Retrovirus Receptor Research Articles

Proteomics Reveals a Physical and Functional Link between Hepatocyte Nuclear Factor 4α and Transcription Factor IID

Proteomic analyses have contributed substantially to our understanding of diverse cellular processes. Improvements in the sensitivity of mass spectrometry approaches are enabling more in-depth analyses of protein-protein networks and, in some cases, are providing surprising new insights into well established, longstanding problems. Here, we describe such a proteomic analysis that exploits MudPIT mass spectrometry and has led to the discovery of a physical and functional link between the orphan nuclear receptor hepatocyte nuclear factor 4alpha (HNF4alpha) and transcription factor IID (TFIID). A systematic characterization of the HNF4alpha-TFIID link revealed that the HNF4alpha DNA-binding domain binds directly to the TATA box-binding protein (TBP) and, through this interaction, can target TBP or TFIID to promoters containing HNF4alpha-binding sites in vitro. Supporting the functional significance of this interaction, an HNF4alpha mutation that blocks binding of TBP to HNF4alpha interferes with HNF4alpha transactivation activity in cells. These findings identify an unexpected role for the HNF4alpha DNA-binding domain in mediating key regulatory interactions and provide new insights into the roles of HNF4alpha and TFIID in RNA polymerase II transcription.

Increased expression of OsSPX1 enhances cold/subfreezing tolerance in tobacco and Arabidopsis thaliana

Low temperature is a major environmental stress for plants. Many important cultivated crops have limited capacity to survive below freezing/subfreezing temperatures. Low inorganic phosphate (Pi) is reportedly important in triggering cold acclimatization. SPX (SYG1/Pho81/XPR1: SYG1, suppressor of yeast gpal; Pho81, CDK inhibitor in yeast PHO pathway; XPR1, xenotropic and polytropic retrovirus receptor) domain proteins have been shown to be involved in the phosphate-related signal transduction and regulation pathways. Recently, Arabidopsis AtSPX family genes have been found to possess diverse functions in plant tolerance to phosphorus starvation, and OsSPX1 is involved in phosphate homeostasis in rice and optimizes growth under phosphate-limited conditions through a negative feedback loop. In this study, our phylogenetic and gene expression profiling approaches identified six rice OsSPX genes up-regulated during cold stress. Transgenic tobacco plants with constitutive expression of OsSPX1 were more tolerant to cold stress than were wild-type plants, and showed better seedling survival and reduced cellular electrolyte leakage. In addition, there was decreased total leaf Pi content and accumulation of free proline and sucrose in transgenic tobacco plants during cold stress. To further establish a cause-and-effect relationship between intracellular Pi level and cold acclimatization in transgenic plants, we generated transgenic Arabidopsis plants with constitutive expression of OsSPX1. Cold stress resulted in reduced leaf Pi levels in Arabidopsis transgenic relative to wild-type plants. From real-time reverse transcriptase-polymerase chain reaction analysis, several Pi starvation-related genes, such as AtSPX1 (orthologue of OsSPX1), PHO2, PLDZ2 and ATSIZ1, showed differential expression between wild-type and transgenic plants during cold stress. Our results indicate that OsSPX1 may play an important role in linking cold stress and Pi starvation signal transduction pathways.

Fibrils of Prostatic Acid Phosphatase Fragments Boost Infections with XMRV (Xenotropic Murine Leukemia Virus-Related Virus), a Human Retrovirus Associated with Prostate Cancer

The xenotropic murine leukemia virus-related virus (XMRV) has recently been detected in prostate cancer tissues and may play a role in tumorigenesis. It is currently unclear how this virus is transmitted and which factors promote its spread in the prostate. We show that amyloidogenic fragments known as semen-derived enhancer of virus infection (SEVI) originating from prostatic acid phosphatase greatly increase XMRV infections of primary prostatic epithelial and stromal cells. Hybrid simian/human immunodeficiency chimeric virus particles pseudotyped with XMRV envelope protein were used to demonstrate that the enhancing effect of SEVI, or of human semen itself, was at the level of viral attachment and entry. SEVI enhanced XMRV infectivity but did not bypass the requirement for the xenotropic and polytropic retrovirus receptor 1. Furthermore, XMRV RNA was detected in prostatic secretions of some men with prostate cancer. The fact that the precursor of SEVI is produced in abundance by the prostate indicates that XMRV replication occurs in an environment that provides a natural enhancer of viral infection, and this may play a role in the spread of this virus in the human population.

