellular organisms. These newly discovered viruses help fill major gaps in the evolutionary history of viruses, revealing a near continuum of diversity among genera, families, and even orders of RNA viruses. Herein, we review some of the recent advances in our understanding of the RNA virosphere that have stemmed from metagenomics, note future directions, and highlight some of the remaining challenges to this rapidly developing field.

Transduction of oncogenes by ALVs and generation of acute transforming viruses is common in natural viral infections. In order to understand the molecular basis for the rapid oncogenicity of Fu-J, an acutely transforming avian leukosis virus isolated from fibrosarcomas in crossbreed broilers infected with subgroup J avian leukosis virus (ALV-J) in China, complete genomic structure of Fu-J virus was determined by PCR amplification and compared with those of Fu-J1, Fu-J2, Fu-J3, Fu-J4, and Fu-J5 reported previously. The results showed that the genome of Fu-J was defective, with parts of gag gene replaced by the complete v-fps oncogene and encoded a 137 kDa Gag-fps fusion protein. Sequence analysis revealed that Fu-J and Fu-J1 to Fu-J5 were related quasi-species variants carrying different lengths of v-fps oncogenes generated from recombination between helper virus and c-fps gene. Comparison of virus carrying v-fps oncogene also gave us a glimpse of the molecular characterization and evolution process of the acutely transforming ALV.

SRC family kinases (SFKs) are known regulators of multiple cellular events, including cell movement, differentiation, proliferation, survival and apoptosis. SFKs are expressed virtually by all mammalian cells. They are non-receptor protein kinases that phosphorylate a variety of cellular proteins on tyrosine, leading to the activation of protein targets in response to environmental stimuli. Among SFKs, SRC, YES and FYN are the ubiquitously expressed and best studied members. In fact, SRC, the prototypical SFK, was the first tyrosine kinase identified in mammalian cells. Studies have shown that SFKs are regulators of cell junctions, and function in endocytosis and membrane trafficking to regulate junction restructuring events. Herein, we briefly summarize the recent findings in the field regarding the role of SFKs in the testis in regulating spermatogenesis, particularly in Sertoli–Sertoli and Sertoli–germ cell adhesion. While it is almost 50 years since the identification of the oncogene v-Src encoded by Rous sarcoma transforming virus, the understanding of SFK involvement during spermatogenesis in the testis remains far behind that in other epithelia and tissues. The goal of this review is to bridge this gap.

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5α ( Ref1 ), showing that XMRV is resistant to TRIM5α restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a skin cancer caused by a gamma- herpesvirus.The virus is a “tumor” transforming virus and thus often leads to cancer in the infected patient. Its association with AIDS over the past two decades has brought increased attention to the roll viral infection plays in the evolution of cancer. KSHV has been shown to interfere in one major pathway that controls cell growth, the apoptotic pathway, important for the maintenance of normal growth and development. Viruses require a live cell for survival and have evolved a mechanism to interfere with apoptosis to create immortal cell hosts. The virus expresses a protein, vBCL-2 that interferes with pro-apoptotic proteins and promotes tumor growth. This viral protein is a functional homology of the human Bcl2 protein and has been shown to interact with several BH3 domain containing family members. Previously we had identified an interaction between the KSHV vBcl2 protein and the human BH3 domain containing protein, BIK...

By means of correlated observations on the roentgenographic and histological structure, in an article written over 50 yr ago, Phemister [1] demonstrated diat osteosarcomas contain tumorous and normal nontumorous bone. Normal growing bone has a trabecular pattern that is specific for each bone and part of a bone as well as a characteristic microscopic palisade of cuboidal basiphilic cells named osteoblasts. Tumorous bone has a disoriented structure widi trabeculae enveloped in a random arrangement of bizarrely shaped anaplastic cells. Tumorous bone grows by proliferation of transformed osteoblasts. The alterations in gene structure and function in the transformed state are obscure, but the involvement is comparable to a mutation. The criteria for cell transformation generally include: loss of cell contact inhibition; altered morphology; increased growdi rate; increased capacity to persist in serial subcultures; chromosomal abnormalities; increased resistance to reinfection by die transforming virus; emergence of new antigens; capacity to form neoplasms [2]. Viral-induced malignant transformation constitutes a heritable change and is accompanied by the loss of regulatory controls of cell growdi. Present knowledge of nonmalignant morphodifferentiation, as well as malignant transformation, is chiefly descriptive. Data on the biochemical controls of morphodifferentiation are lacking. The process beginning widi the fertilized ovum and ending widi the mechanics of unfolding of die structural characteristics, form, and size of functional tissues defies present understanding. The problem is the prerogative of students of dieoretical, as distinguished from experimental, biology.

Eight Chinese field strains of subgroup J avian leukosis viruses (ALV-J) were isolated from broilers or parent stocks during January 1999 to April 2001. One strain, SD9902, was an acute transforming virus, able to induce typical myelocytomatosis in 22-38 days after inoculation of 1-day-old meat-type chicks. The envelope protein and 3'-untranslated region (UTR) of the eight field strains were compared with the U.K. prototype HPRS-103 and several U.S. field strains isolated in 1993-97. All Chinese strains shared an almost identical deletion with the U.S. strain 4817 in the E element region of 3'-UTR when compared with the prototype HPRS-103, indicating that they have a very close phylogenic relationship. Every year, China has to import grandparent stocks of meat-type chickens, mainly from the United States. Chinese isolates should represent a part in the phylogenic tree of U.S. ALV-J evolution. Envelope protein gp85 amino acid sequence analysis demonstrated that, interestingly, all recent Chinese isolates were more closely related to HPRS-103 and the earliest U.S. isolates but not to the late U.S. isolates. The result implies that envelope gp85 may not have diverged from prototype and older strains. It is also possible that some recently imported birds could have been infected by the older viruses that were introduced in the late 1990s.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a transforming virus associated with various cancers, the most common being the skin cancer Kaposi’s sarcoma. KSHV viral proteins are known to disrupt many normal cellular pathways in the human host. One specific target is the apoptotic pathway, or the programed cell death process. The cellular Bcl-2 family proteins, critical components of apoptotic signaling, converge at the mitochondria. This broad family of proteins is comprised of both pro- and anti-apoptotic proteins, sharing homology in four conserved trans-membrane domains, BH1-BH4. KSHV encodes a protein, vBcl2, with functional and sequential homology to the anti-apoptotic Bcl-2 protein. This gene is expressed in the early stages of the viral lytic cycle and has been shown to affect the progression of apoptotic signaling in order to allow for proper viral replication and virion production. We analyzed the interaction between KSHV’s vBcl-2 protein and a human protein, BIK, a member of the BH3 only f...

Avian leukosis virus subgroup J (ALV-J), isolated in the late 1980s, predominantly causes myelocytic myeloid leukosis in meat-type chickens. In the past 10 years, ALV-J infection has become very widespread, causing serious problems to the chicken meat industry. Previously, we have shown that turkey cells can be infected in vitro with Rous sarcoma virus pseudotypes of ALV-J. In this paper, we extend those observations to show that turkey monocyte cultures can be transformed in vitro with acutely transforming ALV-J strain 966. We also show that turkeys are experimentally susceptible to infection with ALV-J prototype strain HPRS-103. However, neoplastic lesions were not observed in these birds, probably due to the short experimental period of 10 weeks. When inoculated into 1-day-old turkey poults, acutely transforming ALV-J strain 966 induced tumours between 3 and 4 weeks after infection. Most of the birds showed tumours involving the liver, with histopathological lesions of myelocytomatosis. The demonstration of the spread of HPRS-103 by contact among turkeys, although observed only at low levels in the present study, stresses the importance of segregation of turkey and chicken breeding operations to avoid the spread of ALV-J infection.

