Evidence against a role for jaagsiekte sheep retrovirus in human lung cancer.

BACKGROUND: Jaagsiekte sheep retrovirus (JSRV) causes a contagious lung cancer in sheep and goats that can be transmitted by aerosols produced by infected animals. Virus entry into cells is initiated by binding of the viral envelope (Env) protein to a specific cell-surface receptor, Hyal2. Unlike almost all other retroviruses, the JSRV Env protein is also a potent oncoprotein and is responsible for lung cancer in animals. Of concern, Hyal2 is a functional receptor for JSRV in humans. RESULTS: We show here that JSRV is fully capable of infecting human cells, as measured by its reverse transcription and persistence in the DNA of cultured human cells. Several studies have indicated a role for JSRV in human lung cancer while other studies dispute these results. To further investigate the role of JSRV in human lung cancer, we used highly-specific mouse monoclonal antibodies and a rabbit polyclonal antiserum against JSRV Env to test for JSRV expression in human lung cancer. JSRV Env expression was undetectable in lung cancers from 128 human subjects, including 73 cases of bronchioalveolar carcinoma (BAC; currently reclassified as lung invasive adenocarcinoma with a predominant lepidic component), a lung cancer with histology similar to that found in JSRV-infected sheep. The BAC samples included 8 JSRV DNA-positive samples from subjects residing in Sardinia, Italy, where sheep farming is prevalent and JSRV is present. We also tested for neutralizing antibodies in sera from 138 Peruvians living in an area where sheep farming is prevalent and JSRV is present, 24 of whom were directly exposed to sheep, and found none. CONCLUSIONS: We conclude that while JSRV can infect human cells, JSRV plays little if any role in human lung cancer.

Retroviral vector production.

In this unit, the basic protocol generates stable cell lines that produce retroviral vectors that carry selectable markers. Also included are an alternate protocol that applies when the retroviral vector does not carry a selectable marker, and another alternate protocol for rapidly generating retroviral vector preparations by transient transfection. A support protocol describes construction of the retroviral vectors. The methods for generating virus from retroviral vector plasmids rely on the use of packaging cells that synthesize all of the retroviral proteins but do not produce replication-competent virus. Additional protocols detail plasmid transfection, virus titration, assay for replication-competent virus, and histochemical staining to detect transfer of a vector encoding alkaline phosphatase.

Disruption of thiamine uptake and growth of cells by feline leukemia virus subgroup A.

Feline leukemia virus (FeLV) is still a major cause of morbidity and mortality in domestic cats and some wild cats despite the availability of relatively effective vaccines against the virus. FeLV subgroup A (FeLV-A) is transmitted in natural infections, and FeLV subgroups B, C, and T can evolve directly from FeLV-A by mutation and/or recombination with endogenous retroviruses in domestic cats, resulting in a variety of pathogenic outcomes. The cell surface entry receptor for FeLV-A is a putative thiamine transporter (THTR1). Here, we have addressed whether FeLV-A infection might disrupt thiamine uptake into cells and, because thiamine is an essential nutrient, whether this disruption might have pathological consequences. First, we cloned the cat ortholog of the other of the two known thiamine transporters in mammals, THTR2, and we show that feline THTR1 (feTHTR1) and feTHTR2 both mediate thiamine uptake, but feTHTR2 does not function as a receptor for FeLV-A. We found that feTHTR1 is widely expressed in cat tissues and in cell lines, while expression of feTHTR2 is restricted. Thiamine uptake mediated by feTHTR1 was indeed blocked by FeLV-A infection, and in feline fibroblasts that naturally express feTHTR1 and not feTHTR2, this blockade resulted in a growth arrest at physiological concentrations of extracellular thiamine. The growth arrest was reversed at high extracellular concentrations of thiamine. Our results show that FeLV-A infection can indeed disrupt thiamine uptake with pathological consequences. A prediction of these experiments is that raising the plasma levels of thiamine in FeLV-infected cats may ameliorate the pathogenic effects of infection.

