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.     

High-yield retroviral production using a temperature-modulated two-stage operation

For clinical trials, large amounts of high-titer retroviral supernatants are required. However, retroviral concentration is relatively low compared with other viral vectors. Moreover, less than half of retroviral vectors suspended in a collected supernatant are infectious because of their short half-lives. In this study, a culture medium of ecotropic retrovirus-producing GP + E86/LNCX cells in tissue culture dishes was circulated through a reservoir, which was arranged with an incubator or ice-bath stage. Titers determined from the retroviral supernatant circulated through an ice-cold reservoir increased for a week from the beginning of retroviral production, while the titers from static production with circulation through the 37 degrees C reservoir reached a plateau after 3 days of retroviral production. After 5 days, 10 times more infectious retroviruses were obtained by circulating and keeping the majority of supernatant longer in the cold reservoir than in the production vessel at 37 degrees C in comparison with the number collected from the static tissue culture dish without circulating the culture medium. Furthermore, the concentration of transduction inhibitors in the supernatant was decreased along with the retardation of retroviral decay at low temperature. The two-stage operation developed in this study should be easily applied to large-scale bioreactors for mass production of high-titer retroviral supernatants.     

Ancient and modern retroviruses

Retroviruses are transmitted in two distinct ways: as infectious particles and as 'endogenous' proviral DNA integrated in the germ line of the host. Modern infectious viruses such as HIV-1 and HIV-2 recently infected mankind from chimpanzee and simian hosts, whereas human endogenous retroviral genomes have been present throughout old world primate evolution. Human T-cell leukemia viruses (HTLV-1 and II) have a much older human provenance than HIV, although new zoonoses from simians may also occur. We have recently characterized new retroviruses in pigs and humans. Porcine endogenous retroviral (PERV) genomes are carried in chromosomal DNA but can be activated to produce virions that are infectious for human cells, which has raised concern over human xenotransplantation using pig tissues. Human retrovirus 5 (HRV-5) is detected as an exogenous genome in association with arthritis and systemic lupus erythematosus.     

Envelope capture by retroviruses

Retroelement transposition is a major source of diversity in genome evolution. Among the retrotransposable elements, the retroviruses are distinct in that their "transposition" extends from their initial host cells to neighboring cells and organisms. A determining step in the conversion of a retrotransposable element into an infectious retrovirus is the acquisition of an envelope glycoprotein, designated Env. Here, we review some examples of envelope "capture" by mammal retroviruses and provide evidence for such a mechanism by HTLV. This phenomenon may explain the notable conservation of env genes observed between phylogenetically distant retroviruses. Elucidation of these recombination processes should help to clarify retroviral phylogeny, better understand retroviral pathogenesis, and may lead to the identification of new retroelements.     

Interplay between endogenous and exogenous human retroviruses

In humans, retroviruses thrive more as symbionts than as parasites. Apart from the only two modern exogenous human retroviruses (human T-cell lymphotropic and immunodeficiency viruses; HTLV and HIV, respectively), ~8% of the human genome is occupied by ancient retroviral DNA [human endogenous retroviruses (HERVs)]. Here, we review the recent discoveries about the interactions between the two groups, the impact of infection by exogenous retroviruses on the expression of HERVs, the effect of HERVs on the pathogenicity of HIV and HTLV and on the severity of the diseases caused by them, and the antiviral protection that HERVs can allegedly provide to the host. Tracing the crosstalk between contemporary retroviruses and their endogenized ancestors will provide better understanding of the retroviral world.     

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.     

