Avian Sarcoma And Leukosis Virus Research Articles

Independent loss events of a functional tetherin gene in galliform birds.

Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Antiviral Activity and Adaptive Evolution of Avian Tetherins.

Tetherin/BST-2 is an antiviral protein that blocks the release of enveloped viral particles by linking them to the membrane of producing cells. At first, BST-2 genes were described only in humans and other mammals. Recent work identified BST-2 orthologs in nonmammalian vertebrates, including birds. Here, we identify the BST-2 sequence in domestic chicken (Gallus gallus) for the first time and demonstrate its activity against avian sarcoma and leukosis virus (ASLV). We generated a BST-2 knockout in chicken cells and showed that BST-2 is a major determinant of an interferon-induced block of ASLV release. Ectopic expression of chicken BST-2 blocks the release of ASLV in chicken cells and of human immunodeficiency virus type 1 (HIV-1) in human cells. Using metabolic labeling and pulse-chase analysis of HIV-1 Gag proteins, we verified that chicken BST-2 blocks the virus at the release stage. Furthermore, we describe BST-2 orthologs in multiple avian species from 12 avian orders. Previously, some of these species were reported to lack BST-2, highlighting the difficulty of identifying sequences of this extremely variable gene. We analyzed BST-2 genes in the avian orders Galliformes and Passeriformes and showed that they evolve under positive selection. This indicates that avian BST-2 is involved in host-virus evolutionary arms races and suggests that BST-2 antagonists exist in some avian viruses. In summary, we show that chicken BST-2 has the potential to act as a restriction factor against ASLV. Characterizing the interaction of avian BST-2 with avian viruses is important in understanding innate antiviral defenses in birds.IMPORTANCE Birds are important hosts of viruses that have the potential to cause zoonotic infections in humans. However, only a few antiviral genes (called viral restriction factors) have been described in birds, mostly because birds lack counterparts of highly studied mammalian restriction factors. Tetherin/BST-2 is a restriction factor, originally described in humans, that blocks the release of newly formed virus particles from infected cells. Recent work identified BST-2 in nonmammalian vertebrate species, including birds. Here, we report the BST-2 sequence in domestic chicken and describe its antiviral activity against a prototypical avian retrovirus, avian sarcoma and leukosis virus (ASLV). We also identify BST-2 genes in multiple avian species and show that they evolve rapidly in birds, which is an important indication of their relevance for antiviral defense. Analysis of avian BST-2 genes will shed light on defense mechanisms against avian viral pathogens.

Reverse Engineering Provides Insights on the Evolution of Subgroups A to E Avian Sarcoma and Leukosis Virus Receptor Specificity.

The initial step of retrovirus entry—the interaction between the virus envelope glycoprotein trimer and a cellular receptor—is complex, involving multiple, noncontiguous determinants in both proteins that specify receptor choice, binding affinity and the ability to trigger conformational changes in the viral glycoproteins. Despite the complexity of this interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins to use a different cellular protein as receptors. The highly homologous subgroup A to E Avian Sarcoma and Leukosis Virus (ASLV) glycoproteins belong to the group of class 1 viral fusion proteins with a two-step triggering mechanism that allows experimental access to intermediate structures during the fusion process. We and others have taken advantage of replication-competent ASLVs and exploited genetic selection strategies to force the ASLVs to naturally evolve and acquire envelope glycoprotein mutations to escape the pressure on virus entry and still yield a functional replicating virus. This approach allows for the simultaneous selection of multiple mutations in multiple functional domains of the envelope glycoprotein that may be required to yield a functional virus. Here, we review the ASLV family and experimental system and the reverse engineering approaches used to understand the evolution of ASLV receptor usage.