Successful regional delivery and long-term expression of a dystrophin gene in canine muscular dystrophy: a preclinical model for human therapies.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle disease caused by mutations in the dystrophin gene. Adeno-associated viral (AAV) vector-mediated gene replacement strategies hold promise as a treatment. Studies in animal models and human trials suggested that immune responses to AAV capsid proteins and transgene products prevented efficient gene therapy. In this study, we used widespread intramuscular (i.m.) injection to deliver AAV6-canine micro-dystrophin (c-µdys) throughout a group of skeletal muscles in dystrophic dogs given a brief course of commonly used immunosuppressants. Robust c-µdys expression was obtained for at least two years and was associated with molecular reconstitution of the dystrophin-glycoprotein complex (DGC) at the muscle membrane. Importantly, c-µdys expression was maintained for at least 18 months after discontinuing immunosuppression. The results obtained in a relevant preclinical model of DMD demonstrate feasibility of widespread AAV-mediated muscle transduction and transgene expression in the presence of transient immunosuppression to achieve molecular reconstitution that can be directly translated to human trials.

No biological evidence of XMRV in blood or prostatic fluid from prostate cancer patients.

BACKGROUND: XMRV (xenotropic murine leukemia virus-related virus) was initially discovered in association with prostate cancer and later with chronic fatigue syndrome (CFS). Its association with CFS is now largely discredited, and current results support a laboratory origin for XMRV with no reproducible evidence for infection of humans. However, some results indicating the presence of XMRV in prostate cancer are difficult to attribute to sample contamination. Here we have sought biological evidence that might confirm the presence of XMRV in prostate cancer samples previously having tested positive. METHODS AND RESULTS: We have tested for infectious XMRV and neutralizing antibodies against XMRV in blood plasma from 29 subjects with prostate cancer, and for infectious XMRV in prostate secretions from another five prostate cancer subjects. Nine of these subjects had previously tested positive for XMRV by PCR or by virus assay. We did not detect XMRV or related retroviruses in any sample, and the neutralizing activities of the plasma samples were all very low, a result inconsistent with XMRV infection of the plasma donors. CONCLUSIONS: We find no evidence for XMRV infection of any human subject tested, either by assay for infectious virus or for neutralizing antibodies. Our results are consistent with the majority of published studies on XMRV, which find that XMRV is not present in humans. The observed low to undetectable XMRV neutralization by human plasma indicates a lack of innate restriction of XMRV replication by soluble factors in human blood.

Xpr1 is an atypical G-protein-coupled receptor that mediates xenotropic and polytropic murine retrovirus neurotoxicity.

Xenotropic murine leukemia virus-related virus (XMRV) was first identified in human prostate cancer tissue and was later found in a high percentage of humans with chronic fatigue syndrome (CFS). While exploring potential disease mechanisms, we found that XMRV infection induced apoptosis in SY5Y human neuroblastoma cells, suggesting a mechanism for the neuromuscular pathology seen in CFS. Several lines of evidence show that the cell entry receptor for XMRV, Xpr1, mediates this effect, and chemical cross-linking studies show that Xpr1 is associated with the Gß subunit of the G-protein heterotrimer. The activation of adenylate cyclase rescued the cells from XMRV toxicity, indicating that toxicity resulted from reduced G-protein-mediated cyclic AMP (cAMP) signaling. Some proteins with similarity to Xpr1 are involved in phosphate uptake into cells, but we found no role of Xpr1 in phosphate uptake or its regulation. Our results indicate that Xpr1 is a novel, atypical G-protein-coupled receptor (GPCR) and that xenotropic or polytropic retrovirus binding can disrupt the cAMP-mediated signaling function of Xpr1, leading to the apoptosis of infected cells. We show that this pathway is also responsible for the classic toxicity of the polytropic mink cell focus-forming (MCF) retrovirus in mink cells. Although it now seems clear that the detection of XMRV in humans was the result of sample contamination with a recombinant mouse virus, our findings may have relevance to neurologic disease induced by MCF retroviruses in mice.