In their classic paper on the identification of the transforming principle of Rous sarcoma virus (RSV) published 1970 in PNAS (1), Peter Duesberg at the University of California, Berkeley, and Peter Vogt, then at the University of Washington, Seattle, drew a seemingly simple yet groundbreaking conclusion. When they analyzed the genomic RNAs of transforming, acutely oncogenic RSV and of transformation-defective (td) mutant derivatives, they found that all transforming virus stocks contained two classes of RNA subunits, a larger one (a) and a smaller one (b), whereas the nontransforming yet replication-competent mutants contained the smaller b subunits only. Duesberg and Vogt concluded that the larger a subunit contained the transforming principle of RSV. Based on this and on subsequent structural comparisons of the a and b subunits of biologically cloned viruses, the transforming principle was defined by the remarkably simple equation a − b = x and was later termed src (for sarcoma). The first biochemical identification of a cancer gene was achieved, initially in a chicken virus. However, the principal proof of a physical underpinning of the cancer gene hypothesis had tremendous impact on a fundamental challenge of medicine, decoding the molecular basis of human carcinogenesis.

Recently, a novel DNA virus has been molecularly cloned from Kaposi's sarcoma (KS) tissue, a tumour common in acquired immune deficiency syndrome (AIDS). Analysis of the viral genome confirms that it is a relative of human herpesviruses and the virus has been designated HHV-8. Epidemiological evidence suggests a strong aetiological link between the presence of HHV-8 DNA and/or antibodies against the virus, and KS. Additional sequence analysis suggests that the HHV-8 genome contains sequences which encode a D type cyclin and a number of other genes potentially implicated in growth deregulation which may be relevant to its proposed role as a transforming virus.

For human B lymphocytes, Epstein-Barr virus (EBV) is a polyclonal activator, inducing both proliferation and /g secretion. It is also a transforming virus capable of generating immortalized B cell lines. These .early and late functions of EBV are not apparently connected. The receptor for EBY CD21, also serves as a receptor for some complement components and is called CR2. This molecule associates with CD19 and TAPA-l on the surface of B cells. This complex is involved in signaling B cells and participates in many responses. We have observed that simultaneous ligation of CD40 and the CD21 complex, by exposure to anti-CD40 MAbs and EBy enhances both the short-term proliferation as well as the long-term transformation rate of human B lymphocytes. B cell proliferation shows synergy between anti-CD40 MAb and EBV CD19 also appears to be involved in the synergistic activation of B cells through CD40 and CD21, since ligation of CD19 with anti­ CD19 MAbs, either prior to or concomitant with exposure to anti-CD40 and EBy markedly inhibits both proliferation and subsequent B cell transformation. These observations do not elucidate the mechanisms of B cell transformation employed by EBV but they do suggest a relationship between early proliferation and later transformation induced by the virus. Anti-CD40 enhances both these effects and anti-CD19 is capable of inhibiting both. [Hum Antibod 1997; 8: 43-47]

The Epstein-Barr virus (EBV) carrier state is characterized by latent infection of the general B-cell pool and by chronic virus replication at oropharyngeal sites. In Caucasian populations, most healthy carriers seem to harbor one dominant transforming virus strain, usually of type I rather than type 2, which persists over time and is detectable both in the blood and in the throat. This finding implies that once the virus carrier state is established, both viral reservoirs are largely if not completely protected from infection with additional strains. However, it is not known which facets of the immune response offer that protection. Here we address this question by a detailed study of EBV carriage in patients T-cell immunocompromised as a result of chronic human immunodeficiency virus (HIV) infection. Resident EBV strains were rescued from blood and from throat washings by using an in vitro transformation assay which aims to minimize bias toward faster-growing transformants; in this way, a mean of 16 independent isolations were made from each of 35 HIV-positive (predominantly male homosexual) patients. These virus isolates were characterized first at the DNA level by PCR amplification across type-specific polymorphisms in the EBNA2 and EBNA3C genes and across the 30-bp deletion and 33-bp repeat loci in the LMP1 gene and then at the protein level by immunoblotting for the strain-specific "EBNAprint" of EBNA1, -2, and -3C molecular weights. By these criteria, 18 of 35 patients harbored only one detectable EBV strain, usually of type 1, as do healthy carriers. However, the other 17 patients showed clear evidence of multiple infection with different EBV strains. In eight cases these strains were of the same type, again usually type 1, and were more often found coresident in throat washings than in the blood. By contrast, a further nine patients gave evidence of coinfection with type 1 and type 2 strains, and in these cases both virus types were detectable in the blood as well as in the throat. Immunological assays on these HIV-positive patients as a group showed a marked impairment of T-cell responses, reflected in reduced levels of EBV-specific cytotoxic T-cell memory, but an elevation of humoral responses, reflected in raised antibody titers to the EBV envelope glycoprotein gp340 and by the maintenance of virus neutralizing antibodies in serum. We infer that selective impairment of the T-cell system predisposes the host to infection with additional exogenously transmitted EBV strains.

Infection of mouse fibroblasts by wild-type polyomavirus results in increased phosphorylation of ribosomal protein S6 (D.A. Talmage, J. Blenis, and T.L. Benjamin, Mol. Cell. Biol. 8:2309-2315, 1988). Here we identify pp70 S6 kinase (pp70S6K) as a target for signal transduction events leading from polyomavirus middle tumor antigen (mT). Two partially transforming virus mutants altered in different mT signalling pathways have been studied to elucidate the pathway leading to S6 phosphorylation. An upstream role for mT-phosphatidylinositol 3-kinase (PI3K) complexes in pp70S6K activation is implicated by the failure of 315YF, a mutant unable to promote PI3K binding, to elicit a response. This conclusion is supported by studies using wortmannin, a known inhibitor of PI3K. In contrast, stable interaction of mT with Shc, a protein thought to be involved upstream of Ras, is dispensable for pp70S6K activation. 250YS, a mutant mT which retains a binding site for PI3K but lacks one for Shc, stimulates pp70S6K to wild-type levels. Mutants 315YF and 250YS induce partial transformation of rats fibroblasts with distinct phenotypes, as judged from morphological and growth criteria. Neither mutant induces growth in soft agar, indicating that an increase in S6 phosphorylation, while necessary for cell cycle progression in normal mitogenesis, is not sufficient for anchorage-independent cell growth. In the polyomavirus systems, the latter requires integration of signals from mT involving both Shc and PI3K.