PH regulation in early endosomes and interferon-inducible transmembrane proteins control avian retrovirus fusion

Enveloped viruses infect host cells by fusing their membranes with those of the host cell, a process mediated by viral glycoproteins upon binding to cognate host receptors or entering into acidic intracellular compartments. Whereas the effect of receptor density on viral infection has been well studied, the role of cell type-specific factors/processes, such as pH regulation, has not been characterized in sufficient detail. Here, we examined the effects of cell-extrinsic factors (buffer environment) and cell-intrinsic factors (interferon-inducible transmembrane proteins, IFITMs), on the pH regulation in early endosomes and on the efficiency of acid-dependent fusion of the avian sarcoma and leukosis virus (ASLV), with endosomes. First, we found that a modest elevation of external pH can raise the pH in early endosomes in a cell type-dependent manner and thereby delay the acid-induced fusion of endocytosed ASLV. Second, we observed a cell type-dependent delay between the low pH-dependent and temperature-dependent steps of viral fusion, consistent with the delayed enlargement of the fusion pore. Third, ectopic expression of IFITMs, known to potently block influenza virus fusion with late compartments, was found to only partially inhibit ASLV fusion with early endosomes. Interestingly, IFITM expression promoted virus uptake and the acidification of endosomal compartments, resulting in an accelerated fusion rate when driven by the glycosylphosphatidylinositol-anchored, but not by the transmembrane isoform of the ASLV receptor. Collectively, these results highlight the role of cell-extrinsic and cell-intrinsic factors in regulating the efficiency and kinetics of virus entry and fusion with target cells.

Intronic deletions of tva receptor gene decrease the susceptibility to infection by avian sarcoma and leukosis virus subgroup A.

The group of avian sarcoma and leukosis virus (ASLV) in chickens contains six highly related subgroups, A to E and J. Four genetic loci, tva, tvb, tvc and tvj, encode for corresponding receptors that determine the susceptibility to the ASLV subgroups. The prevalence of ASLV in hosts may have imposed strong selection pressure toward resistance to ASLV infection, and the resistant alleles in all four receptor genes have been identified. In this study, two new alleles of the tva receptor gene, tvar5 and tvar6, with similar intronic deletions were identified in Chinese commercial broilers. These natural mutations delete the deduced branch point signal within the first intron, disrupting mRNA splicing of the tva receptor gene and leading to the retention of intron 1 and introduction of premature TGA stop codons in both the longer and shorter tva isoforms. As a result, decreased susceptibility to subgroup A ASLV in vitro and in vivo was observed in the subsequent analysis. In addition, we identified two groups of heterozygous allele pairs which exhibited quantitative differences in host susceptibility to ASLV-A. This study demonstrated that defective splicing of the tva receptor gene can confer genetic resistance to ASLV subgroup A in the host.

Defining a Retrovirus Entry Site by Single Particle Tracking

Current knowledge of viral entry pathways and therefore of host factors involved in virus trafficking to the sites of entry is limited. We imaged single Avian Sarcoma and Leukosis Virus (ASLV) co-trafficking with markers for early (Rab5) and late (Rab7) endosomes and visualized the acidification of endosomal lumen and subsequent virus-endosome fusion. The recruitment of Rab5 marker by virus-carrying endosomes usually coincided with acidification of their lumen, which triggered ASLV fusion. Fusion measured by the viral content release occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive), maturing endosomes. Expression of different isoforms of the cognate ASLV receptor (TVA) on target cells mediated virus entry from distinct compartments. In cells expressing the transmembrane TVA950 receptor, ASLV preferentially entered and fused with slowly maturing early endosomes, which accumulated Rab7 after a considerable delay. Viral entry occurred from either slowly or quickly maturing endosomes in cells expressing the GPI-anchored TVA800 receptor, as manifested by ASLV fusion with early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Simultaneous visualization of endosome acidification and viral content release enabled the measurement of the true kinetics of viral fusion with endosomes, which was independent of the receptor isoform. We concluded that the sites of ASLV entry are determined by the kinetic competition between endosome maturation and low pH-dependent fusion. These findings demonstrate the ability of ASLV to enter cells via alternative endocytic pathways and establish infection by fusing with distinct endosomal compartments. This work was partially supported by the NIH R01 AI053668 grant.

Live Cell Imaging of Retroviral Entry

Cellular entry of retroviruses is the first critical stage of retroviral replication. Live cell imaging has been utilized to visualize the dynamics, localization, and kinetics of the viral fusion process. Here, we review the different methodologies used for live cell imaging and how the use of these techniques has better elucidated the viral entry process of avian sarcoma and leukosis virus (ASLV) and human immunodeficiency virus type 1 (HIV-1) as well as cell-to-cell transmission of retroviruses. Although some controversies remain, further development of these techniques will provide new insights into the process and dynamics of retroviral fusion in vivo.

