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Viruses in the family Retroviridae are found in a wide variety of vertebrate hosts. Enveloped virions are 80–100 nm in diameter with an inner core containing the viral genome and replicative enzymes. Core morphology is often characteristic for viruses within the same genus. Replication involves reverse transcription and integration into host cell DNA, resulting in a provirus. Integration into germline cells can result in a heritable provirus known as an endogenous retrovirus. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Retroviridae, which is available at ictv.global/report/retroviridae.

In 1970 the independent and simultaneous discovery of reverse transcriptase in retroviruses (then RNA tumor viruses) by David Baltimore and Howard Temin revolutionized molecular biology and laid the foundations for retrovirology and cancer biology. In this historical review we describe the formulation of the controversial provirus hypothesis by Temin, which ultimately was proven by his discovery of reverse transcriptase in Rous sarcoma virus virions. Baltimore arrived at the same discovery through his studies on replication of RNA-containing viruses, starting with poliovirus and then moving to vesicular stomatitis virus, where he discovered a virion RNA polymerase. Subsequent studies of reverse transcriptase led to the elucidation of the mechanism of retrovirus replication, the discovery of oncogenes, the advent of molecular cloning, the search for human cancer viruses, and the discovery and treatment of HIV/AIDS.

International audience; Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae, and fungi (1, 2). This broad distribution suggests the ancient origin(s) of these viruses, possibly [...]

Murine leukemia viruses (MuLVs) are the prototypical gammaretroviruses, and they have been extensively studied with regard to how they cause disease. Leukemogenesis by two MuLVs is reviewed here: the endogenous Akv MuLV of AKR mice, and exogenous Moloney MuLV. Important features of MuLV leukemogenesis include the in vivo generation of envelope recombinants (MCFs) through recombination with endogenous MuLVs, and induction of preleukemic changes typified by splenic hyperplasia secondary to bone marrow defects. Studies of MuLV leukemogenesis help to frame virological questions about how koala retrovirus (KoRV) may induce neoplastic or other diseases in koalas. Fan, Hung. 2014. Leukemogenesis by murine leukemia viruses: lessons for koala retrovirus (KoRV). In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed. Geoffrey W. Pye, Rebecca N. Johnson and Alex D. Greenwood. Technical Reports of the Australian Museum, Online 24: 83–88. The discovery of koala retrovirus (KoRV) in free-ranging and captive koalas (Phascolarctos cinereus) has been viewed with concern and interest. The primary concern is that KoRV-associated disease such as neoplasms, while yet to be conclusively proven to be KoRV caused, could increase the threats to survival of these animals. In the scientific community there is interest for several reasons: KoRV may be associated with lymphoma in koalas, it appears to be recently introduced into this species, and endogenization is an ongoing process. KoRV infection in koalas may provide an opportunity to study introduction and spread of a gammaretrovirus into a new host species and its accompanying effects. This process has happened in other species, notably mice, but in the more distant past, so some of the processes can only be deduced. At the same time, information learned from the relationship of murine gammaretroviruses and their hosts may provide lessons for understanding the potential relationships of KoRV and disease in koalas. The recent discovery of a second KoRV (KoRV-B) that may be associated with leukemogenicity (Xu et al., 2013) has similarities to oncogenesis in murine leukemia viruses (MuLVs). Leukemogenesis by MuLVs will be summarized here and possible implications to KoRV pathogenesis will be pointed out. Murine leukemia viruses MuLVs were first discovered in inbred mouse strains that had high incidences of leukemia. These studies resulted in isolation of several MuLV strains that cause leukemias of different hematopoietic lineages. For instance Moloney MuLV (M-MuLV) and Gross MuLV induce T-lymphoma, while Friend (F-MuLV) and Rauscher MuLV (R-MuLV) induce erythroleukemia and myeloid leukemia (Fan, 1997). These are the predominant MuLVs used in studies of MuLV leukemogenesis. They are prototypical retroviruses of the gammaretrovirus family. MuLVs can be classified into types based on their envelope proteins and the kinds of cells that they infect, determined by the cell surface proteins that they bind. The leukemogenic MuLVs are mostly ecotropic; they infect cells of mice and rats, but they do not infect most

Pathogenic retroviruses have evolved multiple means for evading host restriction factors such as apolipoprotein B editing complex (APOBEC3) proteins. Here, we show that murine leukemia virus (MLV) has a unique means of counteracting APOBEC3 and other cytosolic sensors of viral nucleic acid. Using virus isolated from infected WT and APOBEC3 KO mice, we demonstrate that the MLV glycosylated Gag protein (glyco-Gag) enhances viral core stability. Moreover, in vitro endogenous reverse transcription reactions of the glyco-Gag mutant virus were substantially inhibited compared with WT virus, but only in the presence of APOBEC3. Thus, glyco-Gag rendered the reverse transcription complex in the viral core resistant to APOBEC3. Glyco-Gag in the virion also rendered MLV resistant to other cytosolic sensors of viral reverse transcription products in newly infected cells. Strikingly, glyco-Gag mutant virus reverted to glyco-Gag–containing virus only in WT and not APOBEC3 KO mice, indicating that counteracting APOBEC3 is the major function of glyco-Gag. Thus, in contrast to the HIV viral infectivity factor protein, which prevents APOBEC3 packaging in the virion, the MLV glyco-Gag protein uses a unique mechanism to counteract the antiviral action of APOBEC3 in vivo—namely, protecting the reverse transcription complex in viral cores from APOBEC3. These data suggest that capsid integrity may play a critical role in virus resistance to intrinsic cellular antiviral resistance factors that act at the early stages of infection.

Retroviruses cause cancers in a variety of animals and humans. Research on retroviruses has provided important insights into mechanisms of oncogenesis in humans, including the discovery of viral oncogenes and cellular proto-oncogenes. The subject of this review is the mechanisms by which retroviruses that do not carry oncogenes (non-acute retroviruses) cause cancers. The common theme is that these tumors result from insertional activation of cellular proto-oncogenes by integration of viral DNA. Early research on insertional activation of proto-oncogenes in virus-induced tumors is reviewed. Research on non-acute retroviruses has led to the discovery of new proto-oncogenes through searches for common insertion sites (CISs) in virus-induced tumors. Cooperation between different proto-oncogenes in development of tumors has been elucidated through the study of retrovirus-induced tumors, and retroviral infection of genetically susceptible mice (retroviral tagging) has been used to identify cellular proto-oncogenes active in specific oncogenic pathways. The pace of proto-oncogene discovery has been accelerated by technical advances including PCR cloning of viral integration sites, the availability of the mouse genome sequence, and high throughput DNA sequencing. Insertional activation has proven to be a significant risk in gene therapy trials to correct genetic defects with retroviral vectors. Studies on non-acute retroviral oncogenesis provide insight into the potential risks, and the mechanisms of oncogenesis.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a lung cancer in sheep known as ovine pulmonary adenocarcinoma (OPA). The disease has been identified around the world in several breeds of sheep and goats, and JSRV infection typically has a serious impact on affected flocks. In addition, studies on OPA are an excellent model for human lung carcinogenesis. A unique feature of JSRV is that its envelope (Env) protein functions as an oncogene. The JSRV Env-induced transformation or oncogenesis has been studied in a variety of cell systems and in animal models. Moreover, JSRV studies have provided insights into retroviral genomic RNA export/expression mechanisms. JSRV encodes a trans-acting factor (Rej) within the env gene necessary for the synthesis of Gag protein from unspliced viral RNA. This review summarizes research pertaining to JSRV-induced pathogenesis, Env transformation, and other aspects of JSRV biology.

Murine leukemia viruses encode a unique form of Gag polyprotein, gPr80 gag or glyco-gag. Translation of this protein is initiated from full-length viral mRNA at an upstream initiation site in the same reading frame as Pr65 gag , the precursor for internal structural (Gag) proteins. Whereas gPr80 gag is evolutionarily conserved among gammaretroviruses, its mechanism of action has been unclear, although it facilitates virus production at a late assembly or release step. Here, it is shown that gPr80 gag facilitates release of Moloney murine leukemia virus (M-MuLV) from cells along an IFN-sensitive pathway. In particular, gPr80 gag -facilitated release occurs through lipid rafts, because gPr80 gag -negative M-MuLV has a lower cholesterol content, is less sensitive to inhibition of release by the cholesterol-depleting agent MβCD, and there is less Pr65 gag associated with detergent-resistant membranes in mutant-infected cells. gPr80 gag can also facilitate the release of HIV-1-based vector particles from human 293T cells.

