Koala retrovirus (KoRV) is in the process of endogenization into the koala ( Phascolarctos cinereus ) genome and is currently spreading through the Australian koala population. Understanding how the koala’s immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala’s immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.

In the neuroendocrine NS‐1 cell line, three separate cAMP signaling pathways mediate growth arrest (via Epac2 ‐‐ p38 MAPK), induction of neuronal genes and cell survival (via PKA ‐‐ CREB), and neurite outgrowth (via NCS‐Rapgef2 ‐‐ ERK) (see Emery et al., J. Biol. Chem. 289:10126–39, 2014). There is no identifiable cross‐talk between these three pathways, thus they are parcellated. These three cAMP signaling pathways are activated by a variety of Gs‐coupled receptors expressed in the CNS, which include the PACAP receptor PAC1 (Emery & Eiden, FASEB J, 26, 3199–3211, 2011) and the D1 dopamine receptor (Jiang et al., eNeuro, ENEURO.0248–17.2017). Therefore, a signaling component unique to one pathway could represent a target for intracellular therapeutics to modulate aspects of neuronal behavior for CNS disorders related to addiction (e.g. D1‐dependent signaling to ERK) and anxiety (e.g. PACAP/PAC1 signaling). Using cell‐based assays to monitor the activation of each of the three pathways, we have sought to identify specific inhibitors and activators of NCS‐Rapgef2.Using a battery of biochemical and high‐content assays that distinguish signaling between the three cAMP sensors (PKA, Epac2 and NCS‐Rapgef2), we found that the adenylate cyclase inhibitor 9‐tetrahydrofuranyladenine (SQ,22536; IC50 for forskolin‐induced cAMP accumulation = 10 μM) also inhibits NCS‐Rapgef2 (IC50 = 170 μM, inhibiting 8‐Br‐cAMP‐induced Elk1 activation, reported in Emery et al., Mol Pharmacol 83, 95–105, 2013). To create compounds with selectivity and greater potency at NCS‐Rapgef2, we generated analogs of 9‐tetrahydrofuranyladenine at several positions of the adenine (6‐amino‐purine) moiety. In NS‐1 cells, we found that the compound N6‐phenyl‐9‐tetrahydrofuranyladenine (EL1101) has greatly reduced activity at adenylate cyclase (IC50 > 300 μM) and no detectable activity, either an agonist antagonist, for PKA‐ and Epac2‐dependent signaling. However, EL1101 inhibts 8‐Br‐cAMP‐induced ERK phosphorylation with a greater potently than 9‐tetrahydrofuranyladenine (IC50 = 10 μM for EL1101). At similar potencies (IC50 = 10 μM), EL1101 also blocked ERK phosphorylation by Gs‐coupled GPCR activation (PACAP/PAC1), adenylate cyclase activation by forskolin, and direct activation of NCS‐Rapgef2 with cell‐permeable cAMP analogs (8‐Br‐cAMP). We also employed a phenotypic high‐content assay (cAMP analog‐dependent neurite outgrowth) and found that EL1101 blocked the neuritogenic effect of cAMP analogs at a similar potency observed in biochemical assays (IC50 = 10 μM). We are currently exploring further derivatization of the 9‐tetrahydrofuranyladenine scaffold to create a sufficiently potent and selective NCS‐Rapgef2 inhibitor for use in vivo to block aspects of D1 dopamine receptor‐dependent signaling caused by psychomotor stimulants that require ERK activation.Support or Funding InformationThis work was supported by National Institute of Mental Health Intramural Research Program Grants 1ZIAMH002386 (to L.E.E.) and 1ZIAMH002592 (to M.V.E.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The beta 1 and beta 2 adrenoceptors (β1 and β2 AR) are GPCRs for the catecholamines norepinephrine and epinephrine. Both of these receptors couple to Gsα. Their activation causes cAMP elevation, which in turn controls cellular signaling through its downstream effectors PKA and the cAMP guanine nucleotide exchange factors (GEFs) Epac1 and Epac2. The β2AR has also been shown to signal via engagement of β‐arrestin, which acts as a scaffold for a variety of signaling proteins, including the MAP kinase ERK. We have recently characterized a neuronal and endocrine‐specific cAMP sensor, a GEF related to Epac 1 and 2, and to the previously characterized non‐cAMP‐activated GEF PDZ‐GEF1, which we have named NCS (neuritogenic cyclic AMP sensor)‐Rapgef2 (a protein product of the Rapgef2 gene). This sensor mediates activation of ERK leading to neuritogenesis in the PC12 and NS‐1 neuroendocrine cell lines (Emery et al., Sci. Signal. 6, ra51, 2013; Emery et al., J. Biol. Chem. 289: 10125, 2014). We have created NS‐1 cell lines stably expressing either β1AR or β2AR, and examined signaling to each of the three cAMP sensors present in these cells following treatment with isoproterenol. In β1AR‐expressing cells, agonist treatment caused activation of all measurable cAMP‐dependent signaling pathways in these cells: Epac2/p38‐dependent growth arrest; PKA‐dependent CREB phosphorylation; and NCS‐Rapgef2/ERK‐dependent neuritogenesis. In contrast, agonist stimulation of β2AR‐expressing cells caused isoproterenol‐initiated Epac2/p38‐dependent growth arrest and PKA‐dependent CREB phosphorylation, but did not couple to NCS‐Rapgef2/ERK‐dependent neuritogenesis. To compare the desensitization profiles of the two receptors, a biosensor that allowed for continuous real‐time cyclic AMP measurements was co‐expressed in each cell line (Emery et al., Peptides, 79: 39, 2016). In β2AR‐expressing cells, the maximal effect of isoproterenol on cAMP was observed after 10 minutes of treatment and decreased rapidly thereafter. In contrast, isoproterenol‐dependent β1AR activation caused persistent cAMP elevation, observed at approximately maximal levels at least 40 minutes following agonist addition. Unlike the mode of ERK phosphorylation observed following β1AR activation (NCS‐Rapgef2‐dependent), ERK activation elicited by β2AR (NCS‐Rapgef2‐independent) most likely occurs in a cellular compartment restricted from transcriptional regulation, which is required for neuritogenesis in this cell type (Ravni et al., Mol. Pharmacol. 73: 1688, 2008). We conclude that there is an inverse relationship between adrenergic receptor desensitization, and engagement of NCS‐Rapgef2 of sufficient duration to support the sustained activation of ERK necessary to promote neuritogenesis in NS‐1 cells. We notice the same inverse relationship in receptors for dopamine (D1) and adenosine (A2A), as well as the neuropeptides PACAP, VIP, and GLP‐1 (PAC1, VPAC1, VPAC2, and GLP‐1R).Support or Funding InformationThis work was supported by NIMH Intramural Research Program Project ZIAMH002386 and by a 2014 NARSAD Young Investigator Grant to A.C.E. from the Brain and Behavior Research Foundation (Grant 21356).