Prototype endogenous avian retroviruses of the genus Gallus.

Ancient endogenous retroviruses (ERVs), designated endogenous avian retrovirus (EAVs), are present in all Gallus spp. including the chicken, and resemble the modern avian sarcoma and leukosis viruses (ASLVs). The EAVs comprise several distinct retroviruses, including EAV-0, EAV-E51 and EAV-HP, as well as a putative member previously named the avian retrotransposon of chickens (ART-CH). Thus far, only the EAV-HP elements have been well characterized. Here, we determined sequences of representative EAV-0 and EAV-E51 proviruses by cloning and data mining of the 2011 assembly of the Gallus gallus genome. Although the EAV-0 elements are primarily deleted in the env region, we identified two complete EAV-0 env genes within the G. gallus genome and prototype elements sharing identity with an EAV-E51-related clone previously designated EAV-E33. Prototype EAV-0, EAV-E51 and EAV-E33 gag, pol and env gene sequences used for phylogenetic analysis of deduced proteins showed that the EAVs formed three distinct clades, with EAV-0 sharing the last common ancestor with the ASLVs. The EAV-E51 clade showed the greatest level of divergence compared with other EAVs or ASLVs, suggesting that these ERVs represented exogenous retroviruses that evolved and integrated into the germline over a long period of time. Moreover, the degree of divergence between the chicken and red jungle fowl EAV-E51 sequences suggested that they were more ancient than the other EAVs and may have diverged through mutations that accumulated post-integration. Finally, we showed that the ART-CH elements were chimeric defective ERVs comprising portions of EAV-E51 and EAV-HP rather than authentic retrotransposons.

Time-Resolved Imaging of Endosome Acidification and Single Retrovirus Fusion with Endosomes

Avian Sarcoma and Leukosis Virus (ASLV) enters cells by receptor-mediated endocytosis followed by low pH-dependent fusion with endosomes. We examined the pH dynamics in virus-carrying endosomes and the relationship between endosome acidification and ASLV fusion in cells expressing alternative receptor isoforms. The pH drop in endosomal lumen was measured by incorporating a genetically encoded pH sensor into the viral membrane. The subsequent single virus fusion was visualized as the release of a fluorescent viral content marker into the cytosol. To our knowledge, this is the first implementation of real-time measurements of endosomal pH dynamics and single virus fusion in the same experiment. We found that the pH in early acidic compartments harboring the virus was broadly distributed, ranging from 5.6 to 6.5. Surprisingly, after initial acidification, the endosomal pH did not decrease further for at as long as we could track the viral particles (up to 20 min). Analysis of the relationship between the endosome motility and lumenal pH showed that cells expressing the transmembrane isoform of the receptor (TVA950) preferentially sorted ASLV to slowly moving, less acidic endosomes. In contrast, ASLV showed no preference for fast vs. slow vesicles in cells expressing the GPI-anchored isoform (TVA800). The rate of fusion (distribution of time intervals between acidification and fusion) was significantly shorter and fusion pores were larger in mobile endosomes compared to more stationary compartments. In addition, TVA950 supported faster fusion than TVA800 in spite of the same average pH values in mobile compartments of these cells. Taken together, our results revealed that the ASLV can be selectively sorted to distinct endosomal pools and that the virus fuses with different kinetics and efficiency with these intracellular compartments. This work was partially supported by NIH AI053668 grant to G.B.M.