All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80 gag ) in addition to the polyprotein precursor of the viral capsid proteins (Pr65 gag ). gPr80 gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65 gag . The role of glyco-Gag in MuLV replication has been unclear, since gPr80 gag -negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80 gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80 gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80 gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80 gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80 gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.

The native envelope gene ( env ) of Jaagsiekte sheep retrovirus (JSRV) also acts as an oncogene. To investigate the mechanism of transformation, we performed yeast 2-hybrid screening for cellular proteins that interact with Env. Among several candidates, we identified mouse or rat zinc finger protein 111 ( zfp111 ). The interaction between Env and Zfp111 was confirmed through in vivo coimmunoprecipitation assays. Knockdown of endogenous Zfp111 caused a decrease in cell transformation by JSRV Env, while overexpression of Zfp111 increased overall Env transformation, supporting a role for Zfp111 in Env transformation. Knockdown of Zfp111 had no effect on the growth rate of parental rat 208F cells, while it decreased the proliferation rate of JSRV-transformed 208F cells, suggesting that JSRV-transformed cells became dependent on Zfp111. In addition, Zfp111 preferentially bound to a higher-mobility form of JSRV Env that has not been described previously. The higher-mobility form of Env (P70 env ) was found exclusively in the nuclear fraction, and size of its polypeptide backbone was the same as that of the cytoplasmic Env polyprotein (Pr80 env ). The differences in glycosylation between the two versions of Env were characterized. These results identify a novel cellular protein, Zfp111, that binds to the JSRV Env protein, and this binding plays a role in Env transformation. These results indicate that JSRV transformation also involves proteins and interactions in the nucleus. IMPORTANCE The envelope protein (Env) of Jaagsiekte sheep retrovirus (JSRV) is an oncogene, but its mechanism of cell transformation is still unclear. Here we identified seven candidate cellular proteins that can interact with JSRV Env by yeast two-hybrid screening. This study focused on one of the seven candidates, zinc finger protein 111 (Zfp111). Zfp111 was shown to interact with JSRV Env in cells and to be involved in JSRV transformation. Moreover, coexpression of JSRV Env and Zfp111 led to the identification of a novel nuclear form of the JSRV Env protein that binds Zfp111. Nuclear Env was found to differ by glycosylation from the cytoplasmic Env precursor to the virion envelope proteins. These results suggest that JSRV Env transformation may involve nuclear events such as an alteration in transcription mediated by Env-Zfp111 interactions.

Background Koala retrovirus (KoRV) is an endogenous and exogenous retrovirus of koalas that may cause lymphoma. As for many other gammaretroviruses, the KoRV genome can potentially encode an alternate form of Gag protein, glyco-gag. Results In this study, a convenient assay for assessing KoRV infectivity in vitro was employed: the use of DERSE cells (initially developed to search for infectious xenotropic murine leukemia-like viruses). Using infection of DERSE and other human cell lines (HEK293T), no evidence for expression of glyco-gag by KoRV was found, either in expression of glyco-gag protein or changes in infectivity when the putative glyco-gag reading frame was mutated. Since glyco-gag mediates resistance of Moloney murine leukemia virus to the restriction factor APOBEC3, the sensitivity of KoRV (wt or putatively mutant for glyco-gag) to restriction by murine (mA3) or human APOBEC3s was investigated. Both mA3 and hA3G potently inhibited KoRV infectivity. Interestingly, hA3G restriction was accompanied by extensive G → A hypermutation during reverse transcription while mA3 restriction was not. Glyco-gag status did not affect the results. Conclusions These results indicate that the mechanisms of APOBEC3 restriction of KoRV by hA3G and mA3 differ (deamination dependent vs. independent) and glyco-gag does not play a role in the restriction. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0193-1) contains supplementary material, which is available to authorized users.

Moloney murine leukemia virus (M-MuLV) lacking the gene for the envelope glycoprotein (env-) was produced in NIH 3T3 cells and investigated using atomic force microscopy (AFM). The particles were compared with similarly produced wild-type virions, some of which had been exposed to a monoclonal antibody against the surface component of the envelope protein (SU protein). The env- particles generally exhibit a distinctly different external appearance suggesting only a low density of associated proteins that have an almost fluid, mechanically unstable character. The weakly associated proteins may be host cell membrane proteins that are incorporated into the viral membrane in place of or in addition to virus envelope protein. The amount of this non-viral protein on virion surfaces appears to vary from negligible in most cases to a substantial complement in others. It seems clear that the presence of the envelope protein, in a mechanical sense, significantly strengthens and stabilizes the virion envelope. Binding of monoclonal antibody to wild-type virions indicates that some particles expose a significant amount of antigen while adjacent virions may not. This suggests that the conformation of the envelope glycoprotein or the disposition of oligosaccharides may be different among particles, on some virions exposing the specific epitope, and others little or none. © 2004 Elsevier Inc. All rights reserved.

NIH 3T3 cells were infected in culture with the oncogenic retrovirus, mouse leukemia virus (MuLV), and studied using atomic force microscopy (AFM). Cells fixed with glutaraldehyde alone, and those postfixed with osmium tetroxide, were imaged under ethanol according to procedures that largely preserved their structures. With glutaraldehyde fixation alone, the lipid bilayer was removed and maturing virions were seen emerging from the cytoskeletal matrix. With osmium tetroxide postfixation, the lipid bilayer was maintained and virions were observable still attached to the cell surfaces. The virions on the cell surfaces were imaged at high resolution and considerable detail of the arrangement of protein assemblies on their surfaces was evident. Infected cells were also labeled with primary antibodies against the virus env surface protein, followed by secondary antibodies conjugated with colloidal gold particles. Other 3T3 cells in culture were infected with MuLV containing a mutation in the gPr80gag gene. Those cells were observed by AFM not to produce normal MuLV on their surfaces, or at best, only at very low levels. The cell surfaces, however, became covered with tubelike structures that appear to result from a failure of the virions to properly undergo morphogenesis, and to fail in budding completely from the cell's surfaces.

A systematic study on generating simian immunodeficiency virus (SIV)-based vectors was carried out. The goal was to generate helper-free, replication-defective SIVmac-based vectors at high titers. The general approach was to cotransfect into human 293T cells a plasmid carrying the vector construct along with two helper plasmids that together expressed the SIVmac virion proteins. Initial vectors carried the bacterial β-galactosidase gene (β-gal). These vectors had a technical difficulty: “pseudotransduction” of β-gal protein produced during the 293T cell transfections. As a result, infection of cultures with these vector stocks also resulted in passive transfer into, and X-gal staining of, cells that had not actually been infected by the vector. A second generation of vectors expressing the enhanced jellyfish green fluorescence protein (EGFP) was not subject to this artifact. A systematic study of the SIVmac-based EGFP vectors was carried out. Helper-free vector stocks were obtained when helper plasmids lacking the SIVmac packaging signals were used. By employing envelope helper plasmids derived from different SIVmac isolates, it was possible to generate SIVmac-based vectors pseudotyped with envelope proteins of different cell tropism. Optimization of vector and helper plasmid structures, transfection conditions, and infection procedures ultimately yielded vector titers in excess of 10 6 /ml.

Many retroviruses that carry oncogenes (acute transforming viruses) are generally replication-defective and therefore require co-infection with a replication competent 'helper' retrovirus for infectivity. The helper virus provides the retroviral proteins necessary for particle production and infection. These include the envelope glycoproteins that specifically bind to cell surface receptors and mediate viral adsorption and entry. Thus, a particular helper virus may influence the nature of disease induced by an oncogene-containing retrovirus due to tissue tropism of the helper. In a previous study, a replication-defective recombinant Moloney murine leukemia virus containing the v-myc oncogene was generated (M-MuLV(myc); Brightman B.K., Pattengale P.K., and Fan H., J Virol 60: 68-81, 1986). When M-MuLV(myc) was inoculated into mice using the non-pathogenic amphotropic murine leukemia virus (Am-MuLV 4070) as a helper, T- and B-lymphoblastic lymphomas resulted with the following two surface phenotypes, namely, (1) Thy 1.2+, B220- and (2) Thy 1.2-, B220+. Thy 1.2 surface antigen is characteristic of cells of the lymphoid lineage, whereas B220 surface antigen is characteristic of cells of the B-lymphoid lineage. In these experiments, to assess the influence of the helper virus on the disease specificity of M-MuLV(myc), two weakly pathogenic ecotropic helper MuLVs that interact with different cell surface receptors than Am-MuLV (Mo+PyF101 and AKV MuLV) were used to pseudotype M-MuLV(myc). In both cases, when inoculated into mice, these pseudotypes induced only T-lymphoblastic lymphoma. These results indicate that for M-MuLV(myc) the types of the tumors induced are influenced by the helper virus utilized, and they suggest that different lymphoid cells may express different levels of retroviral receptors.