We have identified in a neuroendocrine cell line, NS‐1, three separate pathways for cAMP signaling mediated through Epac2 via p38 phosphorylation (subserving growth arrest); through PKA via CREB phosphorylation (subserving neuronal survival and neuron‐specific gene expression); and through the novel cAMP sensor NCS‐Rapgef2 (Emery et al., Sci. Signal. 6,281:ra51, 2013) via ERK phosphorylation (subserving neuritogenesis) (Emery et al., J. Biol. Chem. 289:10126–39, 2014). These pathways are parcellated, exhibiting little if any signaling cross‐talk, and are activated by a variety of CNS Gs‐coupled receptors, including the PAC1 PACAP receptor (Emery & Eiden, FASEB J, 26, 3199–3211, 2011) and the D1 dopamine receptor (Eiden et al., FASEB J, 30, supplement 1, 1265.5, 2016). Thus, points within each pathway may eventually represent targets for intracellular therapeutics aimed at modulation of individual components of neuronal behavior, in the context of CNS disorders relating to addiction and anxiety. Accordingly we have embarked on a drug discovery program, using assays developed in cellula for each pathway, for specific inhibitors of NCS‐Rapgef2.The adenylyl cyclase inhibitor SQ22,536 (9‐tetrahydrofuranyladenine) (IC50 = 10 mM to inhibit forskolin‐induced Elk1 activation) has in addition inhibitory activity against NCS‐Rapgef2 (IC50 = 170 mM to inhibit 8‐Br‐cAMP‐induced Elk1 activation): this work was carried out using a battery of high‐content in cellula assays that distinguish signaling through the three intracellular cAMP sensors NCS‐Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA) (Emery et al., Mol Pharmacol 83, 95–105, 2013). We have now examined the activities of compounds with modified substituents at several positions of adenine (6‐amino‐purine) to enhance selectivity for NCS‐Rapgef2 by decreasing affinity for adenylyl cyclase (AC) without increasing affinity for PKA or Epac. The compound N6‐phenyl‐9‐tetrahydrofuranyladenine is a potent NCS‐Rapgef2 inhibitor (IC50 = 10 mM to inhibit 8‐Br‐cAMP‐dependent ERK phosphorylation) with greatly diminished AC inhibitory activity (IC50 >300 mM for cyclic AMP measurements following 40 min treatment with IBMX + forskolin), without noticeable inhibitory or stimulatory activity at either PKA or Epac. These measurements were carried out in the neuroendocrine pheochromocytoma (PC12)‐derived neuroendocrine cell line NS‐1, engineered to allow high‐content screening for activation and inhibition of AC, PKA, Epac and NCS‐Rapgef2. Exploration of further modification to create a sufficiently potent and selective NCS‐Rapgef2 inhibitor for use in vivo for blockade of D1 dopamine receptor‐dependent effects of psychomotor stimulants that require signaling through ERK are in progress.Support or Funding InformationThis work was supported by the National Institute of Mental Health Intramural Research Program, Projects MH002386 to L.E.E. and MH002592 to M.V.E.