The Epstein-Barr virus (EBV) glycoprotein gp110 has substantial amino acid homology to gB of herpes simplex virus but localizes differently within infected cells and is essentially undetectable in virions. To investigate whether gp110, like gB, is essential for EBV infection, a selectable marker was inserted within the gp110 reading frame, BALF4, and the resulting null mutant EBV stain, B95-110HYG, was recovered in lymphoblastoid cell lines (LCLs). While LCLs infected with the parental virus B95-8 expressed the gp110 protein product following productive cycle induction, neither full-length gp110 nor the predicted gp110 truncation product was detectable in B95-110HYG LCLs. Infectious virus could not be recovered from B95-110HYG LCLs unless gp110 was provided in trans. Rescued B95-110HYG virus latently infected and growth transformed primary B lymphocytes. Thus, gp110 is required for the production of transforming virus but not for the maintenance of transformation of primary B lymphocytes by EBV.

A chimeric construct (VCD) consisting of parts from viral jun, chicken c-jun, and chicken junD was cloned into the replication-competent retroviral RCAS vector. This construct, RCAS-VCD, was found to have a higher focus forming potential in quail fibroblasts than the equivalent construct RCAS-VJ-1, expressing viral jun. DNAs from RCAS-VCD and RCAS-VJ-1 were transfected into primary quail embryo fibroblasts. Cells derived from one RCAS-VCD-induced focus survived cell crisis, which became manifest after 15 passages, and could be expanded into a long-term culture. This cell line, termed VCD, has been passaged for over 100 times so far. The cells grow to very high densities and then pile up into clumps of rounded cells. The culture releases a transforming virus with a titer of 105 FFU/ml, as tested on primary quail embryo fibroblasts. The transformed phenotype of VCD cells was verified by agar colony formation. VCD cells are capable of anchorage-independent growth with a cloning efficiency of 10%. Southern blot analysis of genomic DNA from VCD cells showed proviral integration of the RCAS construct without detectable rearrangements. Northern and Western blot analyses confirmed correct expression from integrated RCAS-VCD of predicted RNAs and of the chimeric Jun(VCD) protein. jun (VCD)-transformed cells provide a constant source of homogeneous cellular material for the investigation of the molecular mechanisms of jun -induced cell transformation and for the identification of direct and indirect targets of Jun protein function.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA 2) is an acidic transcriptional transactivator of virus and cell gene expression and is essential for growth transformation of primary B lymphocytes. EBNA 2 transactivation of response elements (E2REs) can be mediated by interaction with a GTGGGAA-specific DNA-binding factor(s). We now purify the factor by S-Sepharose and EBNA 2 affinity chromatography and identify it as a single 63-kDa protein. The protein is shown to specifically coimmunoprecipitate with EBNA 2 from lymphoblasts transfected with an EBNA 2 FLAG expression vector. Mutation of GTG to TCT in a GTGGGAA motif common to the Cp, LMP2, and LMP1 promoters results in loss of recognition by p63. EBNA 2 amino acids 310-336 are sufficient for p63 binding. The only motif in this 27 amino acid sequence which is common to the EBNA 2 genes of EBV types 1 and 2 is GPPWWPP (I/V) (C/R) DP, which is therefore likely to mediate p63 interaction. Mutation of WW to SS or FF ablates interaction with p63, indicating that both the hydrophobic and aromatic characteristics of WW are essential for its "key" interaction with p63. EBNA 2 with a WW mutated to SS is also unable to marker rescue primary B lymphocyte transforming virus from cells infected with an EBNA 2-deleted virus, while otherwise isogenic wild-type EBNA 2 readily marker rescues transforming virus in parallel experiments. EBNA 2 transactivation through the Cp E2RE is completely abolished by the WW to SS mutation while transactivation of -234 to +40 LMP1 E2RE is only partially affected. These genetic and biochemical experiments support the hypothesis that EBNA 2 WW interaction with a p63 GTGGGAA-binding protein is essential for EBV-mediated cell growth transformation because it specifically associates EBNA 2 with its response elements. This enables the EBNA 2 acidic domain to transcriptionally transactivate specific genes.

Virus from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) can induce rare foci of transformation in rat 208F fibroblasts. Characterization of three such foci revealed that one produced an acutely transforming virus at a high titer. The virus consists of a mutant Nras cDNA from the HT-1080 cells inserted into a retroviral vector (added to the HT-1080 cells as a marker for infection) in place of internal vector sequences. These results show that XMRV can generate acutely transforming viruses at a low rate, as is typical of other replication-competent retroviruses, and reveal the potential for transforming virus contamination of retroviral vectors made from transformed cell lines.

To investigate the cellular mechanism of transformation by retroviruses, we established a system for isolation of cellular and viral mutants for transformation of a rat cell line. Mutagenized untransformed cells of this line were infected with recombinant murine retrovirus containing the src gene of Rous sarcoma virus and the selective marker gene, neo. After reaching confluence, cells transformed by the src gene tend to overgrow and die. Utilizing this property of src transformed rat cells and the selective marker gene, we could easily select untransformed cell clones containing the retrovirus genome. Expression of the src gene product in the flat clones selected was examined by in vitro assay of src kinase activity. To determine whether the mutations of these flat clones were viral or cellular, the susceptibilities of the clones to transformation were examined after superinfection with the wild-type virus and also characterized the retroviruses recovered from these clones. With this system, two novel clones were isolated. One had a defect in viral information affecting the transformed phenotype, but still retained src kinase activity like fully transformed cells. The other showed low src kinase activity but retained wild-type transforming virus, suggesting that a cellular gene involved in viral gene expression was mutated.

Three revenants defective in the ability to form colonies in semisolid medium were isolated from a rat cell line transformed by the E6 and E7 genes of human papillomavirus type 16 (HPV16). These revenants appeared to be defective in a cellular factor(s) necessary for transformation by HPV16-E6E7 genes since they still expressed a comparable amount of HPV16-E6E7 mRNA and E7 protein to the parental cells, harbored rescuable transforming virus, and were resistant to retransformation by HPV16-E6E7 genes. All these reverted phenotypes of the three mutants were recessive on somatic cell hybridization with normal cells, because all the hybrids showed transformed phenotypes.

An Epstein-Barr virus (EBV)-producer line, designated Akata, was established from a Japanese patient with Burkitt's lymphoma. The Akata line possessed the Burkitt's-type chromosome translocation, t(8q- ; 14q+), and was derived from the tumor cell. Akata cells produced a large quantity of transforming virus upon treatment of cells with anti-immunoglobulin antibodies (Takada, 1984). Southern blot analysis of viral DNA indicated that the Akata EBV is nondefective and more representative of wild-type viruses. Akata cells should be useful as a source of EBV.