Integrated into the sheep genome are 15 to 20 copies of type D endogenous loci that are highly related to two exogenous oncogenic viruses, jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV). The exogenous viruses cause infectious neoplasms of the respiratory tract in small ruminants. In this study, we molecularly cloned three intact type D endogenous retroviruses of sheep (enJS56A1, enJS5F16, and enJS59A1; collectively called enJRSV s) and analyzed their genomic structures, their phylogenies with respect to their exogenous counterparts, their capacity to form viral particles, and the expression specificities of their long terminal repeats (LTRs). In addition, the pattern of expression of enJSRV s in vivo was studied by in situ hybridization. All of the three enJSRV proviruses had open reading frames for at least one of the structural genes. In particular, enJS56A1 had open reading frames for all structural genes, but it could not assemble viral particles when highly expressed in human 293T cells. We localized the defect for viral assembly in the first two-thirds of the gag gene by making a series of chimeras between enJS56A1 and the exogenous infectious molecular clone JSRV 21 . Phylogenetic analysis distinguished five ovine type D retroviruses: enJSRV groups A and B, ENTV, and two exogenous JSRV groups (African versus United Kingdom/North America isolates). Transient transfection assays indicated that the LTRs of the three enJSRV s were not preferentially active in differentiated lung epithelial cells. This suggests that the pulmonary tropic JSRV developed from a type D retrovirus that did not have lung specificity. Consistent with this, in situ hybridization of a panel of normal ovine tissues revealed high expression of enJSRV mRNA in the luminal epithelium and glandular epithelium of the uterus; lower expression was localized in the lamina propria of the gut and in the bronchiolar epithelium of the lungs.

Most simple retroviruses induce tumors of a single cell type when infected into susceptible hosts. The SRS 19-6 murine leukemia virus (MuLV), which originated in mainland China, induces leukemias of multiple cellular origins. Indeed, infected mice often harbor more than one tumor type. Since the enhancers of many MuLVs are major determinants of tumor specificity, we tested the role of the SRS 19-6 MuLV enhancers in its broad disease specificity. The enhancer elements of the Moloney MuLV (M-MuLV) were replaced by the 170-bp enhancers of SRS 19-6 MuLV, yielding the recombinants ΔMo+SRS + and ΔMo+SRS − M-MuLV. M-MuLV normally induces T-lymphoid tumors in all infected mice. Surprisingly, when neonatal mice were inoculated with ΔMo+SRS + or ΔMo+SRS − M-MuLV, all tumors were of T-lymphoid origin, typical of M-MuLV rather than SRS 19-6 MuLV. Thus, the SRS 19-6 MuLV enhancers did not confer the broad disease specificity of SRS 19-6 MuLV to M-MuLV. However, all tumors contained ΔMo+SRS M-MuLV proviruses with common enhancer alterations. These alterations consisted of tandem multimerization of a subregion of the SRS 19-6 enhancers, encompassing the conserved LVb and core sites and adjacent sequences. Moreover, when tumors induced by the parental SRS 19-6 MuLV were analyzed, most of the T-lymphoid tumors had similar enhancer alterations in the same region whereas tumors of other lineages retained the parental SRS 19-6 MuLV enhancers. These results emphasize the importance of a subregion of the SRS 19-6 MuLV enhancer in induction of T-cell lymphoma. The relevant sequences were consistent with crucial sequences for T-cell lymphomagenesis identified for other MuLVs such as M-MuLV and SL3-3 MuLV. These results also suggest that other regions of the SRS 19-6 MuLV genome contribute to its broad leukemogenic spectrum.

The nature of Moloney murine leukemia virus (M-MuLV) infection after a subcutaneous (s.c.) inoculation was studied. We have previously shown that an enhancer variant of M-MuLV, Mo+PyF101 M-MuLV, is poorly leukemogenic when used to inoculate mice s.c., but not when inoculated intraperitoneally. This attenuation of leukemogenesis correlated with an inability of Mo+PyF101 M-MuLV to establish infection in the bone marrow of mice at early times postinfection. These results suggested that a cell type(s) is infected in the skin by wild-type but not Mo+PyF101 M-MuLV after s.c. inoculation and that this infection is important for the delivery of infection to the bone marrow, as well as for efficient leukemogenesis. To determine the nature of the cell types infected by M-MuLV and Mo+PyF101 M-MuLV in the skin after a s.c. inoculation, immunohistochemistry with an anti-M-MuLV CA antibody was performed. Cells of developing hair follicles, specifically cells of the outer root sheath (ORS), were extensively infected by M-MuLV after s.c. inoculation. The Mo+PyF101 M-MuLV variant also infected cells of the ORS but the level of infection was lower. By Western blot analysis, the level of infection in skin by Mo+PyF101 M-MuLV was approximately 4- to 10-fold less than that of wild-type M-MuLV. Similar results were seen when a mouse keratinocyte line was infected in vitro with both viruses. Cells of the ORS are a primary target of infection in vivo, since a replication defective M-MuLV-based vector expressing β-galactosidase also infected these cells after a s.c. inoculation.

The complete nucleotide sequence of the genome of Solid-type Reticulum cell Sarcoma 19-6 murine leukemia virus (SRS 19-6 MuLV) was determined. This virus was isolated in mainland China from laboratory mice that had been separated from western mice since the 1930s. The genome is 8,256 nucleotides in length and exhibits a genetic organization characteristic of replication competent MuLVs. Phylogenies constructed from reverse transcriptase (RT) domains showed that SRS 19-6 MuLV is closely related to other MuLV-related retroviruses; however, it has clearly diverged from previously isolated MuLVs. Comparative sequence analysis of the env sequences indicated that SRS 19-6 MuLV encodes a surface (SU) glycoprotein that is related to other ecotropic MuLVs in the VR-A and VR-B variable regions. However, SRS 19-6 MuLV env glycoprotein was distinct from all other MuLVs (ecotropic and non-ecotropic) in the proline-rich hypervariable region. No evidence for recombination with endogenous MuLV env sequences in generation of SRS 19-6 MuLV was observed. Comparisons of long terminal repeat (LTR) sequences revealed that the GV 1.4 molecular clone of Graffi MuLV contained 96% sequence identity to SRS 19-6 MuLV’s LTR with 99% identity when comparisons were restricted to the U3 regions of the two viruses. The consensus enhancer binding motifs contained in the U3 regions of the two viruses were nearly identical. Nevertheless the two viruses have previously been shown to induce distinct patterns of disease. Comparisons between 196 and Graffi GV1.4 MuLVs may provide insights into the mechanisms of disease specificity induced by MuLVs.

The Mo + PyF101 enhancer variant of Moloney murine leukemia virus (M-MuLV) has been very useful in investigating M-MuLV leukemogenesis. When inoculated subcutaneously (s.c.) into neonatal mice, Mo + PyF101 M-MuLV is attenuated for development of disease. Previous studies in mice infected with wild-type M-MuLV have revealed several important preleukemic events, including development of splenic hyperplasia, defects in bone marrow hematopoiesis, and in vivo generation of MCF viruses that arise by recombination in the uninfected mouse. Mo + PyF101 M-MuLV is defective in inducing these effects after s.c. inoculation. In the experiments reported here, a study of Mo + PyF101 M-MuLV infection in rats was carried out. Wild-type M-MuLV is leukemogenic in rats, but infected rats do not form MCF recombinants since they lack the necessary endogenous polytropic envelope sequences. Since Mo + PyF101 M-MuLV's leukemogenic defect is correlated with a failure to generate MCF recombinants, it seemed possible that wild-type M-MuLV might not have a leukemogenic advantage over Mo + PyF101 M-MuLV in rats, where MCF recombinants cannot form. Neonatal Fisher F344 rats were inoculated s.c. or intraperitoneally by wild-type and Mo + PyF101 M-MuLVs. Surprisingly, Mo + PyF101 M-MuLV was completely deficient in leukemogenesis in rats when inoculated by either route while wild-type M-MuLV induced lymphoma with the predicted time course. The leukemogenic defect for Mo + PyF101 M-MuLV resulted from a pronounced defect for establishing infection in rats. Further studies of wild-type M-MuLV in rats indicated that infection was confined almost exclusively to the thymus at early times. In mice wild-type M-MuLV establishes substantial infection in other hematopoietic organs such as spleen and bone marrow as well. Thymic infection was also correlated with a decrease in thymic cellularity at early times.

