A role for endogenous retroviral sequences in the regulation of lymphocyte activation

The genomes of most vertebrates contain numerous retroviral sequences, the great majority of which are non-infectious. These endogenous retroviral sequences are transcribed and translated in many host tissues, and are induced by mitogens. The function, if any, of endogenous retroviruses has been unclear. The transmembrane envelope proteins of some infectious type C retroviruses suppress lymphocyte activation, but it is unknown whether any endogenous type C retroviruses share this suppressive activity. To study the possible effects of murine endogenous retroviral expression, specific antisense oligonucleotides were synthesized complementary to type C retroviral sequences, and were cultured with murine spleen cells. If any of these endogenous retroviruses are suppressing lymphocyte activation, then inhibiting such endogenous retroviral-mediated suppression with antisense might result in lymphocyte stimulation. Three classes of endogenous type C retroviral sequences may be distinguished by antisense oligonucleotides (based on their homology to infectious retroviruses): ecotropic, xenotropic, and mink cell focus-forming (MCF). Antisense oligonucleotides to endogenous MCF envelope gene (env) initiation regions caused: i) doubling or tripling of spleen cell RNA synthesis, and ii) marked increases in lymphocyte surface Ia and Ig expression relative to control oligonucleotides. Antisense oligos to xenotropic or ecotropic env sequences or to endogenous MCF non-envelope sequences had no effect. These data suggest that endogenous MCF sequences exert an inhibitory influence on the murine immune system. Because endogenous MCF expression is inducible by immune stimuli, such expression could constitute an inhibitory feedback circuit that participates in the regulation of immune homeostasis.     

Positive selection of Iris, a retroviral envelope-derived host gene in Drosophila melanogaster

Eukaryotic genomes can usurp enzymatic functions encoded by mobile elements for their own use. A particularly interesting kind of acquisition involves the domestication of retroviral envelope genes, which confer infectious membrane-fusion ability to retroviruses. So far, these examples have been limited to vertebrate genomes, including primates where the domesticated envelope is under purifying selection to assist placental function. Here, we show that in Drosophila genomes, a previously unannotated gene (CG4715, renamed Iris) was domesticated from a novel, active Kanga lineage of insect retroviruses at least 25 million years ago, and has since been maintained as a host gene that is expressed in all adult tissues. Iris and the envelope genes from Kanga retroviruses are homologous to those found in insect baculoviruses and gypsy and roo insect retroviruses. Two separate envelope domestications from the Kanga and roo retroviruses have taken place, in fruit fly and mosquito genomes, respectively. Whereas retroviral envelopes are proteolytically cleaved into the ligand-interaction and membrane-fusion domains, Iris appears to lack this cleavage site. In the takahashii/suzukii species groups of Drosophila, we find that Iris has tandemly duplicated to give rise to two genes (Iris-A and Iris-B). Iris-B has significantly diverged from the Iris-A lineage, primarily because of the "invention" of an intron de novo in what was previously exonic sequence. Unlike domesticated retroviral envelope genes in mammals, we find that Iris has been subject to strong positive selection between Drosophila species. The rapid, adaptive evolution of Iris is sufficient to unambiguously distinguish the phylogenies of three closely related sibling species of Drosophila (D. simulans, D. sechellia, and D. mauritiana), a discriminative power previously described only for a putative "speciation gene." Iris represents the first instance of a retroviral envelope-derived host gene outside vertebrates. It is also the first example of a retroviral envelope gene that has been found to be subject to positive selection following its domestication. The unusual selective pressures acting on Iris suggest that it is an active participant in an ongoing genetic conflict. We propose a model in which Iris has "switched sides," having been recruited by host genomes to combat baculoviruses and retroviruses, which employ homologous envelope genes to mediate infection.     

Localization of the amphotropic murine leukemia virus receptor gene to the pericentromeric region of human chromosome 8.