Working with knowledge gained from pioneers in the RNA tumor virus field (2), the biologic activity of this NZB virus ultimately was demonstrated in 1970 by cultivating NZB mouse embryo cells with hamster or rat tumor cells containing a nonreplicating sequence of the murine sarcoma virus (3). This procedure, using conventional MLV, leads to the rescue of the sarcoma virus genome in the envelope coat of the replication-competent MLV (2). The newly formed transforming virus pseudotype now has the host range of the “helper” MLV. Supernatants from the NZB cell coculture demonstrated the presence of a replicating virus that could induce foci of transformation in rat cells but not mouse embryo cells (3). The NZB MLV host range thus seemed limited to rat cells. Subsequent work demonstrated that the helper MLV visualized in the NZB mouse cells was infectious but was not infectious at all for mouse cells (i.e., complete block). It was infectious only for cells from heterologous species, including those of avian origin (Table 1). Similar viruses were subsequently found in all laboratory strains of Mus musculus domesticus as …

In accordance with earlier studies, we detected higher numbers of Epstein-Barr virus (EBV)-carrying lymphocytes (B-EBV) in the blood of acute infectious mononucleosis (IM) patients and higher amounts of transforming EBV particles in the saliva compared to healthy seropositive individuals. B cells grew in cultures seeded with the low and high density IM lymphocytes. The majority of B cells which grew acquired the infection in vitro (2-step outgrowth), because addition of virus neutralizing antibodies considerably reduced the emergence of lymphoblastoid cell lines (LCLs). Only the minority of the explanted B-EBV cells proliferated. The antiviral drug phosphonoformate (PFA) did not influence the frequency of 2-step LCLs in the IM cultures. This may indicate that a large proportion of EBV carrying B cells have already entered the viral productive cycle in vivo and passed the PFA-sensitive stage at the time of explantation. Earlier experiments with blood of healthy seropositive individuals showed an inhibitory effect of PFA on the generation of LCLs. One healthy individual who entered this study as a control, probably had a reactivated EBV infection as judged by the anti-EA activity in his serum and the high level of virus in his saliva. He had antibodies against EBV nuclear antigens (EBNA), and therefore he did not have a primary infection at the time of the test. Judged by the number of wells with B cell growth, the frequency of virus-carrying B cells in his blood was low. It seems that anti-EBV immunity can control the number of infected B cells in the blood, but does not influence the virus load in the epithelial cells.

Lymphoid cell lines were established which stably carry the Epstein-Barr viral (EBV) BZLF1 gene on an extrachromosomal plasmid. These lines, which spontaneously synthesize the BZLF1 gene product, ZEBRA, were examined for expression of EBV genes which were activated by ZEBRA. Cell lines which acquired oriP plasmids without BZLF1 served as controls. The extent of activation differed among derivatives of four cell lines. X50-7 cells, which harbor a standard latent EBV, could be induced by ZEBRA to produce transforming virus; a cellular subclone of this line was induced to express EBV late antigens but did not release transforming virus. In two other cell lines, Raji and ER, ZEBRA activated only a group of early antigens. Using immunofluorescence and immunoblotting with monoclonal antibodies and Northern analysis five EBV early genes were shown to be induced in cells stably transformed by oriP/BZLF1 plasmids. ZEBRA itself was activated; thus BZLF1 is autostimulatory. Four other activated genes were components of the diffuse (EA-D) and restricted (EA-R) early antigens (BMRF1, BMLF1, BHRF1, and BORF2). Stable cell lines with extrachromosomal BZLF1 expression vectors will ultimately be useful in a variety of experiments designed to study regulation of this gene, to analyze the effects of mutations on ZEBRA protein function, and to define the full spectrum of viral and cellular genes which are activated by and interact with the ZEBRA protein.

A revertant cell line was selected from Moloney sarcoma virus-transformed BALB/c cells after long-term treatment with type I interferon. Despite an actively transcribed and transfectable v-mos gene, these revertant cells were nontumorigenic in nude mice. The functionality of the mos protein was investigated, focusing on the alpha 2(1) collagen promoter regulation, which is known to be affected by mos-induced trans-acting factors. Both in transient expression assays and after stable integration into the cellular genome, the transfected alpha 2(1) collagen promoter fused to the cat reporter gene was activated in the revertant while being downregulated in the original transformed cells. In retransformation assays of the revertant by Moloney sarcoma virus strains homologous to the original transforming virus, no detectable change was noted in the in vitro phenotype or in tumorigenicity. These results reveal that the mos-directed factors were no longer effective on their specific targets. Thus, the R.MSVIF cell could be either an oncoprotein-deficient or a target-related revertant. Attempts at retransformation with unrelated sarcoma viruses bearing v-sis, v-fms, or v-fos oncogenes were also negative. In contrast, tumorigenicity was obtained with the unrelated Kirsten sarcoma virus without any change in the revertant morphology or collagen expression. These findings showed that the common pathway blocked by the reversion and shared by v-mos and v-ras was not required for ras-induced tumorigenicity.

Kaposi's sarcoma (KS) is a very rare tumor except after human immunodeficiency virus type 1 (HIV-1) infection when it becomes common. Most investigators assume that the role of HIV-1 is passive, i.e., via inducing immune deficiency, thereby enhancing cancer development, and specifically, in the case of human herpesvirus 8 (KSHV) by enhancing HHV-8 replication. We suggest that the role of HIV-1 is more active in the disease process by at least two events: 1) promoting an increase in inflammatory cytokines, which through sustained release influences early stage KS by inciting local microinflammatory responses, and 2) by the Tat protein that effects growth of the inflammatory cells. Cultures of all activated endothelial spindle cells, whether hyperplastic or neoplastic, are negative for HHV-8; transmission of HHV-8 does not induce cell growth or transformation; monkeys immune suppressed by simian immunodeficiency virus infection and infected also with HHV-8 do not develop KS; polymerase chain reaction analysis of blood cells shows HHV-8 sequences in monocytes and B cells (about 20% of normal donors in Maryland); M. Starzl showed that early KS has few cells (mostly macrophage) positive for HHV-8, increasing and present in endothelial cells only late in the disease; no increase in HHV-8 has been found in association with progressive immune deficiency; and recent studies in Gambia by others showed that HHV-8 is a very common infection, and though HHV-2 is known to be relatively common, HIV-1 is unusual and so is KS. Collectively, these findings lead me to conclude that there is little evidence that HHV-8 is a transforming virus as has been repeatedly suggested and that its role in KS is more likely to be indirect (like HIV-1), perhaps necessary but hardly likely to be sufficient for KS development.

Avian leukosis virus subgroup J (ALV-J), the most recent member of the avian retroviruses, is predominantly associated with myeloid leukosis in meat-type chickens. We have previously demonstrated that the acutely transforming virus strain 966, isolated from an ALV-J-induced tumor, transformed peripheral blood monocyte and bone marrow cells in vitro and induced rapid-onset tumors, suggesting transduction of oncogenes (L. N. Payne, A. M. Gillespie, and K. Howes, Avian Dis. 37:438–450, 1993). In order to understand the molecular basis for the rapid transformation and tumor induction, we have determined the complete genomic structure of the provirus of the 966 strain. The sequence of the 966 provirus clone revealed that its genome is closely related to that of HPRS-103 but is defective, with the entire pol and parts of the gag and env genes replaced by a 1,491-bp sequence representing exons 2 and 3 of the c- myc gene. LSTC-IAH30, a stable cell line derived from turkey monocyte cultures transformed by the 966 strain of ALV-J, expressed a 72-kDa Gag-Myc fusion protein. The identification of the myc gene in 966 virus as well as in several other ALV-J-induced tumors suggested that the induction of myeloid tumors by this new subgroup of ALV occurs through mechanisms involving the activation of the c- myc oncogene.