The promyelocytic leukemia ( PML ) gene, which encodes a growth- and transformation-suppressor, has been identified at the non-random chromosomal translocation break point t(15;17)(q22;q12) of acute promyelocytic leukemia. To elucidate if PML may play a role in cellular response to DNA damage, PML expression was analyzed by immunofluorescence staining in HeLa cells treated with ionizing radiation (IR) and cisplatin. Our studies demonstrated IR at 20Gy, and cisplatin at 6 μg/ml caused more than 5-10 fold increases in PML protein expression in the PML Oncogenic Domain (POD) by immunofluorescent staining. Northern blotting showed that there was no gross increase in mRNA levels indicating that the induction is a post-transcriptional event. Flow cytometry showed that HeLa cells treated with IR were progressively arrested in G1, which correlates with the optimal expression of PML in the cell cycle. To determine if PML expression was under the control of the tumor suppressor p53, which is known to arrest cells in G1, HeLa cells were transfected with the wild-type p53 gene. PML expression in p53 transduced cells were 5-10 fold higher than the control, indicating that the enhanced expression of PML is apparently dependent on the p53 pathway. These data also indicate that PML may play an important role in cellular response to DNA damage such as DNA repair or apoptosis during G1 arrest.

Background ZASC1 is a zinc finger-containing transcription factor that was previously shown to bind to specific DNA binding sites in the Moloney murine leukemia virus (Mo-MuLV) promoter and is required for efficient viral mRNA transcription (J. Virol. 84:7473-7483, 2010). Methods To determine whether this cellular factor influences Mo-MuLV replication and viral disease pathogenesis in vivo, we generated a ZASC1 knockout mouse model and completed both early infection and long term disease pathogenesis studies. Results Mice lacking ZASC1 were born at the expected Mendelian ratio and showed no obvious physical or behavioral defects. Analysis of bone marrow samples revealed a specific increase in a common myeloid progenitor cell population in ZASC1-deficient mice, a result that is of considerable interest because osteoclasts derived from the myeloid lineage are among the first bone marrow cells infected by Mo-MuLV (J. Virol. 73: 1617-1623, 1999). Indeed, Mo-MuLV infection of neonatal mice revealed that ZASC1 is required for efficient early virus replication in the bone marrow, but not in the thymus or spleen. However, the absence of ZASC1 did not influence the timing of subsequent tumor progression or the types of tumors resulting from virus infection. Conclusions These studies have revealed that ZASC1 is important for myeloid cell differentiation in the bone marrow compartment and that this cellular factor is required for efficient Mo-MuLV replication in this tissue at an early time point post-infection.

While virus growth dynamics have been well-characterized in several infections, data are typically collected once the virus population becomes easily detectable. Earlier dynamics, however, remain less understood. We recently reported unusual early dynamics in an experimental system using adenovirus infection of human embryonic kidney (293) cells. Under identical experimental conditions, inoculation at low infection multiplicities resulted in either robust spread, or in limited spread that eventually stalled, with both outcomes occurring with approximately equal frequencies. The reasons underlying these observations have not been understood. Here, we present further experimental data showing that inhibition of interferon-induced antiviral states in cells results in a significant increase in the percentage of robust infections that are observed, implicating a race between virus replication and the spread of the anti-viral state as a central mechanism. Analysis of a variety of computational models, however, reveals that this alone cannot explain the simultaneous occurrence of both viral growth outcomes under identical conditions, and that additional biological mechanisms have to be invoked to explain the data. One such mechanism is the ability of the virus to overcome the antiviral state through multiple infection of cells. If this is included in the model, two outcomes of viral spread are found to be simultaneously stable, depending on initial conditions. In stochastic versions of such models, the system can go by chance to either state from identical initial conditions, with the relative frequency of the outcomes depending on the strength of the interferon-based anti-viral response, consistent with the experiments. This demonstrates considerable complexity during the early phase of the infection that can influence the ability of a virus to become successfully established. Implications for the initial dynamics of oncolytic virus spread through tumors are discussed., Author Summary We investigate in vitro adenovirus spread starting from the lowest infection multiplicities. This phase of virus dynamics remains poorly understood and is likely critical for ensuring that engineered oncolytic viruses successfully spread and destroy tumors. We find unexpectedly complex dynamics, which are analyzed with a combination of experiments and mathematical models. The experiments indicate that the induction of an interferon-based anti-viral state is a crucial underlying mechanism. The mathematical models demonstrate that this mechanism alone cannot explain the experiments, and that additional mechanisms must be invoked to account for the data. The models suggest that the ability of the virus to overcome the anti-viral state through multiple infection of cells might be one such mechanism.

Characterization of the SRS murine retrovirus complex, derived from the TSZ system of murine leukemia developed in China, was carried out. The initial stock contained XC+, NB-tropic virus (and possibly other viruses), and induced several neoplastic diseases in neonatally inoculated NIH Swiss mice: erythroid leukemia, myeloid leukemia (acute myeloblastic leukemia), and lymphoblastic lymphoma (both B- and T-lymphoid). In addition, approximately 30% of inoculated animals developed central nervous system disease—hindlimb paralysis or semilateral paralysis. Rescue of virus from the spleen of an animal with combined erythroid/myeloid leukemia, followed by endpoint dilution gave two stocks: 19-6 (XC+, NB-tropic) and 19-7 (XC-, NB-tropic). Both stocks induced erythroid and myeloid leukemia, and 19-6 induced CNS symptoms as well. Southern blot analysis indicated that the predominant viruses from the 19-6 and the 19-7 cultures were related, but different in the env region. An infectious molecular clone of provirus from 19-6 cells was obtained. The resulting cloned virus [SRS 19-6 murine leukemia virus (MuLV)] induced four kinds of leukemia: erythroid, myeloid, B-lymphoma, and T-lymphoma; in many cases, more than one tumor type was identified in the same animal. Such a broad spectrum of leukemias induced by a cloned MuLV is unusual. Flaccid hindlimb paralysis induced by SRS 19-6 MuLV could be attributed to meningeal B-lymphoma. Immunofluorescent staining with a panel of env-specific monoclonal antibodies confirmed that the 19-6 and 19-7 viral stocks contained different viruses, which differed from previously characterized MuLVs. The viruses of the SRS complex may provide interesting reagents for investigations of MuLV-induced disease.

We previously described an enhancer variant of Moloney murine leukaemia virus (M-MuLV), delta Mo + SV M-MuLV, in which the enhancers of MuLV have been deleted and replaced with the enhancers of the simian virus 40 (SV40). When this virus is injected into neonatal NIH Swiss mice, pre-B and B-lymphoblastic lymphomas develop with a latency of 17 months. Van Lohuizen et al. (1989) described a line of transgenic mice that carry an activated pim-1 proto-oncogene transgene (E mu pim-1). They also reported that E mu pim-1 transgenic mice show greatly accelerated lymphoma development when infected with wild-type M-MuLV at birth. In these experiments, neonatal E mu pim-1 transgenic mice were infected intraperitoneally with delta Mo + SV M-MuLV. Marked acceleration of T-lymphoid leukaemia was seen. However, 10 of the 11 tumours analysed were found to be negative for the SV40 enhancers, but they still contained M-MuLV DNA as measured by Southern blot analysis. The LTRs on viruses cloned from two such tumours (as well as on virus recovered by infection onto NIH 3T3 cells) were characterized by PCR amplification, molecular cloning and sequence analysis. The LTR's from the two tumours were identical to each other and were distinct from both the delta Mo + SV M-MuLV and wild-type M-MuLV LTRs. However, they were identical to a rearranged solo M-MuLV LTR present in the E mu pim-1 transgene. These results indicate that the recombination in vivo between delta Mo + SV M-MuLV and the E mu pim-1 transgene yielded a replication-competent and pathogenic virus at high efficiency. This is the first report of in vivo recombination between an exogenous MuLV and a solo endogenous LTR.