Lung cancer in mice induced by the jaagsiekte sheep retrovirus envelope protein is not maintained by rare cancer stem cells, but tumorigenicity does correlate with Wnt pathway activation.

JSRV, a simple beta-retrovirus, is the etiologic agent of ovine pulmonary adenocarcinoma, a form of non-small cell lung cancer in sheep and goats. It has been shown that the envelope protein alone is sufficient to induce tumorigenesis in the lungs of mice when delivered via an adeno-associated viral vector. Here, we tested the hypothesis that JSRV envelope-induced tumors are maintained by a small population of tumor-initiating cells, termed cancer stem cells. To test this hypothesis, dissociated cancer cells were sorted from envelope-induced tumors in mouse lung based on the putative stem cell markers Sca-1, CD34, and CD133, the pluripotency-associated transcription factor Oct4, and the level of Wnt signaling. No association with increased tumor-initiating capacity was found with any of the cell-surface markers. In addition, we were unable to detect any evidence of Oct4 expression in tumor-bearing mouse lung. However, tumor cells possessing an active Wnt signaling pathway did show a significant correlation with increased tumor formation upon transplantation. Limiting dilution transplant analysis suggests the existence of a large fraction of cells with the ability to propagate tumor growth, with increasing tumor initiation potential correlating with activated Wnt signaling.

The left half of the XMRV retrovirus is present in an endogenous retrovirus of NIH/3T3 Swiss mouse cells.

Xenotropic murine leukemia virus-related virus (XMRV) is a gammaretrovirus found in association with human prostate cancer and chronic fatigue syndrome, although these associations are controversial. XMRV shows at most 94% identity to known mouse retroviruses. Here we used XMRV-specific PCR to search for a more closely related source of XMRV in mice. While we could not find a complete copy, we did find a 3,600-bp region of XMRV in an endogenous retrovirus present in NIH/3T3 cells. These results show that XMRV has clear ancestors in mice and highlight another possible source of contamination in PCR assays for XMRV.

Jaagsiekte sheep retrovirus and enzootic nasal tumor virus promoters drive gene expression in all airway epithelial cells of mice but only induce tumors in the alveolar region of the lungs.

Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats, while the closely related enzootic nasal tumor virus type 1 (ENTV-1) and ENTV-2 induce tumors in the nasal epithelium of sheep and goats, respectively. When expressed using a strong Rous sarcoma virus promoter, the envelope proteins of these viruses induce tumors in the respiratory tract of mice, but only in the distal airway. To examine the role of the retroviral long terminal repeat (LTR) promoters in determining tissue tropism, adeno-associated virus (AAV) vectors expressing alkaline phosphatase under the control of the JSRV, ENTV-1, or ENTV-2 LTRs were generated and administered to mice. The JSRV LTR was active in all airway epithelial cells, while the ENTV LTRs were active in the nasal epithelium and alveolar type II cells but poorly active in tracheal and bronchial epithelial cells. When vectors were administered systemically, the ENTV-1 and -2 LTRs were inactive in major organs examined, whereas the JSRV showed high-level activity in the liver. When a putative transcriptional enhancer from the 3' end of the env gene was inserted upstream of the JSRV and ENTV-1 LTRs in the AAV vectors, a dramatic increase in transgene expression was observed. However, intranasal administration of AAV vectors containing any combination of ENTV or JSRV LTRs and Env proteins induced tumors only in the lower airway. Our results indicate that mice do not provide an adequate model for nasal tumor induction by ENTV despite our ability to express genes in the nasal epithelium.

Single-strand nicks induce homologous recombination with less toxicity than double-strand breaks using an AAV vector template.