Identification of two distinct structural regions in a human porcine endogenous retrovirus receptor, HuPAR2, contributing to function for viral entry

BackgroundOf the three subclasses of Porcine Endogenous Retrovirus (PERV), PERV-A is able to infect human cells via one of two receptors, HuPAR1 or HuPAR2. Characterizing the structure-function relationships of the two HuPAR receptors in PERV-A binding and entry is important in understanding receptor-mediated gammaretroviral entry and contributes to evaluating the risk of zoonosis in xenotransplantation.ResultsChimeras of the non-permissive murine PAR and the permissive HuPAR2, which scanned the entire molecule, revealed that the first 135 amino acids of HuPAR2 are critical for PERV-A entry. Within this critical region, eighteen single residue differences exist. Site-directed mutagenesis used to map single residues confirmed the previously identified L109 as a binding and infectivity determinant. In addition, we identified seven residues contributing to the efficiency of PERV-A entry without affecting envelope binding, located in multiple predicted structural motifs (intracellular, extracellular and transmembrane). We also show that expression of HuPAR2 in a non-permissive cell line results in an average 11-fold higher infectivity titer for PERV-A compared to equal expression of HuPAR1, although PERV-A envelope binding is similar. Chimeras between HuPAR-1 and -2 revealed that the region spanning amino acids 152–285 is responsible for the increase of HuPAR2. Fine mapping of this region revealed that the increased receptor function required the full sequence rather than one or more specific residues.ConclusionHuPAR2 has two distinct structural regions. In one region, a single residue determines binding; however, in both regions, multiple residues influence receptor function for PERV-A entry.

Receptors for animal retroviruses

Diseases caused by animal retroviruses have been recognized since 19th century in veterinary field. Most livestock and companion animals have own retroviruses. To disclose the receptors for these retroviruses will be useful for understanding retroviral pathogenesis, developments of anti-retroviral drugs and vectors for human and animal gene therapies. Of retroviruses in veterinary field, receptors for the following viruses have been identified; equine infectious anemia virus, feline immunodeficiency virus, feline leukemia virus subgroups A, B, C, and T, Jaagsiekte sheep retrovirus, enzootic nasal tumor virus, avian leukosis virus subgroups A, B, C, D, E, and J, reticuloendotheliosis virus, RD-114 virus (a feline endogenous retrovirus), and porcine endogenous retrovirus subgroup A. Primate lentiviruses require two molecules (CD4 and chemokine receptors such as CXCR4) as receptors. Likewise, feline immunodeficiency virus also requires two molecules, i.e., CD134 (an activation marker of CD4 T cells) and CXCR4 in infection. Gammaretroviruses utilize multi-spanning transmembrane proteins, most of which are transporters of amino acids, vitamins and inorganic ions. Betaretroviruses and alpharetroviruses utilize transmembrane and/or GPI-anchored proteins as receptors. In this review, I overviewed receptors for animal retroviruses in veterinary field.

Stabilization of N-Myc Is a Critical Function of Aurora A in Human Neuroblastoma

In human neuroblastoma, amplification of the MYCN gene predicts poor prognosis and resistance to therapy. In a shRNA screen of genes that are highly expressed in MYCN-amplified tumors, we have identified AURKA as a gene that is required for the growth of MYCN-amplified neuroblastoma cells but largely dispensable for cells lacking amplified MYCN. Aurora A has a critical function in regulating turnover of the N-Myc protein. Degradation of N-Myc requires sequential phosphorylation by cyclin B/Cdk1 and Gsk3. N-Myc is therefore degraded during mitosis in response to low levels of PI3-kinase activity. Aurora A interacts with both N-Myc and the SCF(Fbxw7) ubiquitin ligase that ubiquitinates N-Myc and counteracts degradation of N-Myc, thereby uncoupling N-Myc stability from growth factor-dependent signals.