We recently reported that the adenylate cyclase (AC) inhibitor SQ22,536 (9-tetrahydrofuranyl-adenine) also has inhibitory activity against the neuroendocrine-specific neuritogenic cAMP sensor-Rapgef2 (NCS-Rapgef2), a guanine nucleotide exchanger and activator for the small effector GTPase Rap1. Cell-based assays that distinguish signaling through the three intracellular cAMP sensors NCS-Rapgef2, exchange protein activated by cAMP (Epac), and protein kinase A (PKA), as well as AC, were used. These, collectively, assess the activities of adenine (6-amino-purine) derivatives modified at several positions to enhance selectivity for NCS-Rapgef2 by decreasing affinity for adenylate cyclase (AC), without increasing affinity for PKA or Epac. Testing of each adenine derivative in whole-cell assays incorporates features of cell permeability, target selectivity, and intrinsic potency into a single EC50 or IC50, making robust extrapolation to compound activity in vivo more likely. N6-MBC-cAMP is a selective PKA activator (EC50 = 265 μM) with low efficacy at NCS-Rapgef2. 8-CPT-2'-O-Me-cAMP and ESI-09 are confirmed as Epac-selective, for stimulation and inhibition, respectively, versus both PKA and NCS-Rapgef2. The compound N6-Phe-cAMP is a full agonist of NCS-Rapgef2 (EC50 = 256 μM). It has little or no activity against Epac or PKA. The compound N6-phenyl-9-tetrahydrofuranyladenine is a novel and potent NCS-Rapgef2 inhibitor without activity at PKA, Epac, or ACs, as assayed in the neuroendocrine NS-1 cell line. This line has been engineered to allow high-content screening for activation and inhibition of AC, PKA, Epac, and NCS-Rapgef2 and the cellular activities initiated by these signaling pathway protein components.

SUMMARY Viruses of the subfamily Orthoretrovirinae are defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and “fossil” endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions.

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.

