Maribeth V. Eiden

Early cortical precursors do not undergo LIF‐mediated astrocytic differentiation

Multipotential stem cells have been isolated from the developing and adult CNS. Similar identified factors control the differentiation of these cells. A striking example is the instructive action of CNTF/LIF activating the JAK/STAT pathway to induce astrocytic differentiation in both fetal and adult CNS stem cells. Here we show that E12 cortical precursors express functional LIF receptors but do not exhibit this differentiation response to CNTF/LIF either in explant or in dissociated cell culture. The lack of response to LIF‐induced astrocytic differentiation is maintained in cocultures with LIF responsive cells derived from E15 cortex. This suggests cell intrinsic differences between early and late stage precursors in the interpretation of LIF‐mediated signaling; however, the early nestin‐positive precursor population differentiates into both neurons and neural crest derivatives. These data define differences between CNS stem cells from different stages of cortical development. J. Neurosci. Res. 59:301–311, 2000. Published 2000 Wiley‐Liss, Inc.

A cAMP-Dependent, Protein Kinase A-Independent Signaling Pathway Mediating Neuritogenesis through Egr1 in PC12 Cells

The neurotrophic peptide PACAP (pituitary adenylate cyclase-activating polypeptide) elevates cAMP in PC12 cells. Forskolin and dibutyryl cAMP mimic PACAPs neuritogenic and cell morphological effects, suggesting that they are driven by cAMP. Comparison of microarray expression profiles after exposure of PC12 cells to either forskolin, dibutyryl cAMP, or PACAP revealed a small group of cAMP-dependent target genes. Neuritogenesis induced by all three agents is protein kinase A (PKA)-independent [not blocked by N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89)] and extracellular signal-regulated kinase (ERK)-dependent [blocked by 1,4-diamino-2,3-dicyano-1,4-bis(methylthio) butadiene (U0126)], and therefore cAMP-dependent target genes potentially mediating neuritogenesis were selected for further analysis based on the pharmacological profile of their induction by PACAP (i.e., mimicking that of neuritogenesis). Small interfering RNA (siRNA) targeting one of these genes, Egr1, blocked PACAP-induced neuritogenesis, and siRNA targeting another, Vil2, blocked a component of the cell size increase elicited by PACAP. Neither siRNA blocked PACAPs PKA-dependent antiproliferative effects. PACAP signaling to neuritogenesis was also impaired by dominant-negative Rap1 expression but was not affected by inhibition of protein kinase C (PKC), indicating a G-protein-coupled receptor-mediated differentiation pathway distinct from the one activated by receptor tyrosine kinase ligands such as nerve growth factor (NGF), that involves both Rap1 and PKC. We have thus identified a cAMP-dependent, PKA-independent pathway proceeding through ERK that functions to up-regulate the transcription of two genes, Egr1 and Vil2, required for PACAP-dependent neuritogenesis and increased cell size, respectively. Dominant-negative Rap1 expression impairs both PACAP-induced neuritogenesis and Egr1 activation by PACAP, suggesting that cAMP elevation and ERK activation by PACAP are linked through Rap1.

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.

In Vitro Characterization of a Koala Retrovirus

ABSTRACT Recently, a new endogenous koala gammaretrovirus, designated KoRV, was isolated from koalas. The KoRV genome shares 78% nucleotide identity with another gammaretrovirus, gibbon ape leukemia virus (GALV). KoRV is endogenous in koalas, while GALV is exogenous, suggesting that KoRV predates GALV and that gibbons and koalas acquired the virus at different times from a common source. We have determined that subtle adaptive differences between the KoRV and GALV envelope genes account for differences in their receptor utilization properties. KoRV represents a unique example of a gammaretrovirus whose envelope has evolved to allow for its expanded host range and zoonotic potential.

Changes in viral protein function that accompany retroviral endogenization

Endogenous retroviruses (ERVs) are the remnants of ancient retroviral infections of germ cells and have been maintained in whole or part as heritable genomic elements. The last known endogenization events occurred several million years ago, and therefore stepwise analysis of retroviral endogenization has not been possible. A unique opportunity to study this process became available when a full-length ERV isolated from koalas (KoRV) was shown to have integrated into their germ line within the past 100 years. Even though KoRV shares 78% nucleotide identity with the exogenous and highly infectious gibbon ape leukemia virus (GALV), the infectivity of KoRV, like that of other ERVs, is substantially lower than that of GALV. Differences in the protein coding regions of KoRV that distinguish it from GALV were introduced into the GALV genome, and their functional consequences were assessed. We identified a KoRV gagpol L domain mutation as well as five residues present in the KoRV envelope (env) that, when substituted for the corresponding residues of GALV, resulted in vectors exhibiting substantially reduced titers similar to those observed with KoRV vectors. In addition, KoRV env protein lacks an intact CETTG motif that we have identified as invariant among highly infectious gammaretroviruses. Disruption of this motif in GALV results in vectors with reduced syncytia forming capabilities. Functional assessment of specific sequences that contribute to KoRVs attenuation from a highly infectious GALV-like progenitor virus has allowed the identification of specific modifications in the KoRV genome that correlate with its endogenization.