Gene targeting by homologous recombination (HR) can be induced by double-strand breaks (DSBs), however these breaks can be toxic and potentially mutagenic. We investigated the I-AniI homing endonuclease engineered to produce only nicks, and found that nicks induce HR with both plasmid and adeno-associated virus (AAV) vector templates. The rates of nick-induced HR were lower than with DSBs (24-fold lower for plasmid transfection and 4- to 6-fold lower for AAV vector infection), but they still represented a significant increase over background (240- and 30-fold, respectively). We observed severe toxicity with the I-AniI 'cleavase', but no evidence of toxicity with the I-AniI 'nickase.' Additionally, the frequency of nickase-induced mutations at the I-AniI site was at least 150-fold lower than that induced by the cleavase. These results, and the observation that the surrounding sequence context of a target site affects nick-induced HR but not DSB-induced HR, strongly argue that nicks induce HR through a different mechanism than DSBs, allowing for gene correction without the toxicity and mutagenic activity of DSBs.

Susceptibility of the human retrovirus XMRV to antiretroviral inhibitors.

BACKGROUND: XMRV (xenotropic murine leukemia virus-related virus) is the first known example of an exogenous gammaretrovirus that can infect humans. A limited number of reports suggest that XMRV is intrinsically resistant to many of the antiretroviral drugs used to treat HIV-1 infection, but is sensitive to a small subset of these inhibitors. In the present study, we used a novel marker transfer assay to directly compare the antiviral drug sensitivities of XMRV and HIV-1 under identical conditions in the same host cell type. RESULTS: We extend the findings of previous studies by showing that, in addition to AZT and tenofovir, XMRV and HIV-1 are equally sensitive to AZddA (3'-azido-2',3'-dideoxyadenosine), AZddG (3'-azido-2',3'-dideoxyguanosine) and adefovir. These results indicate that specific 3'-azido or acyclic nucleoside analog inhibitors of HIV-1 reverse transcriptase (RT) also block XMRV infection with comparable efficacy in vitro. Our data confirm that XMRV is highly resistant to the non-nucleoside RT inhibitors nevirapine and efavirenz and to inhibitors of HIV-1 protease. In addition, we show that the integrase inhibitors raltegravir and elvitegravir are active against XMRV, with EC50 values in the nanomolar range. CONCLUSIONS: Our analysis demonstrates that XMRV exhibits a distinct pattern of nucleoside analog susceptibility that correlates with the structure of the pseudosugar moiety and that XMRV is sensitive to a broader range of antiretroviral drugs than has previously been reported. We suggest that the divergent drug sensitivity profiles of XMRV and HIV-1 are partially explained by specific amino acid differences in their respective protease, RT and integrase sequences. Our data provide a basis for choosing specific antiretroviral drugs for clinical studies in XMRV-infected patients.

Acutely transforming retrovirus expressing Nras generated from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV.

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.

Expression of human alpha1-antitrypsin in mice and dogs following AAV6 vector-mediated gene transfer to the lungs.

We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.

An all-feline retroviral packaging system for transduction of human cells.

Abstract The subgroup C feline leukemia virus (FeLV-C) receptor FLVCR is a widely expressed 12-transmembrane domain transporter that exports cytoplasmic heme and is a promising target for retrovirus-mediated gene delivery. Previous studies demonstrated that FeLV-C pseudotype vectors were more efficient at targeting human hematopoietic stem cells than those pseudotyped with gibbon ape leukemia virus (GALV), and thus we developed an all FeLV-C-based packaging system, termed CatPac. CatPac is helper-virus free and can produce higher titer vectors than existing gammaretroviral packaging systems, including systems mixing Moloney murine leukemia virus (MoMLV) Gag-Pol and FeLV-C Env proteins. The vectors can be readily concentrated (>30-fold), refrozen (three to five times), and held on ice (>2 days) with little loss of titer. Furthermore, we demonstrate that CatPac pseudotype vectors efficiently target early CD34(+)CD38(-) stem/progenitor cells, monocytic and erythroid progenitors, activated T cells, mature macrophages, and cancer cell lines, suggesting utility for human cell and cell line transduction and possibly gene therapy.

