Wenqin Xu

An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo

Leukemia and lymphoma account for more than 60% of deaths in captive koalas (Phascolarctos cinereus) in northeastern Australia. Although the endogenizing gammaretrovirus koala endogenous retrovirus (KoRV) was isolated from these koalas, KoRV has not been definitively associated with leukemogenesis. We performed KoRV screening in koalas from the San Diego Zoo, maintained for more than 45 y with very limited outbreeding, and the Los Angeles Zoo, maintained by continuously assimilating captive-born Australian koalas. San Diego Zoo koalas are currently free of malignant neoplasias and were infected with only endogenous KoRV, which we now term subtype “KoRV-A,” whereas Los Angeles Zoo koalas with lymphomas/leukemias are infected in addition to KoRV-A by a unique KoRV we term subtype “KoRV-B.” KoRV-B is most divergent in the envelope protein and uses a host receptor distinct from KoRV-A. KoRV-B also has duplicated enhancer regions in the LTR associated with increased pathology in gammaretroviruses. Whereas KoRV-A uses the sodium-dependent phosphate transporter 1 (PiT1) as a receptor, KoRV-B employs a different receptor, the thiamine transporter 1 (THTR1), to infect cells. KoRV-B is transmitted from dam to offspring through de novo infection, rather than via genetic inheritance like KoRV-A. Detection of KoRV-B in native Australian koalas should provide a history, and a mode for remediation, of leukemia/lymphoma currently endemic in this population.

Genetic Diversity of Koala Retroviral Envelopes

Genetic diversity, attributable to the low fidelity of reverse transcription, recombination and mutation, is an important feature of infectious retroviruses. Under selective pressure, such as that imposed by superinfection interference, gammaretroviruses commonly adapt their envelope proteins to use alternative receptors to overcome this entry block. The first characterized koala retroviruses KoRV subgroup A (KoRV-A) were remarkable in their absence of envelope genetic variability. Once it was determined that KoRV-A was present in all koalas in US zoos, regardless of their disease status, we sought to isolate a KoRV variant whose presence correlated with neoplastic malignancies. More than a decade after the identification of KoRV-A, we isolated a second subgroup of KoRV, KoRV-B from koalas with lymphomas. The envelope proteins of KoRV-A and KoRV-B are sufficiently divergent to confer the ability to bind and employ distinct receptors for infection. We have now obtained a number of additional KoRV envelope variants. In the present studies we report these variants, and show that they differ from KoRV-A and KoRV-B envelopes in their host range and superinfection interference properties. Thus, there appears to be considerable variation among KoRVs envelope genes suggesting genetic diversity is a factor following the KoRV-A infection process.

Primate Gammaretroviruses Require an Ancillary Factor Not Required for Murine Gammaretroviruses To Infect BHK Cells

ABSTRACT BHK cells remain resistant to xenotropic murine retrovirus-related virus (XMRV) or gibbon ape leukemia virus (GALV) infection, even when their respective receptors, Xpr1 or PiT1, are expressed. We set out to determine the stage at which viral infection is blocked and whether this block is mediated by a dominant-negative factor or the absence of a requisite ancillary factor. BHK cells bind neither XMRV nor GALV envelope proteins. BHK cells expressing the appropriate receptors bind XMRV or GALV envelope proteins. BHK cells can be infected by NZB-XMV(New Zealand Black mouse xenotropic murine virus)-enveloped vectors, expressing an envelope derived from a xenotropic retrovirus that, like XMRV, employs Xpr1 as a receptor, and also by vectors bearing the envelope of 10A1 murine leukemia virus (MLV), a murine retrovirus that can use PiT1 as a receptor. The retroviral vectors used in these analyses differ solely in their viral envelope proteins, suggesting that the block to XMRV and GALV infection is mediated at the level of envelope-receptor interactions. N-linked glycosylation of the receptors was not found to mediate resistance of receptor-expressing BHK cells to GALV or XMRV, as shown by tunicamycin treatment and mutation of the specific glycosylation site of the PiT1 receptor. Hybrid cells produced by fusing BHKXpr1 or BHKPiT1 to XMRV- or GALV-resistant cells, respectively, can mediate efficient XMRV or GALV infection. These findings indicate that BHK cells lack a factor that is required for infection by primate xenotropic viruses. This factor is not required for viruses that use the same receptors but were directly isolated from mice.

The host range of gammaretroviruses and gammaretroviral vectors includes post-mitotic neural cells.

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease.