A Cre-loxP based mouse model for conditional somatic gene expression and knock-down in vivo using avian retroviral vectors

Site and time-specific somatic gene transfer using the avian sarcoma-leucosis retrovirus RCAS, has been shown to be a powerful tool to analyze gene function in vivo. RCAS retroviruses transduce only mammalian cells genetically engineered to express the avian retroviral receptor, TVA. Here, we have generated a knock-in mouse line termed LSL-R26Tva-lacZ with concomitant conditional expression of TVA and lacZ by targeting the Rosa26 locus. A loxP flanked transcriptional stop cassette was used for conditional activation of TVA and LacZ expression in a Cre recombinase dependent manner. To demonstrate the ability of this system for site and time-specific somatic gene transfer in vivo, we directed TVA expression to the pancreas. Introduction of an RCAS vector carrying an oncogenic KrasG12D expression cassette induced focal ductal pancreatic lesions that recapitulate human pancreatic intraepithelial neoplasias which progress to pancreatic ductal adenocarcinomas. TVA mediated infection of genetically engineered mice with endogenous expression of KrasG12D in pancreatic progenitor cells using RCAS virus carrying a short hairpin RNA directed against murine TP53, resulted in dramatically enhanced progression to invasive adenocarcinomas. These results show that conditional expression of TVA enables spatio-temporal gene expression and knock-down in a small subset of somatic cells in vivo. Therefore, it closely models carcinogenesis in humans where tumors evolve from somatic gene mutations in developmental normal cells. Combined with the growing number of Cre expression models, RCAS-TVA based gene expression and knock-down systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vitro and in vivo.

Skeletal and hematological anomalies in HYAL2‐deficient mice: a second type of mucopolysaccharidosis IX?

The metabolism of hyaluronan (HA) relies on HA synthases and hyaluronidases, among which hyaluronidase-1 (HYAL1) and -2 (HYAL2) have been proposed as key actors. Congenital HYAL1 deficiency leads to mucopolysaccharidosis IX (MPS IX), a rare lysosomal storage disorder characterized by joint abnormalities. Knowledge of HYAL2 is limited. This protein displays weak in vitro hyaluronidase activity and acts as a receptor for oncogenic ovine retroviruses. We have generated HYAL2-deficient mice through a conditional Cre-lox system. Hyal2(-/-) mice are viable and fertile. They exhibit localized congenital defects in frontonasal and vertebral bone formation and suffer from mild thrombocytopenia and chronic, possibly intravascular, hemolysis. In addition, Hyal2(-/-) mice display 10-fold increases in plasma levels of HA and 2-fold increases in plasma hyaluronidase activity. Globally, there is no HA accumulation in tissues, including bones, but liver sinusoidal cells seem overloaded with undigested HA. Taken together, these elements demonstrate for the first time that murine HYAL2 has a physiological activity in vivo that is relevant for craniovertebral bone formation, maintenance of plasma HA concentrations, and erythrocyte and platelet homeostasis. In addition, the viability of HYAL2-deficient mice raises the possibility that a similar defect, defining a new MPS disorder, exists in humans.

A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors

Site- and time-specific somatic gene transfer by using the avian sarcoma-leukosis retrovirus RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor) has been shown to be a powerful tool to analyze gene function in vivo. RCAS retroviruses that express the avian subgroup A envelope transduce only mammalian cells genetically engineered to express the avian retroviral receptor, tumor virus A (TVA). Here, we generated a knockin mouse line termed LSL-R26(Tva-lacZ) with concomitant conditional expression of TVA and lacZ by targeting the Rosa26 locus. A loxP-flanked transcriptional stop cassette was used for conditional activation of TVA and LacZ expression in a Cre-recombinase-dependent manner. To demonstrate the ability of this system for conditional somatic gene transfer in vivo, we directed TVA expression to the pancreas. Introduction of an RCAS vector with Bryan-RSV polymerase and subgroup A envelope [RCASBP(A)] carrying oncogenic Kras(G12D) induced focal ductal pancreatic lesions that recapitulate human pancreatic intraepithelial neoplasias that progress to pancreatic ductal adenocarcinomas. TVA-mediated infection of genetically engineered mice with endogenous expression of Kras(G12D) in pancreatic progenitor cells by using RCASBP(A) virus carrying a short hairpin RNA directed against murine TP53, resulted in dramatically enhanced progression to invasive adenocarcinomas. These results show that conditional expression of TVA enables spatiotemporal gene expression and knockdown in a small subset of somatic cells in vivo. Therefore, it closely models carcinogenesis in humans where tumors evolve from somatic gene mutations in developmentally normal cells. Combined with the growing number of Cre expression models, RCAS-TVA-based gene expression and knockdown systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vitro and in vivo.