The prostate cancer-associated human retrovirus XMRV lacks direct transforming activity but can induce low rates of transformation in cultured cells.

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 TRIM5alpha (Ref1), showing that XMRV is resistant to TRIM5alpha 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.

Multiple integrated copies and high-level production of the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) from 22Rv1 prostate carcinoma cells.

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, most frequently in humans with a defect in the antiviral defense protein RNase L, suggesting a role for XMRV in prostate carcinogenesis. However, XMRV has not been found in prostate carcinoma cells. Here we show that 22Rv1 prostate carcinoma cells produce high-titer virus that is nearly identical in properties and sequence to XMRV isolated by others and consist primarily of a single clone of cells with at least 10 integrated copies of XMRV, warranting further study of a possible role for XMRV integration in carcinogenesis.

Hyaluronidase 2 and its intriguing role as a cell-entry receptor for oncogenic sheep retroviruses.

Jaagsiekte sheep retrovirus (JSRV) causes lung adenocarcinoma in sheep and goats, while the closely related enzootic nasal tumor virus (ENTV) causes nasal tumors in the same species. The envelope (Env) protein from either virus can transform fibroblasts and epithelial cells in culture, indicating that the Env proteins are responsible for tumorigenesis. However, the primary function of retroviral Env proteins is to mediate virus entry into cells by interacting with specific cell-surface receptors, suggesting that the virus receptor might be a key player in transformation as well. Thus, identification of Hyaluronidase-2 (Hyal2) as the cell-entry receptor for both JSRV and ENTV suggested a role for Hyal2 in oncogenesis. Furthermore, Hyal2 is located in a key lung cancer tumor suppressor locus on chromosome 3p21.3, suggesting that Hyal2 might have a tumor suppressor activity that was disrupted by Env thereby leading to tumorigenesis. However, recent experiments showing that expression of the JSRV or ENTV Env protein in mouse lung can induce lung tumors, even though the viral Env proteins cannot bind to or utilize mouse Hyal2 as a receptor for virus entry into cells, indicate that Hyal2 plays no role in cancer induction by these retroviruses. Hyal2 remains an enigmatic member of the hyaluronidase family given its very low hyaluronidase activity in purified form or when expressed in cultured cells, suggesting that it may have evolved to perform some other as yet unknown function.

Identification of the myelin protein plasmolipin as the cell entry receptor for Mus caroli endogenous retrovirus.

The Asian wild mouse species Mus caroli harbors an endogenous retrovirus (McERV) that is closely related to but distinct from the endogenous retrovirus family defined by the Mus dunni endogenous virus and the Mus musculus endogenous retrovirus. McERV could infect some cell types from humans, dogs, and rats, but not all, and did not infect any mouse cell line tested. Because of its interesting host range and proposed ancestral relationship to primate retroviruses and because none of the entry receptors for this family of retroviruses had been identified, we began a search for the McERV receptor. We determined the chromosomal location of the receptor gene in the human genome by phenotypic screening of the G3 human-hamster radiation hybrid cell line panel and confirmed the localization by assaying for receptor activity conferred by bacterial artificial chromosome (BAC) clones spanning the region. We next localized the gene more precisely in one positive BAC by assaying for receptor activity following BAC digestion with several restriction enzymes that cleaved different sets of genes, and we confirmed that the final candidate gene, plasmolipin (PLLP; TM4SF11), is the novel receptor by showing that the expression of the human PLLP cDNA renders hamster and mouse cells susceptible to McERV infection. PLLP functions as a voltage-dependent potassium ion channel and is expressed primarily in kidney and brain, helping to explain the limited range of cell types that McERV can infect. Interestingly, mouse PLLP also functioned well as a receptor for McERV but was simply not expressed in the mouse cell types that we originally tested.