Episodic diversifying selection shaped the genomes of gibbon ape leukemia virus and related gammaretroviruses

ABSTRACT Gibbon ape leukemia viruses (GALVs) are part of a larger group of pathogenic gammaretroviruses present across phylogenetically diverse host species of Australasian mammals. Despite the biomedical utility of GALVs as viral vectors and in cancer gene therapy, full genome sequences have not been determined for all of the five identified GALV strains, nor has a comprehensive evolutionary analysis been performed. We therefore generated complete genomic sequences for each GALV strain using hybridization capture and high-throughput sequencing. The four strains of GALV isolated from gibbons formed a monophyletic clade that was closely related to the woolly monkey virus (WMV), which is a GALV strain that likely originated in a gibbon host. The GALV-WMV clade in turn formed a sister group to the koala retroviruses (KoRVs). Genomic signatures of episodic diversifying selection were detected among the gammaretroviruses with concentration in the env gene across the GALV strains that were particularly oncogenic and KoRV strains that were potentially exogenous, likely reflecting their adaptation to the host immune system. In vitro studies involving vectors chimeric between GALV and KoRV-B established that variable regions A and B of the surface unit of the envelope determine which receptor is used by a viral strain to enter host cells. IMPORTANCE The gibbon ape leukemia viruses (GALVs) are among the most medically relevant retroviruses due to their use as viral vectors for gene transfer and in cancer gene therapy. Despite their importance, full genome sequences have not been determined for the majority of primate isolates, nor has comprehensive evolutionary analysis been performed, despite evidence that the viruses are facing complex selective pressures associated with cross-species transmission. Using hybridization capture and high-throughput sequencing, we report here the full genome sequences of all the GALV strains and demonstrate that diversifying selection is acting on them, particularly in the envelope gene in functionally important domains, suggesting that host immune pressure is shaping GALV evolution.

Koala Retroviruses: Evolution and Disease Dynamics.

A retroviral etiology for malignant neoplasias in koalas has long been suspected. Evidence for retroviral involvement was bolstered in 2000 by the isolation of a koala retrovirus (KoRV), now termed KoRV-A. KoRV-A is an endogenous retrovirus-a retrovirus that infects germ cells-a feature that makes it a permanent resident of the koala genome. KoRV-A lacks the genetic diversity of an exogenous retrovirus, a quality associated with the ability of a retrovirus to cause neoplasias. In 2013, a second KoRV isolate, KoRV-B, was obtained from koalas with lymphomas in the Los Angeles Zoo. Unlike KoRV-A, which is present in the genomes of all koalas in the United States, KoRV-B is restricted in its distribution and is associated with host pathology (neoplastic disease). Here, our current understanding of the evolution of endogenous and exogenous KoRVs, and the relationship between them, is reviewed to build a perspective on the future impact of these viruses on koala sustainability.

Guanine nucleotide exchange factor Epac2-dependent activation of the GTP-binding protein Rap2A mediates cAMP-dependent growth arrest in neuroendocrine cells

First messenger-dependent activation of MAP kinases in neuronal and endocrine cells is critical for cell differentiation and function and requires guanine nucleotide exchange factor (GEF)-mediated activation of downstream Ras family small GTPases, which ultimately lead to ERK, JNK, and p38 phosphorylation. Because there are numerous GEFs and also a host of Ras family small GTPases, it is important to know which specific GEF–small GTPase dyad functions in a given cellular process. Here we investigated the upstream activators and downstream effectors of signaling via the GEF Epac2 in the neuroendocrine NS-1 cell line. Three cAMP sensors, Epac2, PKA, and neuritogenic cAMP sensor–Rapgef2, mediate distinct cellular outputs: p38-dependent growth arrest, cAMP response element–binding protein–dependent cell survival, and ERK-dependent neuritogenesis, respectively, in these cells. Previously, we found that cAMP-induced growth arrest of PC12 and NS-1 cells requires Epac2-dependent activation of p38 MAP kinase, which posed the important question of how Epac2 engages p38 without simultaneously activating other MAP kinases in neuronal and endocrine cells. We now show that the small GTP-binding protein Rap2A is the obligate effector for, and GEF substrate of, Epac2 in mediating growth arrest through p38 activation in NS-1 cells. This new pathway is distinctly parcellated from the G protein—coupled receptor → Gs → adenylate cyclase → cAMP → PKA → cAMP response element–binding protein pathway mediating cell survival and the G protein—coupled receptor → Gs → adenylate cyclase → cAMP → neuritogenic cAMP sensor–Rapgef2 → B-Raf → MEK → ERK pathway mediating neuritogenesis in NS-1 cells.