Background One of the unique features of gammaretroviruses is that they contain an additional extended form of Gag, glyco-gag, which initiates in the leader sequence. MuLV glyco-gag, gPr80Gag, promotes retrovirus replication and disease progression. Although virtually all infectious MuLVs encode glyco-gag, XMRV (xenotropic murine leukemia virus-related virus) lacks the classical gPr80Gag sequence. We examined XMRV to determine if its leader sequence contains glyco-gag activity, whether the presence of conventional gPr80Gag affects replication of XMRV, and we describe the evolution of glyco-gag-deficient MuLVs in Mus. Results We introduced several mutations disrupting two putative but noncanonical glyco-gag proteins in the leader sequence region in XMRV and found that those mutations did not affect virus release nor susceptibility to the antiviral activity of hA3G (human APOBEC3G). A chimeric XMRV encoding the Moloney MuLV (M-MuLV) leader sequence (MXMRV) demonstrated that M-MuLV glyco-gag facilitated MXMRV release and increased infectivity. Infectivity assays with several cell lines showed that glyco-gag increases XMRV infectivity in all cell lines tested, but the level of this increase varies in different cell lines. Because MuLV glyco-gag counteracts mouse APOBEC3, we investigated whether M-MuLV glyco-gag enhances XMRV infection by counteracting human APOBEC3. Comparison of hAPOBEC3 isoforms expressed in different cell lines indicated that hA3B was the most likely candidate for a restrictive hA3. However over-expression of hA3B showed no enhanced restriction of infection by XMRV compared to MXMRV. Endogenous MuLVs in the sequenced mouse genome were screened for canonical glyco-gag, which was identified in two clades of xenotropic MuLVs (X-MuLVs) and ecotropic MuLVs, but not in other X-MuLVs or in any polytropic MuLVs. Conclusions M-MuLV glyco-gag facilitates XMRV replication, and the leader sequence region in XMRV does not encode proteins equivalent to M-MuLV glyco-gag. The fact that the ability of glyco-gag to enhance XMRV infection varies in different cell lines suggests a glyco-gag sensitive restrictive factor that further reduces XMRV infectivity. The M-MuLV glyco-gag enhancement for XMRV replication is through a hAPOBEC3 independent mechanism. The absence of glyco-gag in MuLVs carried by western European mice suggests that loss of this sequence is a relatively recent event with limited subspecies distribution.

Two independent pathways for gag gene expression exist in Moloney murine leukemia virus (M-MuLV). One begins with Pr65(gag) that is processed and cleaved into the internal structural proteins of the virion. The other pathway begins with the glycosylated gag polyprotein, gPr80(gag). gPr80(gag) consists of Pr65(gag) plus additional N-terminal residues and it is glycosylated. A glycosylated-gag-negative mutant of M-MuLV (Ab-X-MLV) was previously constructed and shown to replicate in tissue culture. To test for the importance of glycosylated gag in vivo, the Ab-X-MLV mutant was inoculated intraperitoneally into newborn NIH Swiss mice. Mutant-infected mice developed typical lymphoblastic lymphomas at rates comparable to wild-type M-MuLV at either high (2 x 10(4) XC pfu/animal) or low (2 x 10(2) XC pfu/animal) doses. However, when viral protein expression was examined in the resultant tumors, six out of six mice showed evidence of virus that had recovered gPr80(gag) expression. These results suggest that glycosylated gag is important for M-MuLV propagation or leukemogenesis in vivo. Copyright 1994 S. Karger AG, Basel

Studies of oncogenic viruses have made seminal contributions to the molecular biology of cancer. Key discoveries include the identification of viral oncogenes and cellular proto-oncogenes, elucidation of signal transduction pathways, and identification of tumor suppressor genes. The origins of cancer virology began almost exactly one hundred years ago with the discovery of avian sarcoma and acute leukemia viruses—RNA-containing viruses of the retrovirus family. The study of animal cancer viruses accelerated beginning in the late 1960s and early 1970s, with the discovery of DNA viruses that could transform cells in culture, and the development of quantitative assays for transformation by DNA and RNA-containing tumor viruses. The discovery of reverse transcriptase in retroviruses in 1970 also greatly accelerated research on these viruses. Indeed RNA and DNA tumor viruses led the way in cancer molecular biology during this era before molecular cloning. It was possible to physically purify virus particles and generate specific hybridization probes for viral DNA and RNA at a time when it was not possible to analyze cellular genes in the same manner. [...]

Murine leukemia viruses (MuLVs) encode two forms of Gag polyprotein: the precursor for the viral core proteins (Pr65gag for Moloney MuLV [M-MuLV]) and a longer glycosylated form (glyco-gag, or gPr80gag). gPr80gag is translated from the same unspliced viral RNA as Pr65gag, from an upstream in-frame CUG initiation codon. As a result, gPr80gag contains 88 unique N-terminal amino acids that include a signal peptide that conducts gPr80gag into the rough endoplasmic reticulum, where it is glycosylated, exported to the cell surface, and cleaved into two proteins of 55 and 40 kDa. The amino-terminal 55-kDa protein remains cell associated with the 88 unique amino acids exposed to the cytosol. We previously showed that gPr80gag facilitates efficient M-MuLV release through lipid rafts. In this report, we found that the unique N-terminal domain of gPr80gag is sufficient to facilitate enhanced M-MuLV particle release from transfected 293T cells. A search for cellular proteins involved in gPr80gag function led to cellular La protein. Overexpression of mouse or human La enhanced M-MuLV particle release in the absence of glyco-gag, and the released virus had a reduced buoyant density characteristic of increased cholesterol content. Moreover, small interfering RNA (siRNA) knockdown of human La abolished glyco-gag enhancement of M-MuLV release. These results implicate La as a cellular protein involved in M-MuLV glyco-gag function. We also found that overexpression of mouse or human La could enhance HIV-1 release in the absence of gPr80gag. Therefore, M-MuLV and HIV-1 may share a pathway for release through lipid rafts involving La., IMPORTANCE Retroviruses cause diseases such as leukemia and AIDS. An important aspect of viral replication is how viruses are released from infected cells. We previously found that a unique protein encoded by murine leukemia viruses (MuLVs), glyco-gag (or gPr80gag), enhances efficient virus release through cholesterol-rich membrane subdomains called lipid rafts. In this study, we found that the N-terminal domain of gPr80gag is sufficient to enhance viral release. A search for cellular proteins that participate in gPr80gag function led to cellular La protein. Overexpression of La phenocopied glyco-gag in enhancing M-MuLV release, and knockdown of La abolished glyco-gag function. M-MuLV glyco-gag also enhanced release of HIV-1, as did overexpression La in the absence of glyco-gag. Thus, M-MuLV and HIV-1 may share a cellular pathway for release through lipid rafts involving La. These results may also be relevant for other viruses that are released through lipid rafts.

The long terminal repeats (LTRs) of defective Moloney Murine Leukemia Virus (M-MuLV) containing the avian v-myc and v-src oncogenes were exchanged for LTRs from murine retroviruses inducing myeloid and erythroid disease, in an attempt to retarget disease specificity. Chimeric MuLVs containing either the Myeloproliferative sarcoma virus or the Rauscher mink cell focus-inducing virus LTRs induced the same disease as the parental viruses, suggesting that for these viruses the v-myc and v-src oncogenes are the major determinants in the disease specificity. However, substitution of the LTRs did affect the efficiency of tumorigenesis.

Retroviruses express Gag and Pol proteins by translation of unspliced genome-length viral RNA. For some retroviruses, transport of unspliced viral RNA to the cytoplasm is mediated by small regulatory proteins such as human immunodeficiency virus Rev, while other retroviruses contain constitutive transport elements in their RNAs that allow transport without splicing. In this study, we found that the betaretrovirus Jaagsiekte sheep retrovirus (JSRV) encodes within the env gene a trans -acting factor (Rej) necessary for the synthesis of Gag protein from unspliced viral RNA. Deletion of env sequences from a JSRV proviral expression plasmid (pTN3) abolished its ability to produce Gag polyprotein in transfected 293T cells, and Gag synthesis could be restored by cotransfection of an env expression plasmid (ΔGP). Deletion analysis localized the complementing activity (Rej) to the putative Env signal peptide, and a signal peptide expression construct showed Rej activity. Two other betaretroviruses, mouse mammary tumor virus (MMTV) and human endogenous retrovirus type K, encode analogous factors (Rem and Rec, respectively) that are encoded from doubly spliced env mRNAs. Reverse transcriptase-PCR cloning and sequencing identified alternate internal splicing events in the 5′ end of JSRV env that could signify analogous doubly spliced Rej mRNAs, and cDNA clones expressing two of them also showed Rej activity. The predicted Rej proteins contain motifs similar to those found in MMTV Rem and other analogous retroviral regulatory proteins. Interestingly, in most cell lines, JSRV expression plasmids with Rej deleted showed normal transport of unspliced JSRV RNA to the cytoplasm; however, in 293T cells Rej modestly enhanced export of unspliced viral RNA (2.8-fold). Metabolic labeling experiments with [ 35 S]methionine indicated that JSRV Rej is required for the synthesis of viral Gag polyprotein. Thus, in most cell lines, the predominant function of Rej is to facilitate translation of unspliced viral mRNA.