Evidence that HIV budding in primary macrophages occurs through the exosome release pathway

Lipid rafts are specialized regions of cell membranes enriched in cholesterol and sphingolipids that are involved in immune activation and signaling. Studies in T-cells indicate that these membrane domains serve as sites for release of human immunodeficiency virus (HIV). By budding through lipid rafts in T-cells, HIV selectively incorporates raft markers and excludes non-raft proteins. This process has been well studied in T-cells, but it is unknown whether lipid rafts serve as budding sites for HIV in macrophages. Recently, we proposed a new model of retroviral biogenesis called the Trojan exosome hypothesis (Gould, S. J., Booth, A., and Hildreth, J. E. K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 10592-10597). This model proposes that retroviruses coopt the existing cellular machinery for exosomal release. Here, we performed the first test designed to differentiate between the lipid raft hypothesis of retroviral biogenesis and the Trojan exosome hypothesis. Using macrophages, we examined the relative abundance of several host proteins on the cell surface, in lipid rafts, and on both HIV particles and exosomes derived from these cells. Our results show significant differences in the abundance of host proteins on the cell surface and in HIV. Moreover, our data demonstrate discordance in the abundance of some proteins in lipid rafts and in HIV. Finally, our data reveal a strong concordance between the host cell protein profile of exosomes and that of HIV. These results strongly support the Trojan exosome hypothesis and its prediction that retroviral budding represents exploitation of a pre-existing cellular pathway of intercellular vesicle trafficking.     

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.     

Structure of Equine Infectious Anemia Virus Matrix Protein

The Gag polyprotein is key to the budding of retroviruses from host cells and is cleaved upon virion maturation, the N-terminal membrane-binding domain forming the matrix protein (MA). The 2.8-A resolution crystal structure of MA of equine infectious anemia virus (EIAV), a lentivirus, reveals that, despite showing no sequence similarity, more than half of the molecule can be superimposed on the MAs of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV). However, unlike the structures formed by HIV-1 and SIV MAs, the oligomerization state observed is not trimeric. We discuss the potential of this molecule for membrane binding in the light of conformational differences between EIAV MA and HIV or SIV MA.     

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.     

Protection against Retrovirus Pathogenesis by SR Protein Inhibitors

Indole derivatives compounds (IDC) are a new class of splicing inhibitors that have a selective action on exonic splicing enhancers (ESE)-dependent activity of individual serine-arginine-rich (SR) proteins. Some of these molecules have been shown to compromise assembly of HIV infectious particles in cell cultures by interfering with the activity of the SR protein SF2/ASF and by subsequently suppressing production of splicing-dependent retroviral accessory proteins. For all replication-competent retroviruses, a limiting requirement for infection and pathogenesis is the expression of the envelope glycoprotein which strictly depends on the host splicing machinery. Here, we have evaluated the efficiency of IDC on an animal model of retroviral pathogenesis using a fully replication-competent retrovirus. In this model, all newborn mice infected with a fully replicative murine leukemia virus (MLV) develop erythroleukemia within 6 to 8 weeks of age. We tested several IDC for their ability to interfere ex vivo with MLV splicing and virus spreading as well as for their protective effect in vivo. We show here that two of these IDC, IDC13 and IDC78, selectively altered splicing-dependent production of the retroviral envelope gene, thus inhibiting early viral replication in vivo, sufficiently to protect mice from MLV-induced pathogenesis. The apparent specificity and clinical safety observed here for both IDC13 and IDC78 strongly support further assessment of inhibitors of SR protein splicing factors as a new class of antiretroviral therapeutic agents.     

An siRNA Screen of Membrane Trafficking Genes Highlights Pathways Common to HIV-1 and M-PMV Virus Assembly and Release

The assembly and release of retroviruses from the host cells requires a coordinated series of interactions between viral structural proteins and cellular trafficking pathways. Although a number of cellular factors involved in retrovirus assembly have been identified, it is likely that retroviruses utilize additional trafficking factors to expedite their assembly and budding that have not yet been defined. We performed a screen using an siRNA library targeting host membrane trafficking genes in order to identify new host factors that contribute to retrovirus assembly or release. We utilized two retroviruses that follow very distinct assembly pathways, HIV-1 and Mason-Pfizer monkey virus (M-PMV) in order to identify host pathways that are generally applicable in retrovirus assembly versus those that are unique to HIV or M-PMV. Here we report the identification of 24 host proteins identified in the screen and subsequently validated in follow-up experiments as contributors to the assembly or release of both viruses. In addition to identifying a number of previously unsuspected individual trafficking factors, we noted multiple hits among proteins involved in modulation of the actin cytoskeleton, clathrin-mediated transport pathways, and phosphoinositide metabolism. Our study shows that distant genera of retroviruses share a number of common interaction strategies with host cell trafficking machinery, and identifies new cellular factors involved in the late stages of retroviral replication.     