Real-Time Imaging of Retrovirus-Endosome Fusion followed by Release and Trafficking of the Viral Core

Binding to a cognate receptor renders the Avian Sarcoma and Leukosis Virus (ASLV) Env glycoprotein competent for undergoing low pH-dependent refolding which lead to fusion with acidic endosomes. Here we took advantage of the ASLV's ability to enter endosomes and maintain infectivity in the presence the NH4Cl, which raises the endosomal pH. This feature allowed capturing receptor-primed viruses in neutral compartments in the presence of NH4Cl and synchronous triggering of fusion by removing the weak base. Such “on-demand” fusion offered several advantages, including the quick and uniform reduction of endosomal pH in all intracellular compartments and precise duration of low pH exposure. The capture/release protocol resulted in a very fast release of a small viral content marker into the cytosol, as visualized by real-time single particle imaging. Moreover, this protocol favored the viral nucleocapsid release which was visualized as spatial separation of the GFP-labeled core from a virus-harboring endosome. Mean square displacement analysis of the core trajectories in the cytosol revealed that these sub-viral particles became much more motile compared to endosomes from which they were released. Importantly, we detected statistically significant differences between the diffusion coefficients of cores released from endosomes in host cells expressing two distinct receptor isoforms. In addition to the difference in motility of sub-viral particles, we also found that the rate of fusion pore dilation (a lag between the loss of a small content marker and release of the viral core) was faster in cell expressing the full-length receptor. Collectively, our data imply that, all things being equal, early endosomes are more conducive to ASLV fusion compared to late endosomes. This work has been supported by the NIH AI053668 grant.<!--Copyright (c) 2006 Microsoft Corporation. All rights reserved.

Intronic deletions that disrupt mRNA splicing of the tva receptor gene result in decreased susceptibility to infection by avian sarcoma and leukosis virus subgroup A.

The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.

Bookmarking the epigenome by retrovirus integration

Integration of retroviruses occurs in most genomic regions with weak but statistical significant preferences for target site sequences. HIV-1 preferentially targets genes, particularly the transcriptionally active ones. Avian sarcoma and leukosis viruses (ASLV) integrate with only slight preference for genes while murine leukemia virus uniquely favors integration in close proximity to upstream or downstream transcription start sites. Genomic features as well as the “epigenetic landscape“ at the site of integration are crucial for the outcome of each integration event, i. e. stable long terminal repeat-driven expression of the provirus or silencing and transcriptional suppression. The current availability of assembled genome sequences with epigenomic characteristics and reliable cloning of retrovirus integration sites enables to study the interplay between transcription signals of the retrovirus and enhancing or suppressive influences of the adjacent cellular DNA. We studied the integration sites of ASLV-derived vectors integrated into normal or DNA methylation-deficient human cells. Integration sites from cell clones containing single provirus were cloned and their genomic features as well as epigenetic landscape were correlated with expression or silencing of the provirus. Dnmt3b turned out to be the major player in silencing the long terminal repeat-driven transcription. Proviruses integrated into H3K4me3-rich CpG islands associated with promoters of active genes display long-term stability of expression and are resistant to the transcriptional silencing after over expression of Dnmt3b. Stability of expression and resistance to the silencing decreases with the distance from transcription start site. By contrast, proviruses integrated into the intergenic regions tend to the spontaneous transcriptional silencing even in DNA methyltransferase-deficient cells.

Hepatitis C Virus Is Primed by CD81 Protein for Low pH-dependent Fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.

Entry Pathways of an Avian Virus into Cells Expressing Transmembrane and GPI-Anchored Receptor Isoforms

Avian Sarcoma and Leukosis Virus (ASLV) entry into cells proceeds through two major steps -priming by cognate receptors on the cell surface followed by low pH-induced fusion with endosomes. Subtype A viruses are able to utilize two naturally occurring isoforms of the TVA receptor for infection, the GPI-anchored (TVA800) and the transmembrane (TVA950) receptors, which are localized to different membrane domains. We monitored the consecutive steps of ASLV entry into cells expressing these receptor isoforms, including endocytosis, delivery into acidic compartments, and fusion with endosomes. Cells expressing TVA950 internalized ASLV much faster than those expressing the alternative receptor. In both cell lines, fusion occurred shortly after internalization, suggesting that virus uptake was the rate-limiting step of entry. We found that ASLV fusion with endosomes proceeded through two relatively long-lived intermediates, a hemifusion-like intermediate and a small fusion pore. The lifetime of these intermediates was dependant on TVA receptor isoforms and their expression levels. TVA950 supported more robust fusion intermediates compared to TVA800. These findings are consistent with ASLV trafficking by via TVA950 to endosomal compartments that are more conducive to fusion. Alternatively, transmembrane receptor might more efficiently prime the viral fusion proteins compared to the GPI-anchored receptor. This work has been supported by the NIH AI053668 grant.