The neuritogenic cAMP sensor (NCS), encoded by theRapgef2gene, 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 aRapgef2cDNA 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 fromRapgef2 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 ofRapgef2. Corticolimbic [e.g., dentate gyrus (DG), basolateral amygdala (BLA)] ERK phosphorylation induced by SKF81297 was significantly attenuated inCamK2α-Cre+/−;Rapgef2cko/ckomale mice. ERK phosphorylation in nucleus accumbens (NAc) MSNs induced by treatment with SKF81297, or the psychostimulants cocaine or amphetamine, was abolished in maleRapgef2cko/ckomice with NAc NCS-Rapgef2-depleting AAV-Synapsin-Cre injections. We conclude that D1-dependent ERK phosphorylation in mouse brain requires NCS-Rapgef2 expression.

Gs‐coupled GPCR signaling in the NS‐1 pheochromocytoma cell line leads to the activation of three distinct cyclic AMP sensors, Epac, PKA and NCS/Rapgef2 mediate distinct cellular outputs: p38‐dependent growth arrest; CREB‐dependent cell survival and neuron‐specific gene expression; and ERK‐dependent neuritogenesis, respectively (Emery et al., Sci. Sig. 6 : ra51, 2013; Emery et al., JBC 289 : 10126, 2014). Expression of D1 dopamine receptors from a stably‐integrated, CMV promoter‐driven expression cassette in NS‐1 cells causes cyclic AMP elevation and activation of all three of these pathways, leading to D1 receptor‐dependent activation of all three downstream pathways, as previously reported for activation of the endogenous PAC1hop receptor by the neuropeptide PACAP. To investigate which of the three pathways might mediate cAMP‐dependent immediate‐early gene (IEG) transcription relevant to neurotransmitter action in vivo, we examined genome‐wide transcriptional regulation by cyclic AMP in NS‐1 cells, using microarray analysis. Treatment of NS‐1 cells for one hour with the cell‐permeant cyclic AMP analog 8‐CPT‐cAMP (100 micromolar), an equipotent activator of Epac2, PKA and NCS/Rapgef2 (Emery et al., JBC 289 : 10126, 2014) resulted in significant up‐regulation of 174 mRNAs. Of these, ten encoded transcription factors, and six of these encoded IEGs: Ier‐3/Egr3, Zif268/Egr1, Nr4a1, Nr4a3, c‐Fos, and JunB. Cyclic AMP‐dependent up‐regulation of the Ier‐3/Egr2 and Zig268/Egr1 genes, but not the Nr1a1, Nr4a3, or JunB genes, was ERK‐dependent (blocked by the MEK inhibitor U0126, at 10 micromolar), while up‐regulation of the c‐Fos gene appeared to be partially ERK‐dependent. These results suggest that regulation of IEG expression by D1 and PAC1 receptor stimulation may involve two cAMP‐dependent pathways, one mediated by NCS/Rapgef2, and the other by either Epac or PKA. These novel signaling pathways may be relevant to IEG regulation by dopamine in the striatum in vivo (Gerfen et al., J. Neurosci. 15: 8167, 1995) and by the stress‐regulatory neuropeptide PACAP in hypothalamus and extended amygdala (Stroth and Eiden, Neuroscience 165: 1025, 2010; Emery and Eiden, FASEB J. 26: 3199, 2012). Support or Funding Information Supported by NIMH‐IRP project ZO1‐MH002386

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.