Publisher Summary Identification of sequences from a new human herpesvirus in tumour tissue from AIDS-related Kaposi's sarcoma (KS) is done using the technique of Representational Difference Analysis (RDA). This technique uses cycles of polymerase chain reaction (PCR) and subtractive hybridization to amplify and enrich low-abundance DNA sequences present in only one of two otherwise identical DNA populations, and is therefore ideal for determining small differences between complex genomes. KS tumour usually appears as brownish-purple lesions, often on the extremities, but may in more aggressive forms of the disease progress to involve organs such as the lungs, lymph nodes, and gastrointestinal tract. The lesions contain many types of cell but are characterized histologically by the presence of elongated spindle cells and irregular slit-like vascular spaces. Seroepidemiology and PCR studies strongly suggest that HHV-8 is the cause of Kaposi's sarcoma and primary effusion lymphoma, and is also associated with multicentric Castleman's disease. Although HHV-8 has not yet been shown to be a transforming virus in vitro, the virus encodes several proto-oncogenes capable of the deregulation of cell cycle control, inhibition of apoptosis and control of growth differentiation, which could contribute to tumour formation.

The rat leukemia virus (RaLV) is an endogenous retrovirus that is spontaneously released by Sprague-Dawley rat embryo cells. The overall structure of the RaLV genome resembles that of other simple, replication-competent retroviruses, but the sequence of the long terminal repeats (LTR) is unique and unrelated to the known retroviruses. Phylogenetically, the RaLV genome appears to be more closely related to the feline leukemia virus group of retroviruses than to the murine leukemia virus group. A remarkable feature of RaLV is that it is capable of transducing a ras proto-oncogene from rat tumor cells in the form of an acutely transforming virus, designated the Rasheed strain of the rat sarcoma virus (RaSV). With the exception of the c- ras sequence, the genomes of both RaLV and RaSV are collinear. The RaSV-encoded oncogene v-Ra- ras expresses a fusion protein with a molecular mass of 29 kDa, and it exhibits a unique structure that has not been described previously for any known virus. The 5′ end of this gene is derived from sequences encoding RaLV matrix followed by 20 bp derived from the U5 region of the RaLV LTR (RS-U5 element) which is joined at its 3′ end to sequences derived from all six (coding and noncoding) exons of the c- ras proto-oncogene at the 3′ end. This recombinational event represents a novel mechanism among the acutely transforming viruses that have been studied.

Cytokine-mediated apoptotic destruction of viral-infected cells, downregulation of virus production and inhibition of anchorage dependent (clonal) cell growth were evaluated using virus-transfected human hepatoblastoma (HepG2) cells. The cytokines evaluated were interferon alpha (IFN-alpha), tumour necrosis factor alpha (TNF-alpha) and thymosin alpha 1 (T alpha 1), all of which have previously been implicated in control of various viral infections. The viruses evaluated were Hepatitis B (HBV) and the transforming virus, SV-40. TNF-alpha-induced apoptosis in the HBV-transfected cell line and the control HepG2 cells but not the HepG2 cells transfected with SV-40 virus. IFN-alpha and T alpha 1 had no effect on apoptosis. TNF-alpha also prevented the clonal growth of the HBV-HepG2 and control HepG2 but enhanced the growth of the SV-40-transfected HepG2 cells. IFN-alpha inhibited the clonal growth of all three cell lines in contrast to T alpha 1 which inhibited the clonal growth of only the HBV-transfected cells. Although TNF-alpha, IFN-alpha, and T alpha 1 when given alone did not significantly inhibit HBV-DNA production in the culture supernatant from HBV-HepG2 cells, the combination of T alpha 1 and IFN-alpha resulted in a statistically significant inhibition of virus production. These studies demonstrate that HepG2 cells transfected with HBV and SV-40 are useful for defining the mechanisms of cytokine activity. The HBV-transfected cells are especially useful in defining possible in vivo differences in responses to cytokines with respect to HBV production, apoptosis and clonal cell growth. Multiple mechanisms through which different cytokines can influence HBV infection and hepatoblastoma growth were identified and the importance of defining effective combinations to improve therapy in vivo demonstrated.

The tissue tropism was studied for the HPRS-103 strain of avian leukosis virus, which belongs to a new envelope subgroup, designated J. Studies were conducted in blood monocyte and bone marrow cell cultures and in chickens from six lines that had been shown previously to differ in susceptibility to induction by this virus of myeloid leukosis and other tumors. Using an immunohistochemical technique to detect expression of viral group-specific antigen (Gag) in various tissues, we detected no major differences among the six lines of chickens at 3 and 7 weeks of age following infection as embryos. Thus, Gag expression did not correlate with differences in tumor susceptibility. Of the tissues examined, greatest Gag expression was observed in cells specific to the adrenal gland, heart, kidney, and proventriculus and especially in smooth muscle cells and connective tissue. After infection of 1-day-old chicks, greater tissue expression was observed in line 21 chicks, which mostly developed a tolerant viremic infection, than in Brown Leghorn chicks, which developed virus-neutralizing antibodies. An acutely transforming virus, strain 966, derived from HPRS-103-induced myeloid leukosis, showed a tropism similar to HPRS-103. The HPRS-103 strain showed a lower propensity to replicate in the medullary region of the lymphoid follicles of the bursa of Fabricius than did the RAV-1 strain of subgroup A avian leukosis virus. This low bursal tropism may be a factor in why HPRS-103 does not induce lymphoid leukosis. The HPRS-103 and 966 virus replicated in blood monocyte cultures from chickens from the six lines, indicating a tropism for the myelomonocytic cell lineage. In comparison, as previously reported, RAV-1 did not replicate well in the monocyte cultures, whereas RAV-2, a subgroup B avian leukosis virus, did replicate. The tropism of HPRS-103 for monocytes may relate to its ability to cause myeloid leukosis. Monocyte and bone marrow cell cultures from the six lines ranked similarly in differences in susceptibility to transformation by 966 virus and showed evidence that their relative susceptibilities correlated with susceptibility of chickens from these lines to induction of myeloid leukosis by HPRS-103, suggesting common tissue-specific viral and host factors involved in oncogenesis by these two viruses.

Hepatitis B virus (HBV) DNA integrates into the host DNA and shows a series of potentially oncogenetic properties, but HBV is not an acutely transforming virus, because HCC develops decades after infection. Other factors, namely cirrhosis, inflammation, alcohol intake, and viral superinfections, could promote the oncogenetic process induced by HBV-DNA integration. We studied the impact of HDV infection in the pathogenesis of HCC in 62 consecutive patients. Their mean age was 59 years (range 25-75 years), 54 were male and eight female; 58 had cirrhosis. The findings suggest that HBsAg-positive patients with HDV superinfection developed cirrhosis and HCC at an earlier age than HBsAg carriers without HDV infection. HDV appears to represent a "promotion" factor for HCC in subjects with an oncogenic risk induced by HBV. A long-lasting necroinflammatory lesion of the liver substained by productive HBV and HDV infections may be a major pathogenetic mechanism.