Mammalian Models Based on RCAS-TVA Technique

The retroviral vector (RCAS) has been widely used in avian system to study development and diseases, but is not suitable for mammals which do not produce the retrovirus receptor TVA. In this review, we trace the current uses of RCAS-TVA approach in mammalian system with improved strategies, including generation of tv-a transgenic mice, use of soluble TVA receptor and retroviral receptor-ligand fusion proteins, improvement of RCAS vectors, and compare a series of mammalian models in variant studies of gene function, development, oncogenesis and gene therapy. All those studies demonstrate that the RCAS-TVA based mammalian models are powerful tools for understanding the mechanisms and target treating of human diseases.

RCAS/SCL-TVA Animal Model Allows Targeted Delivery of Polyoma Middle T Oncogene to Vascular Endothelial Progenitors In vivo and Results in Hemangioma Development

To recapitulate the generation of cancer stem cells in the context of an intact animal using a retroviral vector capable of in vivo delivery of oncogenes to primitive endothelial and hematopoietic stem cells. Targeting of these progenitors was achieved using transgenic mice in which the avian TVA retroviral receptor was placed under the control of the stem cell leukemia (scl/tal-1) gene promoter and SCL +19 enhancer. Injection of an avian retrovirus encoding polyoma middle T (PyMT), an oncogene that transforms endothelial cells, caused rapid lethality in all SCL-TVA mice but not in control TVA(-) littermates. The infected animals exhibited hemorrhagic foci in several organs. Histopathologic analysis confirmed the presence of hemangiomas and the endothelial origin of the PyMT-transformed cells. Surprisingly, the transformed endothelial cells contained readily detectable numbers of TVA(+) cells. By contrast, normal blood vessels had very few of these cells. The presence of TVA(+) cells in the lesions suggests that the cells originally infected by PyMT retained stem cell characteristics. Further analysis showed that the tumor cells exhibited activation of the phosphatidylinositol 3-kinase/Akt and S6/mammalian target of rapamycin pathways, suggesting a mechanism used by PyMT to transform endothelial progenitors in vivo. We conclude that this experimental system can specifically deliver oncogenes to vascular endothelial progenitors in vivo and cause a fatal neoplastic disease. This animal model should allow the generation of endothelial cancer stem cells in the natural environment of an immunocompetent animal, thereby enabling the recapitulation of genetic alterations that are responsible for the initiation and progression of human malignancies of endothelial origin.

Functional hierarchy of two L domains in porcine endogenous retrovirus (PERV) that influence release and infectivity

The porcine endogenous retrovirus (PERV) Gag protein contains two late (L) domain motifs, PPPY and P(F/S)AP. Using viral release assays we demonstrate that PPPY is the dominant L domain involved in PERV release. PFAP represents a novel retroviral L domain variant and is defined by abnormal viral assembly phenotypes visualized by electron microscopy and attenuation of early PERV release as measured by viral genomes. PSAP is functionally dominant over PFAP in early PERV release. PSAP virions are 3.5-fold more infectious in vitro by TCID50 and in vivo results in more RNA positive tissues and higher levels of proviral DNA using our human PERV-A receptor (HuPAR-2) transgenic mouse model [Martina, Y., Marcucci, K.T., Cherqui, S., Szabo, A., Drysdale, T., Srinivisan, U., Wilson, C.A., Patience, C., Salomon, D.R., 2006. Mice transgenic for a human porcine endogenous retrovirus receptor are susceptible to productive viral infection. J. Virol. 80 (7), 3135–3146]. The functional hierarchies displayed by PERV L domains, demonstrates that L domain selection in viral evolution exists to promote efficient viral assembly, release and infectivity in the virus–host context.

The GLN Family of Murine Endogenous Retroviruses Contains an Element Competent for Infectious Viral Particle Formation

Several families of endogenous retroviruses (ERVs) have been identified in the mouse genome, in several instances by in silico searches, but for many of them it remains to be determined whether there are elements that can still encode functional retroviral particles. Here, we identify, within the GLN family of highly reiterated ERVs, one, and only one, copy that encodes retroviral particles prone to infection of mouse cells. We show that its envelope protein confers an ecotropic host range and recognizes a receptor different from mCAT1 and mSMIT1, the two previously identified receptors for other ecotropic mouse retroviruses. Electron microscopy disclosed viral particle assembly and budding at the cell membrane, as well as release of mature particles into the extracellular space. These particles are closely related to murine leukemia virus (MLV) particles, with which they have most probably been confused in the past. This study, therefore, identifies a new class of infectious mouse ERVs belonging to the family Gammaretroviridae, with one family member still functional today. This family is in addition to the two MLV and mouse mammary tumor virus families of active mouse ERVs with an extracellular life cycle.