Cyclic AMP elevation is well known to cause dividing neuroendocrine (e.g. PC12) cells to differentiate through a process that includes growth arrest, neurite extension, and induction of neuronal genes. Using the PC12 subclone NS‐1, we have previously shown that downstream of cAMP, signaling for differentiation is mediated by three insulated signaling pathways, initiated by cAMP activation of Epac2/Rapgef4, NCS/Rapgef2, and PKA, which regulate the activation of p38 MAP kinase, ERK, and CREB, respectively. These, in turn, are individually necessary for growth arrest (Epac2/Rapgef4), neurite extension (NCS/Rapgef2), and neuron‐specific gene expression (PKA) (Emery et al., 2014, J Biol Chem, 289:10126–39). Specific inhibitors and activators of PKA, Epac, and NCS/Rapgef2 would be useful to further explore the unique physiological functions of these three cAMP sensors in the CNS. Several compounds that show differential activity with respect to PKA and Epac exist, however, the selectivity of these agents across all three neuroendocrine cAMP sensors is not yet known. The goal of the work reported here was to identify cAMP analogs that provide specific activation and inhibition of each of these cAMP sensors. To this end, we have used a medium‐throughput assay platform that allows for quantitative measurement of the phosphorylation status of ERK, CREB, and p38 MAPK, which serve as read‐outs for activation of NCS/Rapgef2, PKA, and Epac2, respectively. 8‐CPT‐cAMP was found to be a full agonist with a similar potency at each sensor, while 8‐CPT‐2′‐O‐Me‐cAMP was highly specific for Epac relative to PKA (as reported by Enserink et al., 2002, Nat Cell Biol, 4:901–6) and also NCS/Rapgef2. We examined previously characterized PKA site‐specific agonists, i.e. compounds that stimulate PKA holoenzyme activity only when applied in combination, to determine if any of these provide SAR entree to further development of Epac or NCS/Rapgef2‐specific agonists. Our preliminary evidence suggests that several B site‐specific compounds, when administered singly, show significant activity at NCS/Rapgef2 without stimulating either Epac or the PKA holoenzyme. None of the cyclic nucleotide antagonists (Rp diastereomeric cAMP analogs) that we tested effectively inhibited cAMP‐dependent NCS/Rapgef2 signaling. Based on our previous work indicating that the nucleoside adenylate cyclase inhibitor SQ22,536 inhibits NCS/Rapgef2 (Emery et al., 2013, Mol Pharmacol, 83:95–105), along with new insights into structure‐activity relationships for NCS/Rapgef2 gleaned from our survey of B‐site‐specific activators, we propose that the development of a cell‐permeant, highly specific inhibitor of NCS/Rapgef2 is feasible.Support or Funding InformationSupported by NIMH‐IRP project ZO1‐MH002386

ABSTRACT Gibbon ape leukemia virus (GALV) and koala retrovirus (KoRV) most likely originated from a cross-species transmission of an ancestral retrovirus into koalas and gibbons via one or more intermediate as-yet-unknown hosts. A virus highly similar to GALV has been identified in an Australian native rodent ( Melomys burtoni ) after extensive screening of Australian wildlife. GALV-like viruses have also been discovered in several Southeast Asian species, although screening has not been extensive and viruses discovered to date are only distantly related to GALV. We therefore screened 26 Southeast Asian rodent species for KoRV- and GALV-like sequences, using hybridization capture and high-throughput sequencing, in the attempt to identify potential GALV and KoRV hosts. Only the individuals belonging to a newly discovered subspecies of Melomys burtoni from Indonesia were positive, yielding an endogenous provirus very closely related to a strain of GALV. The sequence of the critical receptor domain for GALV infection in the Indonesian M. burtoni subsp. was consistent with the susceptibility of the species to GALV infection. The second record of a GALV in M. burtoni provides further evidence that M. burtoni , and potentially other lineages within the widespread subfamily Murinae , may play a role in the spread of GALV-like viruses. The discovery of a GALV in the most western part of the Australo-Papuan distribution of M. burtoni , specifically in a transitional zone between Asia and Australia (Wallacea), may be relevant to the cross-species transmission to gibbons in Southeast Asia and broadens the known distribution of GALVs in wild rodents. IMPORTANCE Gibbon ape leukemia virus (GALV) and the koala retrovirus (KoRV) are very closely related, yet their hosts neither are closely related nor overlap geographically. Direct cross-species infection between koalas and gibbons is unlikely. Therefore, GALV and KoRV may have arisen via a cross-species transfer from an intermediate host whose range overlaps those of both gibbons and koalas. Using hybridization capture and high-throughput sequencing, we have screened a wide range of rodent candidate hosts from Southeast Asia for KoRV- and GALV-like sequences. Only a Melomys burtoni subspecies from Wallacea (Indonesia) was positive for GALV. We report the genome sequence of this newly identified GALV, the critical domain for infection of its potential cellular receptor, and its phylogenetic relationships with the other previously characterized GALVs. We hypothesize that Melomys burtoni , and potentially related lineages with an Australo-Papuan distribution, may have played a key role in cross-species transmission to other taxa.