Recently several groups have demonstrated cytomegalovirus (CMV) protein and DNA in a large majority of glioblastomas (GBMs), though the exact role of CMV in tumors remains controversial. Although CMV is capable of activating several oncogenic pathways, it has never been shown to be a transforming virus. Because of this, we hypothesize that CMV infection can modify the rate of gliomagenesis in the context of genetic mutations known to predispose to tumor formation. To test this, we used the Smith strain mouse CMV (MCMV) to infect Mut3 (GFAP-cre; Nf1loxP/+; Trp53-/+) mice that develop normally but eventually develop spontaneous gliomas at an adult age After intraperitoneal viral inoculation, mice exhibited multisystemic infection throughout the body, including the brain. Mut3 mice infected perinatally (103 plaque forming units) developed gliomas significantly sooner than mock-infected Mut3 mice. MCMV-infected mice developed GBMs (WHO grade IV) at a higher rate than less severe tumors, whereas, mock-infected mice developed less severe gliomas (WHO grade III). Since MCMV preferentially infected the neural stem cell (NSC) niche, an area shown to generate gliomas, we interrogated changes in signaling in this population via microarray of cultured NSCs in MCMV- versus Mock-infected mice. Ingenuity Pathway Analysis identified a PDGF-B network as the highest scoring differentially regulated network between MCMV-and Mock-infected neurospheres. In vitro infection of mouse tumorspheres and human GBM tumorspheres with CMV validated the observed upregulation of PDGF-B, a molecule shown to cause gliomas de novo. Additionally, in vitro infection of human glioma stem cells with HCMV (Towne strain) or exogenous PDGF-B activated STAT3, a key regulator of gliomas. In mice infected with MCMV, PDGF-B is upregulated in CA2/3 neurons and STAT3 is activated in the subventricular zone and dentate gyrus suggesting a paracrine effect of MCMV-induced PDGF-B. Also, infected human brain tumor stem cells demonstrated an increase in proliferation via flow cytometry. Taken together, our data suggest that CMV in gliomas may accelerate GBM progression by activating PDGF-B/STAT3 signaling in the NSC population. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4815. doi:1538-7445.AM2012-4815

Bryostatin 1 is a novel anti-tumor agent currently undergoing clinical trial. We investigated the effect of this drug on B-lymphocyte cell lines that carry the Epstein-Barr virus and found that it induces these latently infected cells into the production of transforming virus particles over a wide range of concentrations. These results may have clinical implications, particularly with regard to the use of the drug in the immunocompromised patient.

The immortalization and transformation of cultured human cells has far-reaching implications for both cell and cancer biology. Human cell transformation studies will increase our understanding of the mechanisms underlying carcinogenesis and differentiation. The neoplastic process can now be studied in a model human cell culture system. The accompanying biochemical and genetic changes, once identified, will help define the relationship between malignancy and differentiation. The present studies indeed demonstrate that the neoplastic process can now be studied in a human cell model system. Primary human cells treated with various carcinogens became immortalized in culture but were not tumorigenic. Additional exposure to either retroviruses, chemical carcinogenes or X-ŗay irradiation to these cells induced morphological alterations associated with the acquisition of neoplastic properties. These findings demonstrate the malignant transformation of human primary cells in culture by the combined action of either a DNA transforming virus and a retrovirus or a DNA virus and a chemical or X-ray irradiation, and support an multistep process for neoplastic conversion. It has been known that normal human cells in culture are remarkably resistant to experimentally induced tumorigenicity. However, as shown above, normal human cells could now be transformed into tumorigenic cells.

Epstein-Barr virus (EBV) isolates can be broadly classified as type 1 or type 2 on the basis of allelic polymorphism of the virus-encoded nuclear antigens EBNAs 2, 3a, 3b and 3c, and individually identified based on Mr values of their EBNA proteins (EBNA type). Here we have used this natural heterogeneity amongst isolates to re-examine the question of EBV persistence in vivo, asking in particular whether virus carriage in oropharyngeal epithelium and/or in B lymphoid tissues involves infection with a single or with multiple virus strains. Firstly, 76 healthy virus carriers were classified into serotype groups on the basis of preferential antibody reactivity to type 1 EBNAs (serotype 1) or to type 2 EBNAs (serotype 2); 60 of the 76 donors were serotype 1, four of the 76 donors were serotype 2 and 12 of the 76 donors were anti-EBNA 2, 3a, 3b, 3c antibody-negative and therefore could not be serotyped. Representative donors from each group were then selected for virus isolations from blood (by spontaneous in vitro transformation) and from throat washings (by cord blood cell transformation). All 13 serotype 1 donors tested and six of seven non-serotypeable donors gave a type 1 virus isolate, whereas all four serotype 2 donors and one of the seven non-serotypeable donors gave a type 2 isolate. Multiple transforming virus isolates from any one donor, whether from blood or throat washings, were all of the one strain characteristic of that particular donor; sequential isolations showed retention of the same strain over several years. Finally, throat washing samples from these same donors were examined for amplifiable EBV DNA in the polymerase chain reaction using EBV type-specific oligonucleotide primers and probes derived from the polymorphic EBNA 2 and EBNA 3c loci. The results were consistent with earlier virus isolation studies, each individual donor showing amplification either of type 1 or type 2 sequences. We conclude that multiple EBV infections must occur rarely, if at all, in healthy virus carriers; EBV persistence in vivo is characterized by dominance of a single transforming virus strain.

The mechanisms underlying interactions between viruses and carcinogens are only beginning to be understood. Peripheral blood lymphocytes (PBLs) separated from patients with acquired immunodeficiency syndrome (AIDS) are a relevant model to study chemical carcinogen-EBV synergism, due to the high degree of EBV reactivation and potential for EBV-associated malignant lymphomas in patients with AIDS. To better understand chemical carcinogen-EBV synergism at a molecular level, we examined the organization of the terminal region of EBV genome carried by LCLs established from patients with AIDS. For this study we chose LCLs established from PBLs treated with aflatoxin B1 (AFB1), a mycotoxin and known carcinogen. From an analysis of the EcoR1 Dhet terminal region, we found the majority of the LCLs established spontaneously to be clonal in origin, but individual lineages established from the same patient often carried EBV episomal circular forms of the genome with varying numbers of fused 500-base pair terminal repeats. Linear forms of the EBV genome with varying numbers of fused 3’ and 5’ 500-base pair terminal repeats were only detected in spontaneously derived LCLs which were established from carcinogen-treated PBLs. The presence of linear EBV DNA correlated with the presence of extracellular transforming virus. LCLs established from PBLs separated from patients with AIDS following addition of exogenous EBV (B95.8 or FF41–1) most often carried episomal circular forms of EBV genome with a terminal region characteristic of the infecting viral strain. LCLs derived from EBV-infected PBLs treated with AFB1 often carried variation of the parent episomal circular form differing in terminal region varying by number of fused terminal repeats. In addition, various forms of linear EBV DNA varying by the number of fused 3’ terminal repeats could be detected in these LCLs. These results show that the chemical carcinogen AFB1 directly interact with EBV and drive EBV-infected lymphocytes toward a semi-permissive infection. Furthermore, the permissive state of an LCL correlates with changes in the organization of the terminal region of the EBV genome and provides a model to explore how chemical carcinogens enhance EBV-induced transformation and EBV replication, and induce malignant conversion of EBV-carrying LCLs.