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus causing a contagious lung cancer of sheep. JSRV encodes unspliced and spliced viral RNAs, among which unspliced RNA encodes Gag and Pol proteins and a singly spliced mRNA encodes Env protein. In another study we found that JSRV encodes a regulatory protein, Rej, that is responsible for synthesis of Gag polyprotein from unspliced viral RNA. Rej is encoded in the 5′ end of env , and it enhances nuclear export or accumulation of cytoplasmic unspliced viral RNA in 293T cells but not in most other cell lines (A. Hofacre, T. Nitta, and H. Fan, J. Virol. 83:12483-12498, 2009). In this study, we found that mutations in the 3′ end of env in the context of a cytomegalovirus-driven full-length JSRV expression construct abolished Gag protein synthesis and released viruses in 293T cells. These mutants also showed deficits in accumulation of unspliced viral RNA in the cytoplasm. These mutants defined a Rej-responsive element (RejRE). Inhibition of CRM1 but not Tap function prevented nuclear export/accumulation of cytoplasmic unspliced RNA in 293T cells, similarly to other complex retroviruses that express analogous regulator proteins (e.g., human immunodeficiency virus Rev). Structural modeling of the RejRE with Zuker M-fold indicated a region with a predicted stable secondary structure. Mutational analysis in this region indicated the importance of both secondary structures and primary nucleotide sequences in a central stem-bulge-stem structure. In contrast to 293T cells, mutations in the RejRE did not affect the levels of cytoplasmic unspliced RNA in 293 cells, although the unspliced RNA showed partial degradation, perhaps due to lack of translation. RejRE-containing RNA relocalized Rej protein from the nucleus to the cytoplasm in 293 and rat 208F cells, suggesting binding of Rej to the RejRE.

It is now well established that proteins belonging to the APOBEC3 (A3) family are potent restriction factors of viruses and retrotransposable elements (6, 16, 46). Several mechanisms have been proposed for the antiviral activity of APOBEC3. Most, if not all, family members have cytidine deaminase (CDA) activity. As for human immunodeficiency virus type 1 (HIV-1) lacking the viral infectivity factor (vif) gene, human APOBEC3G (hA3G) protein is incorporated into budding HIV-1 virions and deaminates cytidine to uridine residues during reverse transcription. This causes hypermutation in the nascent retroviral DNA and restricts infection through increased degradation of the DNA and the generation of nonfunctional proviral genomes (16, 26). This mechanism is counteracted by Vif, which binds and degrades hA3G via the ubiquitin/proteasome pathway in virus-producing cells, thereby preventing it from being packaged (27, 47, 49, 55). A3 proteins also inhibit replication by an undefined CDA-independent mechanism(s) (33, 48). A3 proteins packaged into virions have been shown to inhibit the accumulation of HIV-1 reverse transcription products, plus-strand DNA transfer, and provirus integration, at least in tissue culture cells (18, 24, 29). In addition to A3 proteins inhibiting infection when packaged into virions, there is mounting evidence that these cellular proteins can restrict incoming virus particles. For example, hA3G expressed in recipient resting CD4+ T cells and mature dendritic cells (mDCs) functions as a postentry, Vif-resistant restriction for HIV infection (7, 39). Several A3 proteins are also expressed at higher levels in cells that restrict HIV-1 infection, such as monocytes and mDCs (36, 37, 39). Indeed, hA3G levels increase during dendritic cell (DC) maturation ex vivo, and treatment of several different cell types with alpha interferon (IFN-α) leads to higher levels of hA3G expression (5, 37, 39, 43, 50). Unlike humans, whose genome carries seven A3 genes, mice have a single mA3 gene, and although the mouse protein restricts HIV-1 infection and is a functional CDA, the domain organization of this single gene is different from any of the human genes (8, 14, 23). Previously, we showed that mice with targeted deletion of the mA3 gene supported higher levels of infection by the betaretrovirus mouse mammary tumor virus (MMTV) than their wild-type counterparts (35). Moreover, we showed that this restriction was independent of A3 CDA activity. More recently, several groups have demonstrated that mA3 inhibits infection by murine leukemia virus and that this also occurs without cytidine deamination (3, 42). Thus, although both MMTV and murine leukemia virus are infectious in mice, it is likely that endogenous A3 plays a role in limiting infection and thereby pathogenesis. In vivo, MMTV first infects DCs at the site of infection, which then carry the virus to lymph nodes (9). Infected DCs produce virus and, because they are professional antigen-presenting cells, present the MMTV-encoded superantigen (Sag) to T cells expressing Sag-specific T-cell receptor Vs chains (41). This results in the stimulation of Sag-reactive T cells, proliferation of B and T cells, and subsequent amplification of infection. MMTV's interactions with DCs also induce their maturation and migration, causing increased surface expression of costimulatory molecules and the MMTV entry receptor transferrin receptor 1 (TfR1) (4, 9, 28). Although MMTV infection of DCs leads to maturation and increased TfR1 expression on the cell surface, MMTV nonetheless infects immature DCs (iDCs) more efficiently than it infects mDCs (51). In this study, we examined whether this preferential replication pattern was due to differential restriction of MMTV by mA3 in iDCs versus that in mDCs. We demonstrate that virion-packaged and DC-intrinsic mA3 function together to restrict MMTV infection ex vivo and in vivo and that this restriction is not due to cytidine deamination. We also show that DC activation and maturation increase mA3 levels in target cells and that this increase results in reduced viral infection ex vivo and in vivo. These studies point to the importance of examining the role of host antiviral mechanisms using natural pathogens and their hosts.

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-lymphoblastic lymphoma 2 to 4 months after inoculation. Enhancer sequences in the U3 region of the M-MuLV long terminal repeat, primarily the 75-bp tandem repeats, strongly influence the disease specificity and latency of M-MuLV. We investigated the role of GC-rich sequences downstream of the tandem repeats in the disease specificity of M-MuLV. A recombinant M-MuLV lacking 23 bases of a GC-rich sequence (-174 to -151), Delta 27A M-MuLV, was tested for pathogenesis in neonatal NIH Swiss mice. Delta 27A M-MuLV induced disease with a longer latency than did M-MuLV (7 versus 3 months) in greater than 85% of inoculated mice. More interestingly, this virus showed an expanded repertoire of hematopoietic diseases. Molecular analyses and histopathologic examinations indicated that while 39% of mice inoculated with Delta 27A M-MuLV developed T-cell lymphoblastic lymphoma typical of wild-type M-MuLV, the majority developed acute myeloid leukemia, erythroleukemia, or B-cell lymphoma. Viral DNA corresponding to Delta 27A M-MuLV was detectable in most of the tumors analyzed. These findings indicate that the GC-rich region significantly influences the disease specificity and latency of M-MuLV.

We previously described a preleukemic state induced by Moloney murine leukemia virus (Mo-MuLV) characterized by hematopoietic hyperplasia in the spleen. Further experiments suggested that splenic hyperplasia results from inhibitory effects in the bone marrow, leading to compensatory extramedullary hematopoiesis. An enhancer variant of Mo-MuLV, Mo + PyF101 Mo-MuLV, fails to induce preleukemic hyperplasia and has greatly reduced leukemogenicity, indicating the importance of this state to efficient leukemogenesis. An alternative method for induction of preleukemic hyperplasia was sought. Treatment of mice with 89Sr causes specific ablation of bone marrow hematopoiesis and compensatory extramedullary hematopoiesis in spleen and nodes. NIH Swiss mice were inoculated neonatally with Mo + PyF101 Mo-MuLV and treated with 89Sr at 6 weeks of age. Approximately 85% developed lymphoid leukemia with a time course resembling that caused by wild-type Mo-MuLV. In contrast, very few animals treated with Mo + PyF101 Mo-MuLV or 89Sr alone developed disease. In approximately one-third of cases, the Mo + PyF101 Mo-MuLV proviruses were found at common sites for wild-type Mo-MuLV-induced tumors (c-myc, pvt-1, and pim-1), indicating that this virus is capable of performing insertional activation in T-lymphoid cells. These results support the proposal that splenic hyperplasia results from inhibitory effects in the bone marrow. They also indicate that Mo + PyF101 Mo-MuLV is blocked in early and not late events in leukemogenesis.