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.     

Retroviral integration profiles: their determinants and implications for gene therapy

Retroviruses have often been used for gene therapy because of their capacity for the long-term expression of transgenes via stable integration into the host genome. However, retroviral integration can also result in the transformation of normal cells into cancer cells, as demonstrated by the incidence of leukemia in a recent retroviral gene therapy trial in Europe. This unfortunate outcome has led to the rapid initiation of studies examining various biological and pathological aspects of retroviral integration. This review summarizes recent findings from these studies, including the global integration patterns of various types of retroviruses, viral and cellular determinants of integration, implications of integration for gene therapy and retrovirus-mediated infectious diseases, and strategies to shift integration to safe host genomic loci. A more comprehensive and mechanistic understanding of retroviral integration processes will eventually make it possible to generate safer retroviral vector platforms in the near future.     

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.     

Functions of early (AP-2) and late (AIP1/ALIX) endocytic proteins in equine infectious anemia virus budding

The proline-rich L domains of human immunodeficiency virus 1 (HIV-1) and other retroviruses interact with late endocytic proteins during virion assembly and budding. In contrast, the YPDL L domain of equine infectious anemia virus (EIAV) is apparently unique in its reported ability to interact both with the mu2 subunit of the AP-2 adaptor protein complex and with ALG-2-interacting protein 1 (AIP1/Alix) protein factors involved in early and late endosome formation, respectively. To define further the mechanisms by which EIAV adapts vesicle trafficking machinery to facilitate virion production, we have examined the specificity of EIAV p9 binding to endocytic factors and the effects on virion production of alterations in early and late endocytic protein expression. The results of these studies demonstrated that (i) an approximately 300-residue region of AIP1/Alix-(409-715) was sufficient for binding to the EIAV YPDL motif; (ii) overexpression of AIP1/Alix or AP-2 mu2 subunit specifically inhibited YPDL-mediated EIAV budding; (iii) virion budding from a replication-competent EIAV variant with its L domain replaced by the HIV PTAP sequence was inhibited by wild type or mutant mu2 to a level similar to that observed when a dominant-negative mutant of Tsg101 was expressed; and (iv) overexpression or siRNA silencing of AIP1/Alix and AP-2 revealed additive suppression of YPDL-mediated EIAV budding. Taken together, these results indicated that both early and late endocytic proteins facilitate EIAV production mediated by either YPDL or PTAP L domains, suggesting a comprehensive involvement of endocytic factors in retroviral assembly and budding that can be accessed by distinct L domain specificities.     

Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance

Integrase interactor 1 (INI1)/hSNF5 is a host factor that directly interacts with human immunodeficiency virus type 1 (HIV-1) integrase and is incorporated into HIV-1 virions. Here, we show that while INI1/hSNF5 is completely absent from purified microvesicular fractions, it is specifically incorporated into HIV-1 virions with an integrase-to-INI1/hSNF5 stoichiometry of approximately 2:1 (molar ratio). In addition, we show that INI1/hSNF5 is not incorporated into related primate lentiviral and murine retroviral particles despite the abundance of the protein in producer cells. We have found that the specificity in incorporation of INI1/hSNF5 into HIV-1 virions is directly correlated with its ability to exclusively interact with HIV-1 integrase but not with other retroviral integrases. This specificity is also reflected in our finding that the transdominant mutant S6, harboring the minimal integrase interaction domain of INI1/hSNF5, blocks HIV-1 particle production but not that of the other retroviruses in 293T cells. Taken together, these results suggest that INI1/hNSF5 is a host factor restricted for HIV-1 and that S6 acts as a highly specific and potent inhibitor of HIV-1 replication.