Ubiquitination and Degradation of Tal1/SCL Are Induced by Notch Signaling and Depend on Skp2 and CHIP

Notch signaling controls diverse eukaryotic differentiation processes in multiple cell types, thus demanding versatile tools with which Notch triggers downstream events. Ubiquitin-mediated proteolysis has previously been shown to be one such tool with which Notch regulates the turnover of the basic helix-loophelix transcription factor, E47. Here, we show that Notch signaling also accelerated the degradation of Tal1/SCL (T cell acute leukemia 1/stem cell leukemia) protein, a basic helix-loop-helix protein involved in the development of hematopoietic, vascular, and neuronal tissues. Notch-induced Tal1/SCL degradation was mediated by ubiquitination and proteasomes. The sequence responsible for Tal1 degradation was localized to a region in the C terminus of Tal1, which is evolutionarily conserved, thus suggesting a functional significance. Analogous to the situation for E47, Notch-induced Tal1/SCL degradation not only required Skp2, a substrate-binding subunit of SCF ubiquitin ligase complexes, but also relied on CHIP, a chaperone-binding protein with a ubiquitin ligase activity. In contrast to the fact that the N-terminal tetratricopeptide region (TPR) domain of CHIP is necessary and sufficient for E47 ubiquitination and degradation, CHIP promoted Tal1 degradation with both chaperone binding and ubiquitin ligase activities, which are mediated by its TPR domain and U box, respectively. Although the TPR domain was not involved in Tal1/SCL binding, it was required for enhancing its degradation. Likewise, the ubiquitin ligase activity of CHIP was dispensable for Tal1/SCL binding but essential for degradation. These findings provide both novel mechanistic insights into the operation of cullin-based ubiquitin ligase complexes and potential means by which Notch and Tal1/SCL regulate eukaryotic development.

Phenotypic Analysis ofDlx5 Overexpression in Post-natal Bone

Dlx5 plays an important role in the embryonic development of mineralized tissues. We hypothesized that Dlx5 also functions in regulating post-natal bone formation in mice. To prove this hypothesis, we infected 5-day-old bone sialoprotein (BSP)/avian retroviral receptor gene (TVA) transgenic mice with replication-competent retroviral vectors expressing wild-type Dlx5 (RCAS-Dlx5WT) and mutated Dlx5 at arginine (R) 31 of its homeodomain (RCAS-Dlx5RH). Immunohistochemistry indicated that RCAS-Dlx5WT increased BSP and osteopontin (OPN) expression, whereas it decreased that of osteocalcin (OC). RCAS-Dlx5RH mediated opposite effects. Semi-quantitative RT-PCR confirmed these results. Ex vivo overexpression of RCAS-Dlx5WT in BSP/TVA calvarial cells promoted, whereas that of RCAS-Dlx5RH inhibited, mineralized nodule formation as compared with that in control cells. Our results suggest that Dlx5 promotes expression of early markers of osteogenic differentiation and increases mineralization post-natally.

A novel Flk1-TVA transgenic mouse model for gene delivery to angiogenic vasculature

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.

An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors: Dong B, Kim S, Hong S, Das Gupta J, Malathi K, Klein EA, Ganem D, Derisi JL, Chow SA, Silverman RH, Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH

We recently reported identification of a previously undescribed gammaretrovirus genome, xenotropic murine leukemia virus-related virus (XMRV), in prostate cancer tissue from patients homozygous for a reduced activity variant of the antiviral enzyme RNase L. Here we constructed a full-length XMRV genome from prostate tissue RNA and showed that the molecular viral clone is replication-competent. XMRV replication in the prostate cancer cell line DU145 was sensitive to inhibition by IFN-β. However, LNCaP prostate cancer cells, which are deficient in JAK1 and RNase L, were resistant to the effects of IFN-β against XMRV. Furthermore, DU145 cells rendered deficient in RNase L with siRNA were partially resistant to IFN inhibition of XMRV. Expression in hamster cells of the xenotropic and polytropic retrovirus receptor 1 allowed these cells to be infected by XMRV. XMRV provirus integration sites were mapped in DNA isolated from human prostate tumor tissue to genes for 2 transcription factors (NFATc3 and CREB5) and to a gene encoding a suppressor of androgen receptor transactivation (APPBP2/PAT1/ARA67). Our studies demonstrate that XMRV is a virus that has infected humans and is susceptible to inhibition by IFN and its downstream effector, RNase L.