Retroviruses are associated with a variety of diseases including an array of malignancies, immunodeficiencies and neurological disorders. In particular, studies of oncogenic retroviruses established fundamental principles of modern molecular cancer biology. Studies of avian Rous sarcoma virus (RSV) led to the discovery of the viral oncogene src, and this was followed by the discovery of other viral oncogenes in retroviruses of mammals including rodents, cats, monkeys and so forth. Studies of the viral oncogenes in turn led to the discovery of cellular proto-oncogenes in the host genome; cellular oncogenes have been shown to be activated in a variety of human cancers, including those with no viral involvement. Oncogenic animal retroviruses can be divided into two groups based on their mechanisms of tumourigenesis, acute transforming retroviruses and nonacute retroviruses. Acute transforming retroviruses are typically replication defective and they induce tumours rapidly due to expression of their viral oncogenes. Nonacute retroviruses are replication competent and they induce tumours with longer latencies, by activating cellular proto-oncogenes in the tumour cells; this results from insertion of proviral DNA in the vicinity of the activated proto-oncogene. More recently, human T-cell leukaemia virus type I (HTLV-I) was discovered as an etiological agent of human cancer (adult T-cell leukaemia [ATL]); this virus also encodes regulatory genes some of which are important for its oncogenic potential. Most recently, the retroviral structural protein Envelope (Env) has been shown to be directly involved in oncogenic transformation for certain retroviruses. Env-induced transformation is a new paradigm for retroviral oncogenesis. In this review, we will summarise research on retrovirus oncogenic transformation over the past 100 years since the first published report of an oncogenic virus with particular attention to Env-induced transformation.

Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor virus (ENTV), and endogenous sheep retroviruses (ESRVs) are highly related sheep betaretroviruses that display different expression profiles in vivo. JSRV and ENTV are expressed in lungs and nasal adenocarcinomas, respectively, while ESRVs are primarily expressed in the reproductive tract of ewes. Evidence suggests that the cell tropism of JSRV, ENTV, and ESRVs is due to the transcriptional specificity of the LTRs. We have previously found several enhancer elements in the JSRV LTR that are important for lung-specific expression, including binding sites for the lung-specific transcription factor HNF-3beta, as well as binding sites for the ubiquitously expressed transcription factors C/EBP and NF-I. In this study, we have aligned the U3 regions of JSRV, ENTV, and several ESRVs in order to compare the transcriptional enhancer elements of JSRV that are conserved or absent in ESRV and ENTV. All three JSRV U3 sequences examined contain two conserved HNF-3 binding sites, while the ENTV and ESRV U3 regions are not predicted to bind this transcription factor. In addition, the C/EBP binding site is interrupted in the ESRV LTRs, but conserved in the ENTV LTRs. Some enhancer elements are conserved between JSRV and ENTV, but a reporter vector carrying the ENTV-1 LTR showed less activity than a JSRV LTR-driven reporter vector in a lung epithelial cell line. These studies support the importance of LTR enhancer elements in the respective tissue specificities of these exogenous and endogenous betaretroviruses.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a transmissible lung cancer in sheep. One of the unique features of this virus is that in infected animals, the only tissues that show expression of the virus are the tumor cells in the lung. We previously showed that the JSRV long terminal repeat (LTR) is preferentially active in murine lung epithelial cell lines (MLE-15 and mtCC1-2). To further explore the tissue specificity, we inserted the JSRV enhancer sequences from the U3 region of the LTR into a Moloney murine leukemia virus (M-MuLV) LTR lacking its own enhancer sequences, to give the chimeric LTR DeltaMo + JS. Transient transfection assays indicated that the DeltaMo + JS LTR is5-fold more active in lung epithelial cell lines than in non-lung lines, compared to the wild-type M-MuLV LTR. This was due to preferential activity of the JSRV enhancers in lung epithelial cells. Moreover, M-MuLV driven by the DeltaMo + JS LTR was3 logs more infectious in MLE-15 cells compared to non-lung cell lines. This chimeric virus may facilitate investigations of the tissue-specificity of JSRV.

Retroviral pseudotyping for gene transfer applications endeavors to alter vector tropism and maintain a suitable titer. We investigated the compatibility of the Jaagsiekte sheep retrovirus (JSRV) envelope glycoprotein with the feline immunodeficiency virus (FIV) vector. A construct consisting of the minimal JSRV env coding region expressed from a standard mammalian expression plasmid generated FIV vector titers of approximately 10(4) TU/ml following standard triple transfection, collection of supernatants, and concentration by centrifuge. Interestingly, retention of the native proviral 5' and 3' flanking regions surrounding the JSRV env resulted in exceptional titers of approximately 10(8) TU/ml following the same viral preparation. To discern the regions necessary to achieve this 10,000-fold increase in titer, additional constructs were designed and tested. Our results indicate that the enhanced vector titer correlates with an increase in steady-state levels of envelope RNA that results from a combination of RNA splicing and stability, leading to increased envelope protein production. Expression of four other glycoproteins in an expression plasmid retaining the enhancing elements from the JSRV proviral sequence increased FIV vector titers from 0- to 100-fold. These novel data demonstrate that optimization of the envelope expression construct can profoundly influence titers for lentivirus vectors.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of a contagious lung cancer of sheep, ovine pulmonary adenocarcinoma (OPA). In this study, we characterized the virus-specific RNAs in 293T cells transiently transfected with a human cytomegalovirus promoter-driven JSRV expression plasmid, in productively infected OHH1.LU deer lung cells, and in OPA tumors from field isolates. Typical unspliced (presumably for gag, pro, and pol) and singly spliced env mRNAs were detected. In addition, six other virus-specific RNAs were detected that resulted from the use of alternate splice acceptor sites and two premature polyadenylation sites (located in gag and in env). The orf-x gene of the virus appears to be expressed from two singly spliced subgenomic mRNAs of 3.2 kb that would encode an independent orf-x protein of 179 amino acids. In addition, the results suggested that there may also be an internal promoter for orf-x.

Our goal was to develop a system to study proteins that associate in vivo with the Moloney murine leukemia virus (M-MuLV) enhancer elements by the isolation of intact proviral chromatin. The M-MuLV long terminal repeats (LTRs) contain tandemly repeated transcriptional enhancer sequences consisting of smaller motifs that bind cellular DNA-binding proteins implicated in transcriptional regulation. The M-MuLV enhancers are also important for disease specificity and latency of disease induction. To enrich for proviral chromatin containing M-MuLV LTR sequences, an affinity purification scheme was employed that relies on the affinity of bacterial Lac repressor protein for Lac operator (LacO) DNA sequences. An infectious M-MuLV recombinant was constructed that contains bacterial LacO sequences inserted into a nonessential region downstream from the 5' LTR of the virus (M-MuLV-LacO). Nuclei from M-MuLV-LacO-infected cells were digested with PvuII (which will liberate an LTR fragment containing LacO sequences), and digested chromatin was leached from the nuclei in hypotonic buffer. M-MuLV-LacO chromatin was then recovered by binding to an affinity matrix consisting of a beta-galactosidase-Lac repressor fusion protein anchored to acrylamide beads by an anti-beta-galactosidase monoclonal antibody [7]. Specifically bound chromatin was eluted under physiological conditions by incubation with the galactose analog isopropyl-beta-D-thiogalactopyranoside. Southern blot analysis confirmed the specific enrichment of M-MuLV proviral chromatin by this method.

Cell tropism of human and simian immunodeficiency viruses (HIV and SIV, respectively) is governed in part by interactions between the viral envelope protein and the cellular receptors. However, there is evidence that envelope-host cell interactions also affect postentry steps in viral replication. We used a helper-free replication-defective SIV macaque (SIVmac)-based retroviral vector carrying the enhanced jellyfish green fluorescent protein inserted into thenefregion (V1EGFP) to examine SIV tropism in a single cycle of infection. Vector stocks containing envelope proteins from three different SIVmac clones, namely, SIVmac239 (T-lymphocyte tropic [T-tropic]), SIVmac316 (macrophage tropic [M-tropic]), and SIVmac1A11 (dualtropic), were tested. SIVmac239 replicates efficiently in many human T-cell lines, but it does not efficiently infect primary rhesus macrophages. Conversely, SIVmac316 efficiently infects primary macrophages, but it does not replicate in Molt4-Clone8 (M4C8) T cells. SIVmac1A11 replicates efficiently in both cell types. When primary macrophages were infected with V1EGFP pseudotyped by SIVmac316 or SIVmac1A11 envelopes, the infection was substantially (ca. 200- to 300-fold) more efficient than for the SIVmac239 pseudotype. Thus, in primary macrophages, a major component of M versus T tropism involves relatively early events in the infection cycle. Quantitative PCR studies indicated that synthesis and transport of vector DNA into the nucleus were similar for macrophages infected with the clone 239 and 316 pseudotypes, suggesting that the restriction for SIVmac239 infection is after reverse transcription and nuclear import of viral DNA. When the same vector pseudotypes were used to infect M4C8 cells, they all showed approximately equivalent infectivities, even though replication-competent SIVmac316 does not continue to replicate in these cells. Therefore, in M4C8 cells, restriction involves a late step in the infection cycle (after proviral integration and expression). Thus, depending on the cell type infected, envelope-dependent cell interactions that govern SIV M and T tropism may involve different steps in infection.

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s.c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s.c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.

Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphoma with a mean latency of 3 to 4 months. During the preleukemic period (4 to 10 weeks postinoculation) a marked decrease in thymic size is apparent for M-MuLV-inoculated mice in comparison to age-matched uninoculated mice. We were interested in studying whether the thymic regression was due to an increased rate of thymocyte apoptosis in the thymi of M-MuLV-inoculated mice. Neonatal NIH/Swiss mice were inoculated subcutaneously (s.c.) with wild-type M-MuLV (approximately 10 5 XC PFU). Mice were sacrificed at 4 to 11 weeks postinoculation. Thymic single-cell suspensions were prepared and tested for apoptosis by two-parameter flow cytometry. Indications of apoptosis included changes in cell size and staining with 7-aminoactinomycin D or annexin V. The levels of thymocyte apoptosis were significantly higher in M-MuLV-inoculated mice than in uninoculated control animals, and the levels of apoptosis were correlated with thymic atrophy. To test the relevance of enhanced thymocyte apoptosis to leukemogenesis, mice were inoculated with the Mo+PyF101 enhancer variant of M-MuLV. When inoculated intraperitoneally, a route that results in wild-type M-MuLV leukemogenesis, mice displayed levels of enhanced thymocyte apoptosis comparable to those seen with wild-type M-MuLV. However, in mice inoculated s.c., a route that results in attenuated leukemogenesis, significantly lower levels of apoptosis were observed. This supported a role for higher levels of thymocyte apoptosis in M-MuLV leukemogenesis. To examine the possible role of mink cell focus-forming (MCF) recombinant virus in raising levels of thymocyte apoptosis, MCF-specific focal immunofluorescence assays were performed on thymocytes from preleukemic mice inoculated with M-MuLV and Mo+PyF101 M-MuLV. The results indicated that infection of thymocytes by MCF virus recombinants is not required for the increased level of apoptosis and thymic atrophy.

We previously showed that neonatal mice inoculated with Moloney murine leukemia virus (M-MuLV) exhibit a preleukemic state characterized by splenomegaly and increased numbers of hematopoietic progenitors. An M-MuLV variant with greatly reduced leukemogenic potential, Mo+PyF101 M-MuLV, does not generally induce this preleukemic state. In order to investigate the mechanism involved in M-MuLV induction of preleukemic hyperplasia, we tested the CFU-mixed myeloid and erythroid (CFUmix) from M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice for the presence of virus by antibody staining and for the release of infectious virus. The majority of CFUmix colonies from both M-MuLV- and Mo+PyF101 M-MuLV-inoculated mice contained infectious virus even though M-MuLV-inoculated mice showed elevated levels of CFUmix while the Mo+PyF101 M-MuLV-inoculated mice did not. This indicates that direct infection of hematopoietic progenitors was not sufficient to induce hyperplasia. Rather, hematopoietic hyperplasia may result indirectly from infection of some other cell type.

Viruses and cancer have had a long history, beginning from the first discoveries of cancer viruses in animals in the late 19th century. Animal cancer viruses have provided great insights into fundamental processes of cancer, such as the discovery of cellular protooncogenes and tumor suppressor proteins (1). More recently, the involvement of viruses in human cancer has been established (2). It is now estimated that 20–25% of human cancers worldwide have a known viral etiology (2) (Table 1), although, given the multifactorial nature of carcinogenesis, other factors also contribute. In some cases, the viruses directly promote tumor development through expression of viral protein(s) that alter the growth properties of the tumor cell. For instance, the E6 and E7 proteins of oncogenic human papillomaviruses (HPVs) bind and inactivate cellular tumor suppressor proteins (3). In other cases viruses may indirectly cause cancer; the tumor cells themselves may not express the virus. Hepatitis B and C viruses appear to cause liver cancer by inducing chronic destruction and compensatory replacement of hepatocytes; the continual hepatocyte division may establish a cellular environment where other changes lead to cancer. In addition, human immunodeficiency virus (HIV) that causes AIDS indirectly causes cancer by crippling immune defenses against cancers induced by Kaposi's sarcoma herpes virus/HHV-8, Epstein–Barr virus, and HPV 6. Viral involvements in human cancer have practical implications. First, prevention of infection may reduce the risk of developing cancer; vaccines for oncogenic HPV have recently been approved for prevention of cervical cancer. Second, mechanistic studies may identify viral or cellular targets for cancer treatment or prevention. In this issue of PNAS, Dong et al. (4) provide evidence for a new human retrovirus associated with familial prostate cancer (PC).

Moloney murine leukemia virus is a prototypical simple retrovirus that has been an extremely useful model for leukemogenesis. Important steps in leukemogenesis include proviral activation of cellular proto-oncogenes, generation of mink cell focus-inducing recombinants, and early (preleukemic) virus-induced changes in hematopoiesis.

We recently showed that different routes of inoculation affect the leukemogenicity of the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV). Intraperitoneal (i.p.) inoculation of neonatal mice with Mo+PyF101 M-MuLV greatly enhanced its leukemogenicity compared with subcutaneous (s.c.) inoculation. We previously also suggested that the leukemogenicity defect of Mo+PyF101 M-MuLV when inoculated s.c. may result from the inability of this virus to form env gene recombinant (mink cell focus-inducing [MCF]) virus. In this study, virus present in end-stage tumors and in preleukemic animals inoculated i.p. by Mo+PyF101 M-MuLV was characterized. In contrast to s.c. inoculation, all tumors from i.p.-inoculated mice contained high levels of recombinant MCF virus. Furthermore, Southern blot analyses demonstrated that the majority of the tumors contained altered Mo+PyF101 M-MuLV long terminal repeats. The U3 regions from several tumors with altered long terminal repeats were cloned by PCR amplification. Sequence analyses indicated that the M-MuLV 75-bp tandem repeat in the enhancer region was triplicated. This amplification was also previously observed in mice infected s.c. with a pseudotypic mixture of Mo+PyF101 M-MuLV and Mo+PyF101 MCF virus. The enhancer triplication was an early event, and it occurred within 2 weeks postinfection. Recombinant MCF viruses were not detected by Southern blot analyses until 4 weeks postinfection. Thus, the M-MuLV enhancer triplication event was initially important for efficient propagation of ecotropic Mo+PyF101 M-MuLV. The increased leukemogenicity following i.p. inoculation could be explained if the triplication enhances Mo+PyF101 M-MuLV replication in the bone marrow and bone marrow infection is required for recombinant MCF virus formation.

We previously showed that the Mo+PyF101 variant of Moloney murine leukemia virus (M-MuLV) is poorly leukemogenic when inoculated subcutaneously (s.c.) into neonatal mice. We recently found that intraperitoneal (i.p.) inoculation of neonatal mice with the same virus significantly enhanced its leukemogenicity. In this study, infections of neonatal mice by the two different routes of inoculation were compared. We studied replication of the virus in vivo to identify critical preleukemic events. These would be observed in mice inoculated i.p. by Mo+PyF101 M-MuLV but not when inoculation was s.c. Infectious center assays indicated that regardless of the route of inoculation, Mo+PyF101 M-MuLV showed delayed infection of the thymus compared with wild-type M-MuLV. On the other hand, i.p.-inoculated mice showed more rapid appearance of infectious centers in the bone marrow than did s.c.-inoculated animals. Thus, the enhanced leukemogenicity of i.p. inoculation correlated with efficient early infection of the bone marrow and not with early infection of the thymus. These results suggest a role for bone marrow infection for efficient leukemogenesis in Mo+PyF101 M-MuLV-infected mice. Consistent with this notion, if bone marrow infection was decreased by injecting 10- to 12-day-old animals i.p., leukemogenicity resembled that of s.c. inoculation. Thus, two cell types that are critical for the induction of efficient leukemia were implicated. One cell delivers virus from the site of s.c. inoculation (the skin) to the bone marrow and is apparently restricted for Mo+PyF101 M-MuLV replication. The second cell is in the bone marrow, and its early infection is required for efficient leukemogenesis.

Historically, retroviruses were first isolated and studied because they efficiently cause tumors in experimental and domestic animals (Rous, 1911; Ellerman and Bang, 1908). Work was particularly intense during the 1970s, under the “War on Cancer” and the Special Virus Cancer Program. Important discoveries from re-trovirology such as oncogenes and proto-oncogenes (Bishop, 1987) have had profound implications for understanding tumorigenesis, including for spontaneous human tumors. Retroviruses remain some of the best model systems for studying carcinogenesis in the whole organism, since they induce tumors with extremely high efficiency. The focus of this chapter will be retroviral carcinogenesis in small animal models. Major attention will be paid to nonacute retroviruses, since they induce disease through multiple steps, with analogies to non-virus-induced cancer as well.