EVALUATION OF CAPTIVE GIBBONS (HYLOBATES SPP., NOMASCUS SPP., SYMPHALANGUS SPP.) IN NORTH AMERICAN ZOOLOGICAL INSTITUTIONS FOR GIBBON APE LEUKEMIA VIRUS (GALV)

Abstract:  This study evaluated 79 captive gibbons (Hylobates, Nomascus, and Symphalangus spp.) within 30 North American zoological institutions for evidence of exposure to and possible infection with gibbon ape leukemia virus (GALV). Enzyme-linked immunosorbent assays (ELISAs) on gibbon serum samples revealed the presence of antibodies against GALV antigens in 28% of animals, indicating previous exposure or possibly protective immunity to GALV. Virus detection in gibbon blood or serum using polymerase chain reaction (PCR) or co-culture of gibbon peripheral blood mononuclear cells with human cells was negative for all samples submitted. The majority (19/27, 70%) of animals with reported health conditions were clinically healthy at the time of sample collection. Historically accrued clinical data were used to assess association of diseases in gibbons antibody positive for GALV. The results suggest captive gibbons could mount an immune response to GALV and show no evidence of infection. There was no association with neoplastic conditions in seropositive animals. The potential role of gibbons as a reservoir for GALV and the role of GALV as an epizoonotic-zoonotic agent or as a contributor to gibbon ape morbidity and mortality are not substantiated by the study findings.

C-terminal amidation of PACAP-38 and PACAP-27 is dispensable for biological activity at the PAC1 receptor.

PACAP-27 and PACAP-38 are the exclusive physiological ligands for the mammalian PAC1 receptor. The role of C-terminal amidation of these ligands at that receptor was examined in neuroendocrine cells expressing the PAC1 receptor endogenously and in non-neuroendocrine cells in which the human and rat PAC1 receptors were expressed from stable single-copy genes driven by the CMV promoter, providing stoichiometrically appropriate levels of this Gs-coupled GPCR in order to examine the potency and intrinsic activity of PACAP ligands and their des-amidated congeners. We found that replacement of the C-terminal glycine residues of PACAP-27 and -38 with a free acid; or extension of either peptide with the two to three amino acids normally found at these positions in PACAP processing intermediates in vivo following endoproteolytic cleavage and after exoproteolytic trimming and glycine-directed amidated, were equivalent in potency to the fully processed peptides in a variety of cell-based assays. These included real-time monitoring of cyclic AMP generation in both NS-1 neuroendocrine cells and non-neuroendocrine HEK293 cells; PKA-dependent gene activation in HEK293 cells; and neuritogenesis and cell growth arrest in NS-1 cells. The specific implications for the role of amidation in arming of secretin-related neuropeptides for biological function, and the general implications for neuropeptide-based delivery in the context of gene therapy, are discussed.

NCS-Rapgef2, the Protein Product of the Neuronal Rapgef2 Gene, Is a Specific Activator of D1 Dopamine Receptor-Dependent ERK Phosphorylation in Mouse Brain

Visual Abstract The neuritogenic cAMP sensor (NCS), encoded by the Rapgef2 gene, links cAMP elevation to activation of extracellular signal-regulated kinase (ERK) in neurons and neuroendocrine cells. Transducing human embryonic kidney (HEK)293 cells, which do not express Rapgef2 protein or respond to cAMP with ERK phosphorylation, with a vector encoding a Rapgef2 cDNA reconstituted cAMP-dependent ERK activation. Mutation of a single residue in the cyclic nucleotide-binding domain (CNBD) conserved across cAMP-binding proteins abrogated cAMP-ERK coupling, while deletion of the CNBD altogether resulted in constitutive ERK activation. Two types of mRNA are transcribed from Rapgef2 in vivo. Rapgef2 protein expression was limited to tissues, i.e., neuronal and endocrine, expressing the second type of mRNA, initiated exclusively from an alternative first exon called here exon 1’, and an alternative 5’ protein sequence leader fused to a common remaining open reading frame, which is termed here NCS-Rapgef2. In the male mouse brain, NCS-Rapgef2 is prominently expressed in corticolimbic excitatory neurons, and striatal medium spiny neurons (MSNs). Rapgef2-dependent ERK activation by the dopamine D1 agonist SKF81297 occurred in neuroendocrine neuroscreen-1 (NS-1) cells expressing the human D1 receptor and was abolished by deletion of Rapgef2. Corticolimbic [e.g., dentate gyrus (DG), basolateral amygdala (BLA)] ERK phosphorylation induced by SKF81297 was significantly attenuated in CamK2α-Cre+/−; Rapgef2cko/cko male mice. ERK phosphorylation in nucleus accumbens (NAc) MSNs induced by treatment with SKF81297, or the psychostimulants cocaine or amphetamine, was abolished in male Rapgef2cko/cko mice with NAc NCS-Rapgef2-depleting AAV-Synapsin-Cre injections. We conclude that D1-dependent ERK phosphorylation in mouse brain requires NCS-Rapgef2 expression.