The host range of retroviruses is determined primarily by the presence of specific receptors on target cells which are recognized by the retroviral envelope glycoprotein. Somatic cell hybrids have been used to determine the chromosomal locations of several retroviral receptors in mice prior to their molecular cloning. Here we report that by using human-Chinese hamster somatic cell hybrids and a retroviral vector, we have mapped the receptor for the amphotropic murine leukemia virus to the pericentromeric region of human chromosome 8.     

Polycation-mediated enhancement of retroviral transduction efficiency depends on target cell types and pseudotyped Env proteins: implication for gene transfer into neural stem cells

Polycations such as polybrene (PB) are routinely used for most retroviral vector-mediated gene transfer studies because they can increase the infectivity of retroviruses. However, it was not systematically determined if addition of the polycation is an essential prerequisite for all retroviral transductions. To test this, we measured the effects of the polycation on transduction efficiency using various combinations of target cells and pseudotyped viral envelope (Env) proteins. Here, we show polycations do not always increase retroviral transduction efficiency and that their enhancing effect depends on both the type of target cells and Env proteins. The findings presented here also suggest that high transduction rates can be achieved in primary neural stem cells in vitro and in vivo by choosing an appropriate Env protein for pseudotyping without using polycations which are potentially toxic to primary cells and may change the intrinsic characteristics of cells.     

Syncytins - retroviral envelope genes captured for the benefit of placental development

In the past, germline infections by retroviruses have led to vertical transmission of "endogenized" retroviruses. Escaping genetic drift, some of the viral genes have been conserved until now because of beneficial effects on their host. Here we present the syncytin genes that encode envelope proteins from endogenous retroviruses. Syncytins have inherited fusogenic properties from their infectious ancestor and are specifically expressed in the placenta. Both properties have suggested their involvement in the formation of the syncytiotrophoblast, a multinucleated layer that mediates feto-maternal exchanges in the placenta. The capture of syncytin genes occurred on several independent occasions during evolution of mammals. Knock-out experiments of syncytins in the mouse definitively confirmed the role of these genes in placentation. Finally, a second function for syncytins, i.e. an immunosuppressive activity, could contribute to materno-fetal immune tolerance. This constitutes a remarkable example of convergent evolution where the properties of retroviral envelope genes are subverted to play a major physiological role.     

Characterization of Resistance to Rhabdovirus and Retrovirus Infection in a Human Myeloid Cell Line

Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1.     

Incorporation of Fowl Plague Virus Hemagglutinin into Murine Leukemia Virus Particles and Analysis of the Infectivity of the Pseudotyped Retroviruses

We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and a lacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.     

Antibody binding in proximity to the receptor/glycoprotein complex leads to a basal level of virus neutralization

Hypothetically, antibodies may neutralize enveloped viruses by diverse mechanisms, such as disruption of receptor binding, interference with conformational changes required for virus entry, steric hindrance, or virus aggregation. Here, we demonstrate that retroviral infection mediated by the avian sarcoma-leukosis virus (ASLV-A) envelope glycoproteins can be neutralized by an antibody directed against a functionally unimportant component of a chimeric receptor protein. Thus, the binding of an antibody in proximity to the retroviral envelope glycoprotein-receptor complex, without binding to the entry machinery itself, results in neutralization. This finding provides additional support for the hypothesis that steric hindrance is sufficient for antibody-mediated neutralization of retroviruses.     