Cellular Transcription Factor ZASC1 Regulates Murine Leukemia Virus Transcription

To identify cellular processes involved in retroviral infection, we employed a high-volume forward genetic screen of insertionally mutagenized somatic cells using a murine leukemia virus (MLV) vector. This approach identified a clonal cell line that exhibited approximately 10-fold reduced gene expression from MLV vectors following infection despite supporting normal levels of MLV reverse transcription and integration. The defect in this cell line was specific for the MLV long terminal repeat (LTR) promoter, as normal levels of reporter gene expression were obtained from both an internal cytomegalovirus (CMV) promoter contained within an LTR-defective MLV vector and LTR expression from an avian sarcoma and leukosis virus (ASLV) vector. Complementation and shRNA knockdown experiments demonstrated that the defective gene in these cells is ZASC1 (ZNF639), a transcription factor with strong links to cancer and inherited ataxias. We demonstrated that ZASC1 is a sequence-specific DNA binding protein with three closely related binding sites located within the MLV LTR promoter, but it does not bind to the ASLV promoter. Mutating these putative ZASC1 binding sites significantly reduced levels of MLV gene expression. While wild-type ZASC1 activated expression from the MLV promoter, a green fluorescent protein-ZASC1 fusion protein showed dominant-negative inhibition of MLV gene expression. These studies identify the cellular transcription factor ZASC1 as an activator of MLV gene expression and provide tools that should be useful in studying the links between ZASC1 and human diseases.

Diverse Enveloped Viruses Fail to Undergo Complete Fusion with the Cell Plasma Membrane

Recent work from our laboratory (Miyauchi et al. 2009, Cell 137:433-444) provided functional evidence for HIV-1 entry via an endocytic pathway. In contrast, HIV-1 fusion with the plasma membrane (PM) did not proceed beyond the lipid mixing step. We have therefore assessed the ability of other enveloped viruses to fuse with the PM of permissive cells. Imaging of single amphotropic Murine Leukemia Virus particles revealed complete endoplasmic fusion with occasional lipid mixing at the PM. Next, we tested whether low pH-dependent viruses can be forced to fuse with the PM by lowering the external pH. When retroviral cores pseudotyped with the Semliki Forest Virus E1E2 glycoproteins were bound to cells and exposed to acidic pH, lipid mixing, but not viral content transfer was observed. Likewise, Avian Sarcoma and Leukosis Virus (ASLV) that enters target cells via a receptor-mediated endocytosis followed by low pH-induced fusion with endosomes failed to release its content at the PM upon reducing the pH. These results demonstrate a surprisingly strong preference for endosomal fusion among pH-dependent and pH-independent viruses, suggesting the existence of a fusion block at the PM and/or the involvement of an endosomal factor(s) facilitating the viral fusion. Consistent with the latter mechanism, inhibition of the dynamin function blocked endosomal fusion of HIV-1, ASLV and several other viruses. Together, these results are consistent with the involvement of cellular factors in facilitating the viral entry via endosomes. Supported by NIH grants GM054787 and AI053668.

A Charged Second-Site Mutation in the Fusion Peptide Rescues Replication of a Mutant Avian Sarcoma and Leukosis Virus Lacking Critical Cysteine Residues Flanking the Internal Fusion Domain