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.

The neuritogenic cAMP sensor (NCS), the protein product of the Rapgef2 gene, couples GPCR‐Gs‐dependent elevation of cAMP to ERK activation required for neuritogenesis, while additional aspects of cAMP‐dependent cell differentiation are mediated through PKA (pro‐survival effects) and Epac2 (growth arrest) (Emery et al., JBC 289: 10126, 2014). Functional full‐length Rapgef2 is expressed in cell lines of neuroendocrine lineage, but not in several non‐neuroendocrine cell lines commonly used for high‐throughput GPCR ligand and antagonist screening (Emery et al., Sci. Sig. 6: ra51, 2013). In a series of NS‐1 cell sublines into which Gs‐coupled GPCRs were introduced as single‐copy genes under the control of the CMV promoter, some (e.g. ADCYAP1R1, ADBR1, DRD1), but not others (ADBR2, ADORA2A), engage NCS/Rapgef2 and induce neuritogenesis when stimulated with the appropriate ligand. All are coupled to adenylate cyclase and cause stimulation of Epac and PKA. To determine the mechanism of coupling specificity between GPCRs and NCS/Rapgef2, we are evaluating the membranous adenylate cyclase (AC) specificity for NCS/Rapgef2 coupling. In PC12 cells, which express ACs 3, 4, 6, 7, and 9, PACAP‐initiated signaling through ADCYAP1R1 (cAMP elevation, ERK phosphorylation, and neuritogenesis) is greatly attenuated by knockdown of AC6, but not AC7, achieved by stable lentiviral‐mediated shRNA expression, with no significant effects of either knock‐down on forskolin‐induced downstream cAMP‐dependent signaling events (ERK activation and neuritogenesis). These data suggest that the ADCYAP1R1 (PAC1) receptor is preferentially coupled to AC6, and this AC preference is linked to engagement of NCS/Rapgef2, ERK activation, and neuritogenesis.

PC12 cells express five adenylate cyclase (AC) isoforms, most abundantly AC6 and AC7. These two ACs were individually silenced using lentiviral short hairpin RNAs, which lead to a decrease (≥80%) of the protein product of each transcript. These stable PC12 sublines were then used to examine potential AC isoform preference for signaling through a family B G protein–coupled receptor (GPCR). Cells were challenged with the endogenous agonist of the pituitary adenylate cyclase–activating polypeptide type I receptor (PAC1), pituitary adenylate cyclase–activating polypeptide (PACAP)-38, or the diterpene forskolin as an AC-proximal control. Intracellular cAMP levels were elevated by forskolin about equally in wild-type, AC6, and AC7 knockdown cells. The ability of PACAP-38 and forskolin to activate three cAMP sensors downstream of AC [protein kinase A (PKA), exchange protein activated by cAMP (Epac) 2/Rapgef4, and neuritogenic cAMP sensor (NCS)/Rapgef2] was examined by monitoring the phosphorylation status of their respective targets, cAMP response element–binding protein, p38, and extracellular signal-regulated kinase. Forskolin stimulation of each downstream target of cAMP was unaffected by knockdown of either AC6 or AC7. PACAP-38 activation of all downstream targets of cAMP was unaffected by AC7 knockdown, but abolished following AC6 knockdown. Membrane cholesterol depletion with methyl-β-cyclodextrin mimicked the effects of AC6 silencing on PACAP signaling, without attenuating forskolin signaling. These data suggest that vicinal constraint of the GPCR PAC1 and AC6 determines the exclusive requirement for this AC in PACAP signaling, but that the coupling of the cAMP sensors PKA, Epac2/Rapgef4, and NCS/Rapgef2, to their respective downstream signaling targets, determines how cAMP signaling is parcellated to physiologic responses, such as neuritogenesis, upon GPCR-Gs activation in neuroendocrine cells.

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.

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.

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.