Effect of hypotonic stress on retroviral transduction

Short half-life has long been known as a main barrier for retroviral gene delivery due to quick degradation that seriously limited application of retrovirus-mediated methodology in the clinical use. To circumvent this challenge, many physical and chemical approaches have been developed to maximize contact opportunity of retroviruses and cells before viral vectors decay. However, most of methods are not easy to be followed due to complicated equipment settings and/or long procedures. In this study, we introduced an easy, cost-effective, efficient, and scalable strategy to enhance retroviral transduction by hypo-osmotic stress. It has been demonstrated that under hypotonic exposure, cell membrane is permeabilized to allow numerous exterior molecules accessing to cytoplasm through an intensive endocytosis, yielding high efficiency of cellular uptake. We hypothesized this hypotonic stress-induced internalization may provide a unique opportunity of cell entry for retroviruses without the need of receptor binding, and thus overcome the insufficient transduction rate due to loss of envelope protein. Indeed, our results showed that with assistance of hypotonic stress, retroviral transduction rates dramatically increased about 5.6- and 17.7-fold using fresh and decayed retroviruses, respectively, in comparison with corresponding groups without hypotonic stress. In summary, hypotonic stress was shown as a promising tool for enhancement of retroviral transduction efficiency without limitation of short half-life.     

Immunomodulation by peptide analogs of retroviral envelope protein

The mechanism by which retroviral proteins exert their immunosuppressive influence has remained enigmatic. Early studies have demonstrated that retroviral infection suppresses cellular and humoral immune responses. A hydrophilic 26 amino acid region of the otherwise hydrophobic transmembrane envelope protein of murine and feline leukemia viruses, p15E, is conserved among the transmembrane envelope proteins of numerous animal retroviruses (e.g. murine, feline, bovine and simian) as well as in human T-cell leukemia virus, and to a lesser extent, in human immunodeficiency virus (HIV). We evaluated the immunomodulatory properties of various synthetic retroviral envelope peptides synthesized as overlapping fragments to this conserved sequence. We report that two small peptides inhibit human mixed lymphocyte reaction (MLR), interleukin-2 (IL-2) and tumor necrosis factor (TNF-alpha) production. These peptides did not affect human natural killer (NK) cell cytotoxicity in vitro, and nitric oxide (NO) production in mouse macrophage cells, RAW264.7. Our observations suggests immunomodulatory potential of two retroviral peptide analogs.     

Effects of cell cycle status on early events in retroviral replication

The study of retroviruses over the last century has revealed a wide variety of disease-producing mechanisms, as well as apparently harmless interactions with animal hosts. Despite their potential pathogenic properties, the intrinsic features of retroviruses have been harnessed to create gene transfer vectors that may be useful for the treatment of disease. Retroviruses, as all viruses, have evolved to infect specific cells within the host, and such specificities are relevant to both pathogenesis and retrovirus-based vector design. The majority of cells of an animal host are not progressing rapidly through the cell cycle, and such a cellular environment appears to be suboptimal for replication of all retroviruses. Retrovirus-based vectors can therefore be restricted in many important target cells, such as post-mitotic differentiated cells or stem cells that may divide only infrequently. Despite intense interest, our understanding of how cell cycle status influences retroviral infection is still quite limited. In this review, we focus on the importance of the cell cycle as it relates to the early steps in retroviral replication. Retroviruses have been categorized based on their abilities to complete these early steps in non-cycling cells. However, all retroviruses are subject to a variety of cell cycle restrictions. Here, we discuss such restrictions, and how they may block retroviral replication, be tolerated, or overcome.     

Activation of a cell entry pathway common to type C mammalian retroviruses by soluble envelope fragments

Mutations that negatively or positively affect the fusion properties of murine leukemia viruses (MLVs) have been found within all subdomains of their SU (surface) and TM (transmembrane) envelope units. Yet, the interrelations between these different regions of the envelope complex during the cell entry process are still elusive. Deletion of the histidine residue of the conserved PHQV motif at the amino terminus of the amphotropic or the ecotropic MLV SU resulted in the AdelH or the MOdelH fusion-defective mutant envelope, respectively. These delH mutant envelopes are incorporated on retroviral particles at normal densities and normally mediate virion binding to cells expressing the retroviral receptors. However, both their cell-cell and virus-cell fusogenicities were fully prevented at an early postbinding stage. We show here that the fusion defect of AdelH or MOdelH envelopes was also almost completely reverted by providing either soluble SU or a polypeptide encompassing the receptor-binding domain (RBD) to the target cells, provided that the integrity of the amino-terminal end of either polypeptide was preserved. Restoration of delH envelope fusogenicity was caused by activation of the target cells via specific interaction of the latter polypeptides with the retrovirus receptor rather than by their association with the delH envelope complexes. Moreover crossactivation of the target cells, leading to fusion activation of AdelH or MOdelH envelopes, was achieved by polypeptides containing various type C mammalian retrovirus RBDs, irrespective of the type of entry-defective glycoprotein that was used for infection. Our results indicate that although they recognize different receptors for binding to the cell surface, type C mammalian retroviruses use a common entry pathway which is activated by a conserved feature of their envelope glycoproteins.     