We read with interest the recent article in the Journal by Strickler et al. (1), in which the authors performed a retrospective age–period–cohort analysis to estimate the incidence of pleural mesothelioma in individuals potentially exposed to simian virus 40 (SV40) -contaminated poliovirus vaccines. The authors concluded that “Age-specific trends in U.S. pleural mesothelioma incidence rates are not consistent with an effect of exposure to SV40-contaminated poliovirus vaccine” and suggested that “monitoring of vaccine-exposed cohorts should continue.” We wish to raise two issues about this and similar studies (2–4) that address “exposure” to SV40contaminated polio vaccines and the incidence of human cancers. First, although a cohort study is recognized as the best design to identify incidence and natural history of disease (with a known exposure) and may be used to assess multiple outcomes after a single exposure (5), the prevalence of SV40 infections in the human population is not known, and the available data about prevalence are limited and inconclusive (2–4). Second, an important confounding factor in the epidemiologic assessment of malignant mesothelioma is that the actual number of people infected with SV40 through the use of contaminated polio vaccines is not known and may be less than the “exposed” population size assumed in the study by Strickler et al. (1). For example, in the United States, not all vaccine lots were contaminated with SV40, formalin inactivation was expected to reduce the titer of live SV40 in the lots that were contaminated, and successful infection rates by live SV40 are unknown (2–4). Therefore, an inability to identify the population that was actually infected with SV40 through the use of contaminated polio vaccines precludes a meaningful calculation of cancer incidence in relation to exposure to those vaccines. Furthermore, the Institute of Medicine (IOM) Immunization Safety Review Committee found that epidemiologic studies of cancer rates in people potentially “exposed” to SV40-contaminated vaccines, such as the analysis by Strickler et al. (1), are inadequate to evaluate a causal relationship between exposure and disease (2). These limitations led the IOM to “not recommend additional epidemiological studies of people potentially exposed to contaminated polio vaccine” (2). However, the IOM concluded that “the biological evidence is strong that SV40 is a transforming virus” and that “the biological evidence is of moderate strength that SV40 exposure could lead to cancer in humans under natural conditions” (2). Future studies are needed to determine the prevalence of SV40 infections in different human populations. The IOM recognized that gaps in our understanding of the pathogenesis of SV40 in humans are important and recommended “targeted biological research,” including “further study of the transmissibility of SV40 in humans” (2).

The nucleic acid sequence of the 3' region of human T-cell leukemia virus type II (HTLV-II) proviral DNA was determined using a HTLV-II proviral clone that could be recovered as infectious, transforming virus. The sequence data indicate a region of unknown function of approximately equal to 1.6 kilobase pairs in the 3' region, analogous to the X region previously identified in human T-cell leukemia virus type I (HTLV-I). Three overlapping open reading frames are present in the X region of HTLV-II. One of these open reading frames, Xc, is most likely to encode a protein product, because it has greater predicted amino acid sequence homology (78%) with the X-IV region of HTLV-I and a greater percentage of its base differences with X-IV at the third nucleotide position of codons than do the other open reading frames. Sequences of the X-region that include the open reading frames are conserved in two deletion mutants of HTLV-II, which are associated with a subline of Mo cells with a decreased dependence on fetal bovine serum.

Epstein-Barr virus (EBV) isolates show sequence divergence in the BamHI YH region of the genome which encodes the nuclear antigen EBNA 2, a protein thought to be involved in the initiation of virus-induced B-cell transformation; type A isolates (such as B95-8 EBV) encode a 82- to 87-kilodalton EBNA 2A protein, whereas type B isolates (such as AG876 EBV) encode an antigenically distinct 75-kilodalton EBNA 2B protein. In the present work 12 type A isolates and 8 type B isolates have been compared for their ability to transform resting human B cells in vitro into permanent lymphoblastoid cell lines. Although the kinetics of initial focus formation was not markedly dependent upon the EBNA 2 type of the transforming virus, on subsequent passage type A virus-transformed cells (type A transformants) yielded cell lines much more readily than did type B transformants. Direct comparison between the two types of transformant revealed clear differences in several aspects of growth phenotype. Compared with type A transformants, cell lines established with type B virus isolates consistently displayed an unusual growth pattern with poor survival of individual cells shed from lymphoblastoid clumps, a lower growth rate and a greater sensitivity to seeding at limiting dilutions, and a significantly lower saturation density that could not be corrected by supplementation of the medium with culture supernatant containing B-cell growth factors. This is the first direct evidence that, in EBV-transformed B-cell lines, the EBNA 2 protein plays a continuing role in determining the cellular growth phenotype.

Summary Chromosome-mediated transfer of murine leukaemia (MuLV) and murine sarcoma (MuSV) virus genetic information to uninfected recipient cells was investigated. Metaphase chromosomes from AKR MuLV-infected SC-1 mouse cells were incubated with NIH/3T3 cells. After several passages (1 to 3 weeks), infectious virions exhibiting reverse transcriptase activity and the characteristic host range of ecotropic, N-tropic AKR virus appeared in the supernatant fluids of the treated cells. Restriction endonuclease analysis of genomic DNA from transfected cells indicated that AKR proviral DNA was associated with the high molecular weight DNA of the host. These results demonstrate that the AKR MuLV genome can be stably transferred to uninfected recipient cells via isolated metaphase chromosomes. Although AKR virions are not able to infect heterologous cells, chromosome-mediated transfection resulted in the establishment of productive AKR MuLV infection in mink cells. Thus, the use of chromosomes to transfer virus genes can circumvent the natural host restriction barrier. In other experiments, it was shown that normal NIH/3T3 cells were transformed after exposure to metaphase chromosomes isolated from an MuSV-infected, non-producer line. Foci were detected 14 to 21 days after chromosome treatment and were shown to contain true viral transformants since transforming virus was produced after superinfection with MuLV.

Four continuous lines of RSV-transformed quail cells were established; QERC-31F and QERC-31N cells derived from quail embryo cells and PERP and PERY cells from adult quail peritoneal macrophages. Marked morphological difference was noted between QERC-31F and QERC-31N; the former showed fusiform shape and the latter nodular shape. Both PERP and PERY showed macrophage-like morphology with phagocytic capacity. All four cell lines contained gs antigen and gp 85. Production of transforming virus was found in QERC-31N, PERP and PERY. In spite of failure in production of transforming virus, EQRC-31F was demonstrated to produce C-type particles by electron microscopy and to contain tumor-specific surface antigen by in vivo immunization and in vitro microcytotoxicity tests.

We have constructed deletions within the region of cloned Rous sarcoma virus DNA coding for the N-terminal 30 kilodaltons of p60src. Infectious virus was recovered after transfection. Deletions of amino acids 15 to 149, 15 to 169, or 149 to 169 attenuated but did not abolish transforming activity, as assayed by focus formation and anchorage-independent growth. These deletions also had only slight effects on the tyrosine kinase activity of the mutant src protein. Deletion of amino acids 169 to 264 or 15 to 264 completely abolished transforming activity, and src kinase activity was reduced at least 10-fold. However, these mutant viruses generated low levels of transforming virus by recombination with the cellular src gene. The results suggest that as well as previously identified functional domains for p60src myristylation and membrane binding (amino acids 1 to 14) and tyrosine kinase activity (amino acids 250 to 526), additional N-terminal sequences (particularly amino acids 82 to 169) can influence the transforming activity of the src protein.