Distribution of human endogenous retrovirus type W receptor in normal human villous placenta

The fusion of trophoblast cells into the villous syncytiotrophoblast is crucial for appropriate placental function and fetal development. Fusion occurs following the interaction of syncytin-1, an envelope protein of the endogenous retrovirus HERV-W, and the RD114/mammalian type D retrovirus receptor (RDR/ASCT2) on adjacent cell membranes. This process must be tightly regulated in order to maintain the proliferative pool of cytotrophoblast cells as well as the function of the syncytia. We sought to investigate whether syncytial fusion of placental cytotrophoblast cells may be regulated via modulation of RDR/ASCT2 expression. Expression of RDR/ASCT2 in term and first trimester villous placenta was assessed along with a number of molecular markers using immunofluorescent staining. In a complementary approach, Western blotting was used to investigate RDR/ASCT2 expression in a panel of choriocarcinoma cell lines before and after stimulation of fusion. Villous placental RDR/ASCT2 expression was found to be restricted to the cytotrophoblast compartment, being largely absent in the syncytiotrophoblast. Local variations in RDR/ASCT2 expression were not associated with the proliferative status of cytotrophoblast cells. RDR/ASCT2 expression was also shown to be down-regulated in BeWo choriocarcinoma cells after stimulation of syncytial fusion. This first report of the localisation and distribution of RDR/ASCT2 in human placental villi suggests that the fusion of placental trophoblast cells is not regulated by local or temporal variations of RDR/ASCT2 expression in villous cytotrophoblast cells.

Identification of a retroviral receptor used by an envelope protein derived by peptide library screening.

This study demonstrates the power of a genetic selection to identify a variant virus that uses a new retroviral receptor protein. We screened a random peptide library within the receptor-binding domain of a feline leukemia virus retroviral Envelope (FeLV Env) protein for productive infection of feline AH927 cells. One variant, A5, obtained with altered tropic properties acquired the ability to use the solute carrier protein family 35 member F2 (SLC35F2) as a receptor. The SLC35F2 protein is a presumed transporter of unknown function predicted to encode 8 to 10 transmembrane-spanning regions and is not homologous to any identified retroviral receptor. Expression of the feline SLC35F2 cDNA in nonpermissive cells renders the cells susceptible to infection by A5 virus, with remarkably high titers in the range of 10(5) infectious units per ml. The human SLC35F2 ORF also functioned as the retroviral receptor, albeit at lower efficiency than the feline homologue. The successful selection of a novel molecule, the SLC35F2 transporter/channel-type protein, as a receptor by the FeLV Env backbone suggests that multipass transmembrane proteins may be particularly suited for use in productive viral entry and fusion. The analysis of retroviral Env libraries randomized in the receptor-binding domain offers a viable means to develop viral vectors targeted to specific cell types in the absence of known targeting ligands.

An infectious retrovirus susceptible to an IFN antiviral pathway from human prostate tumors

We recently reported identification of a previously undescribed gammaretrovirus genome, xenotropic murine leukemia virus-related virus (XMRV), in prostate cancer tissue from patients homozygous for a reduced activity variant of the antiviral enzyme RNase L. Here we constructed a full-length XMRV genome from prostate tissue RNA and showed that the molecular viral clone is replication-competent. XMRV replication in the prostate cancer cell line DU145 was sensitive to inhibition by IFN-beta. However, LNCaP prostate cancer cells, which are deficient in JAK1 and RNase L, were resistant to the effects of IFN-beta against XMRV. Furthermore, DU145 cells rendered deficient in RNase L with siRNA were partially resistant to IFN inhibition of XMRV. Expression in hamster cells of the xenotropic and polytropic retrovirus receptor 1 allowed these cells to be infected by XMRV. XMRV provirus integration sites were mapped in DNA isolated from human prostate tumor tissue to genes for two transcription factors (NFATc3 and CREB5) and to a gene encoding a suppressor of androgen receptor transactivation (APPBP2/PAT1/ARA67). Our studies demonstrate that XMRV is a virus that has infected humans and is susceptible to inhibition by IFN and its downstream effector, RNase L.