The entry process of the avian sarcoma and leukosis virus (ASLV) family of retroviruses requires first a specific interaction between the viral surface (SU) glycoproteins and a receptor on the cell surface at a neutral pH, triggering conformational changes in the viral SU and transmembrane (TM) glycoproteins, followed by exposure to low pH to complete fusion. The ASLV TM glycoprotein has been proposed to adopt a structure similar to that of the Ebola virus GP2 protein: each contains an internal fusion peptide flanked by cysteine residues predicted to be in a disulfide bond. In a previous study, we concluded that the cysteines flanking the internal fusion peptide in ASLV TM are critical for efficient function of the ASLV viral glycoproteins in mediating entry. In this study, replication-competent ASLV mutant subgroup A [ASLV(A)] variants with these cysteine residues mutated were constructed and genetically selected for improved replication capacity in chicken fibroblasts. Viruses with single cysteine-to-serine mutations reverted to the wild-type sequence. However, viruses with both C9S and C45S (C9,45S) mutations retained both mutations and acquired a second-site mutation that significantly improved the infectivity of the genetically selected virus population. A charged-amino-acid second-site substitution in the TM internal fusion peptide at position 30 is preferred to rescue the C9,45S mutant ASLV(A). ASLV(A) envelope glycoproteins that contain the C9,45S and G30R mutations bind the Tva receptor at wild-type levels and have improved abilities to trigger conformational changes and to form stable TM oligomers compared to those of the C9,45S mutant glycoprotein.

The Mechanism of Budding of Retroviruses From Cell Membranes.

Retroviruses have evolved a mechanism for the release of particles from the cell membrane that appropriates cellular protein complexes, referred to as ESCRT-I, -II, -III, normally involved in the biogenesis of multivesicular bodies. Three different classes of late assembly (L) domains encoded in Gag, with core sequences of PPXY, PTAP, and YPXL, recruit different components of the ESCRT machinery to form a budding complex for virus release. Here, we highlight recent progress in identifying the role of different ESCRT complexes in facilitating budding, ubiquitination, and membrane targeting of avian sarcoma and leukosis virus (ASLV) and human immunodeficiency virus, type 1 (HIV-1). These findings show that retroviruses may adopt parallel budding pathways by recruiting different host factors from common cellular machinery for particle release.

Transduction of Rhesus Macaque Hematopoietic Stem and Progenitor Cells with Avian Sarcoma and Leukosis Virus Vectors

Genome-wide integration site analyses showed that Moloney murine leukemia virus (MoMLV)- and lentivirus-derived vectors integrate preferentially into the coding regions of genes, posing a risk of insertional mutagenesis. Avian sarcoma and leukosis viruses (ASLVs) were previously reported to have a weak preference for gene-coding regions in a cell line study as compared with human immunodeficiency virus and MoMLV; however, thus far these vectors have not been studied for their potential efficacy in transduction of hematopoietic progenitor and stem cells. In this study we investigated for the first time the ability of ASLV-derived RCAS (replication-competent ALV LTR [avian leukosis virus long terminal repeat] with a splice acceptor) vectors to transduce rhesus macaque hematopoietic progenitors and long-term repopulating cells, in an autologous transplantation model. RCAS vectors can efficiently and stably transduce rhesus macaque CD34+ hematopoietic progenitor cells with an efficiency of transduction of up to 34% ex vivo. In two animals transplanted with RCAS vector-transduced autologous CD34+ cells, highly polyclonal hematopoietic reconstitution with sustained gene-marking levels in myeloid and lymphoid lineages was observed up to 18 months post-transplantation. These findings are encouraging and suggest that this vector system should be explored and further optimized for gene therapy applications targeting hematopoietic stem and progenitor cells.

The hr1 and Fusion Peptide Regions of the Subgroup B Avian Sarcoma and Leukosis Virus Envelope Glycoprotein Influence Low pH-Dependent Membrane Fusion

The avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) is activated to trigger fusion by a two-step mechanism involving receptor-priming and low pH fusion activation. In order to identify regions of ASLV Env that can regulate this process, a genetic selection method was used to identify subgroup B (ASLV-B) virus-infected cells resistant to low pH-triggered fusion when incubated with cells expressing the cognate TVB receptor. The subgroup B viral Env (envB) genes were then isolated from these cells and characterized by DNA sequencing. This led to identification of two frequent EnvB alterations which allowed TVB receptor-binding but altered the pH-threshold of membrane fusion activation: a 13 amino acid deletion in the host range 1 (hr1) region of the surface (SU) EnvB subunit, and the A32V amino acid change within the fusion peptide of the transmembrane (TM) EnvB subunit. These data indicate that these two regions of EnvB can influence the pH threshold of fusion activation.