Retroviral infection of hES cells produces random-like integration patterns

Retroviral integration provides us with a powerful tool to realize prolonged gene expressions that are often critical to gene therapy. However, the perturbation of gene regulations in host cells by viral genome integration can lead to detrimental effects, yielding cancer. The oncogenic potential of retroviruses is linked to the preference of retroviruses to integrate into genomic regions that are enriched in gene regulatory elements. To better navigate the double-edged sword of retroviral integration we need to understand how retroviruses select their favored genomic loci during infections. In this study I showed that in addition to host proteins that tether retroviral pre-integration complexes to specific genomic regions, the epigenetic architecture of host genome might strongly affect retroviral integration patterns. Specifically, retroviruses showed their characteristic integration preference in differentiated somatic cells. In contrast, retroviral infections of hES cells, which are known to display decondensed chromatin, produced random-like integration patterns lacking of strong preference for regulatory-element-rich genomic regions. Better identification of the cellular and viral factors that determine retroviral integration patterns will facilitate the design of retroviral vectors for safer use in gene therapy.     

Crystal structure of a pivotal domain of human syncytin-2, a 40 million years old endogenous retrovirus fusogenic envelope gene captured by primates

HERV-FRD is a human endogenous retrovirus that entered the human genome 40 million years ago. Its envelope gene, syncytin-2, was diverted by an ancestral host most probably because of its fusogenic property, for a role in placenta morphogenesis. It was maintained in a functional state in all primate branches as a bona fide cellular gene, submitted to a very low mutation rate as compared to infectious retrovirus genomes. The structure of the syncytin-2 protein thus provides a good insight into that of the oldest mammalian retroviral envelope. Here, we report the crystal structure of a central fragment of its "fossil" ectodomain, allowing a remarkable superposition with the structures of the corresponding domains of present-day infectious retroviruses, in spite of a more than 60% divergent sequence. These results suggest the existence of a unique structural solution selected by these proteins for their fusogenic function.     

Mapping of receptor binding domains in the envelope protein of spleen necrosis virus.

Spleen necrosis virus (SNV) is an amphotropic retrovirus originally isolated from a duck. Although of avian origin, it also replicates on some mammalian cells. SNV-derived retroviral vectors work with high efficiency and have a high potential for various gene transfer applications. However, little is known about the envelope-receptor interactions of this virus. We constructed a series of recombinant envelope proteins to characterize the SU peptide of SNV. We found that, in contrast to the envelope proteins of other retroviruses, truncated envelope proteins of SNV are transported to the cell surface. Surprisingly, particles displaying truncated envelope proteins can still infect cells, although at reduced efficiencies. Furthermore, these proteins can confer partial superinfection interference. Our data suggest that peptides throughout SU are involved in envelope-receptor interactions. To more precisely determine the localization of the main receptor binding domain, point mutations were introduced at certain regions of the SNV SU which are highly conserved among retroviruses belonging to the same receptor interference group. We identified one point mutation in the middle of SU (position 192) which drastically reduced infectivity and strongly reduced the ability to confer superinfection interference. The level of expression was not abolished, and translocation to the cell membrane of the mutant envelope occurred efficiently. This indicates that amino acid 192 may be directly involved in receptor binding.