A focus-forming virus previously isolated from a BALB/c mouse hemangiosarcoma has been shown to be replication defective. Analysis of individual BALB/c mouse sarcoma virus (BALB-MSV) nonproducer transformants for expression of helper virus-coded proteins revealed genetically stable variants that expressed two, three, or all four gag gene products in the absence of detectable helper viral env gene expression. The type-specific antigenic determinants of helper viral proteins encoded by the BALB-MSV genome and by the B-tropic virus isolated from the BALB-MSV stock were demonstrated to be indistinguishable from those of BALB:virus-1, a known endogenous virus of BALB/c cells. These findings imply that a BALB/c endogenous virus was involved in the generation of BALB-MSV. By the same immunological approach, the presence of at least a portion of the Moloney-MuLV gag gene has been identified in two other transforming viruses--Moloney-MSV and Abelson lymphosarcoma virus--previously isolated from the BALB/c strain. The tissue culture properties of cells transformed by these defective viruses were also shown to be distinguishable. These findings indicate that transforming virus isolates of the same inbred strain differ in their transforming activities as well as in the helper viral sequences stably associated with their genomes.

In studies of the viral and cellular functions involved in expression of transformation by murine sarcoma virus, selective methods have led to the isolation of morphologic revertants following mitomycin C mutagenization of nonproductively transformed mouse cells. The revertants exhibit normal growth properties, yet still contain the sarcoma virus. Further, they are as susceptible as normal cells to exogenous sarcoma virus infection. In the present studies, these revertants are shown to contain levels of sarcoma viral RNA quantitatively and qualitatively indistinguishable from that present in the parental transformed clone. Following rescue with helper leukemia virus, they release low levels of wild-type transforming virus and a large excess of transformation-defective sarcoma virus as measured by molecular hybridization. The defective viruses can be transmitted to new cells in the absence of morphologic alteration. These results provide strong evidence that the revertants contain mutant viruses defective in transforming functions. The release of wild-type sarcoma virus by cells in a revertant culture appears to occur concomitantly with the spontaneous appearance of retransformed cells. This suggests that the reversion of mutant virus to wild-type within the cell occurs as a result of reversion of a point mutation in the integrated sarcoma viral genome. The present sarcoma virus mutants appear to be the first obtained by spontaneous or chemically-induced genetic alteration of stably integrated virus in eucaryotic cells.

The Sloan-Kettering viruses (SKVs) are replication-defective retroviruses that transform avian cells in vitro. Each of the three SKV isolates is a mixture of viruses with genomes ranging in size from 4.1 to 8.9 kilobases (kb) with a predominant genome of 5.7 kb. Using a cDNA representing a sequence, v-ski, that is SKV specific and held in common by the multiple SKV genomes, we generated a restriction map of the 5.7-kb SKV genome and molecularly cloned a ski-containing fragment from SKV proviral DNA. Southern hybridization and sequence analysis showed that the cloned DNA fragment consisted of the 1.3-kb ski sequence embedded in the p19gag sequence and followed by the remaining 5' half of the gag gene and small portions of both the pol and env genes. A large deletion encompassing the 3' half of gag and the 5' 80% of pol was mapped to a position about 1 kb downstream from the 3' ski-gag junction. To determine whether the cloned ski sequence had transforming activity, the ski-containing fragment and a cloned Rous-associated virus 1 (RAV-1) genome were used to construct an analog of the 5.7-kb SKV genome, RAV-SKV. Cotransfection of chicken embryo cells with RAV-SKV and RAV-1 yielded foci of transformed cells whose morphology was identical to that induced by the natural SKVs. The transformed transfected cells produced transforming virus with a 5.7-kb ski-containing genome and synthesized a gag-containing polyprotein of 110 kilodaltons (kDa). Several nonproducer clones of RAV-SKV-transformed cells were analyzed, and most were found to synthesize a 5.7-kb SKV RNA and a 110-kDa polyprotein. One clone was found to contain an 8.9-kb SKV RNA, and this clone synthesized a 125-kDa polyprotein. Since both the 5.7- and 8.9-kb genomes and the 110- and 125-kDa polyproteins had been identified in studies on the natural SKVs, the present results not only demonstrate the transforming activity of these individual SKVs but also suggest mechanisms for their generation.

We report that fibroblast lines derived from mouse (NIH 3T3) and mink (CCL-64) can be transformed via transfection with the total cellular DNA extracted from cells transformed with feline sarcoma virus. Replication of helper virus is not required in the recipient cell. Transformation of recipient cells is observed when the donor DNA is extracted from either virus producer or nonproducer transformed cell lines. A transforming virus can be rescued from nonproducer transformed recipients upon superinfection with a replication competent helper virus.

Inoculation of a cotton-topped marmoset with cell-free EBV, strain B95-8, resulted in the induction of lymphomas in liver, spleen, and mesenteric lymph nodes. Continous suspension cell lines were established from tumor tissue from each of these three sites. Cells of each line were positive for intracellular EBV antigens and EB nuclear antigen and released low levels of transforming virus. Early after initial culturing each line had a high frequency of cells (84-85%) with receptors for activated complement but these receptors were no longer evident on retesting several months later. All three lines initially showed high levels of cells which stained for surface lambda light chain and gamma and mu heavy chains. Cells of all three lines showed a decrease in gamma chain staining and the lymph-node tumor line showed reductions in surface staining for mu and lambda chains as well after prolonged cultivation. Clonal cells of the three lines resembled their respective parental lines in surface markers. Cytogenetically, all the lines were hypodiploid, 2n = 45, and several karyotypes from all three lines showed the loss of a medium-sized metacentric chromosome. A marker chromosome with an extra band in the centromeric region was apparent in the lymph-node tumor line. These results indicate that cells of individual tumors in a cotton-topped marmoset with multiple EBV-induced lymphomas are similar to each other in a number of characteristics although a certain degree of divergence was observed. Thus, it is possible that EBV-induced lymphomas in cotton-topped marmosets may have a common cell origin as is the case with Burkitt lymphoma in humans.

Epstein-Barr virus (EBV) is a ubiquitous transforming virus of the herpes group showing tropism for B lymphocytes. Primary infection in normal hosts results in a transient lymphoproliferative disorder, acute infectious mononucleosis (IM), that is restricted by cytotoxic and suppressive lymphocytes. However, in the immunodeficient host, EBV-induced lymphoproliferation may behave in a biologically malignant fashion. Patients with primary immunodeficiencies and those with immune incompetence resulting from suppressive therapy in allograft transplantation or infection with human immunodeficiency virus (HIV) have EBV-related illness ranging from fulminant mononucleosis and invasive polyclonal B cell hyperplasia to monoclonal B cell malignancies. While the direct link between EBV and malignant B cell proliferation in these patients has not been elucidated, the association has been increasingly recognized with improved techniques of viral detection. Clinical management can be guided by the location and extent of tumor, histologic features, and clonality. Regional and node-based polyclonal proliferations may respond to prompt reduction of immunosuppressive therapy and efforts to interrupt the replicative cycle with antiviral agents. Systemic cytotoxic therapy often leads to further immunosuppression and should be reserved for patients with progressive disease, advanced visceral involvement, and monoclonal lymphoid malignancies.