Identification of Envelope Determinants of Feline Leukemia Virus Subgroup B That Permit Infection and Gene Transfer to Cells Expressing Human Pit1 or Pit2

ABSTRACT The retroviral vector systems that are in common use for gene therapy are designed to infect cells expressing either of two widely expressed phosphate transporter proteins, Pit1 or Pit2. Subgroup B feline leukemia viruses (FeLV-Bs) use the gibbon ape leukemia virus receptor, Pit1, as a receptor for entry. Our previous studies showed that some chimeric envelope proteins encoding portions of FeLV-B could also enter cells by using a related receptor protein, Pit2, which serves as the amphotropic murine leukemia virus receptor (S. Boomer, M. Eiden, C. C. Burns, and J. Overbaugh, J. Virol. 71:8116–8123, 1997). Here we show that an arginine at position 73 within variable region A (VRA) of the FeLV-B envelope surface unit (SU) is necessary for viral entry into cells via the human Pit2 receptor. However, C-terminal SU sequences have a dominant effect in determining human Pit2 entry, even though this portion of the protein is outside known receptor binding domains. This suggests that a combination of specific VRA sequences and C-terminal sequences may influence interactions between FeLV-B SU and the human Pit2 receptor. Binding studies suggest that the C-terminal sequences may affect a postbinding step in viral entry via the Pit2 receptor, although in all cases, binding of FeLV-B SU to human Pit2 was weak. In contrast, neither the arginine 73 nor specific C-terminal sequences are required for efficient binding or infection with Pit1. Taken together, these data suggest that different residues in SU may interact with these two receptors. The specific FeLV-Bs described here, which can enter cells using either human Pit receptor, may be useful as envelope pseudotypes for viruses used in gene therapy.

Reassessing the role of region A in Pit1-mediated viral entry.

ABSTRACT The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.

Subcellular Redistribution of Pit-2 P i Transporter/Amphotropic Leukemia Virus (A-MuLV) Receptor in A-MuLV-Infected NIH 3T3 Fibroblasts: Involvement in Superinfection Interference

ABSTRACT Amphotropic murine leukemia virus (A-MuLV) utilizes the Pit-2 sodium-dependent phosphate transporter as a cell surface receptor to infect mammalian cells. Previous studies established that infection of cells with A-MuLV resulted in the specific down-modulation of phosphate uptake mediated by Pit-2 and in resistance to superinfection with A-MuLV. To study the mechanisms underlying these phenomena, we constructed plasmids capable of efficiently expressing ɛ epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of ɛ-epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent Pi uptake. This increase in Piuptake was specifically blocked by A-MuLV infection but not by infection with ecotropic MuLV (E-MuLV) (which utilizes a cationic amino acid transporter, not Pit-2, as a cell surface receptor). These data, together with the finding that the tagged Pit-2 transporters retained their A-MuLV receptor function, indicate that the insertion of epitope tags does not affect either retrovirus receptor or Pitransporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both ɛ-epitope- and GFP-specific antibodies as well as with Pit-2 antiserum. Both the epitope- and GFP-tagged transporters showed almost exclusive plasma membrane localization when expressed in NIH 3T3 cells, as determined by laser scanning confocal microscopy. Importantly, when NIH 3T3 cells expressing these proteins were productively infected with A-MuLV, the tagged transporters and receptors were no longer detected in the plasma membrane but rather were localized to a punctate structure within the cytosolic compartment distinct from Golgi, endoplasmic reticulum, endosomes, lysosomes, and mitochondria. The intracellular Pit-2 pool colocalized with the virus in A-MuLV-infected cells. A similar redistribution of the tagged Pit-2 proteins was not observed following infection with E-MuLV, indicating that the redistribution of Pit-2 is not directly attributable to general effects associated with retroviral infection but rather is a specific consequence of A-MuLV–Pit-2 interactions.

Comparison of cDNAs encoding the gibbon ape leukaemia virus receptor from susceptible and non-susceptible murine cells

The gibbon ape leukaemia virus (GaLV) family of type C retroviruses consists of five closely related viral isolates, GaLV SF, GaLV SEATO, GaLV Br, GaLV H and simian sarcoma-associated virus. The cDNA encoding the human receptor for GaLV SEATO had previously been isolated. We now demonstrate that all of the above GaLVs can use the human form of the GaLV receptor to infect cells. All murine cells analysed to date have been found to be resistant to infection by GaLVs owing to the absence of a functional GaLV receptor. We have now identified a murine cell line which is unique in its susceptibility to GaLV infection. This cell line was established from a Japanese feral mouse, Mus musculus molossinus. We cloned and sequenced the cDNA for the receptor expressed in these cells and compared it to the cDNA for the GaLV receptor expressed in resistant murine cells such as NIH 3T3 (derived from M. m. musculus) and MDTF (derived from M. dunni tail fibroblasts). The crucial region for GaLV infection (the fourth extracellular domain) from the functional M. m. molossinus GaLV receptor is quite divergent from the same region of the M. m. musculus and M. dunni proteins, but similar to that of the functional human GaLV receptor. These results confirm the importance of the amino acids of this region in GaLV receptor function.

Rapgef2 Connects GPCR-Mediated cAMP Signals to ERK Activation in Neuronal and Endocrine Cells

The guanine nucleotide exchange factor Rapgef2 links cAMP and ERK signaling to mediate neuritogenesis. Bridging the Gap Between cAMP and ERK The process by which neuronal cells extend neurite projections (neuritogenesis) is activated by G protein–coupled receptor (GPCR) signaling and depends on the production of the second messenger cyclic adenosine monophosphate (cAMP) and activation of the mitogen-activated protein kinase ERK. Whether the cAMP and ERK signaling pathways operate in parallel or whether ERK activation occurs in response to cAMP generation has been unclear. Emery et al. isolated the Rap guanine nucleotide exchange factor Rapgef2 as a cAMP-binding protein in neuroendocrine cells and showed that it was activated in response to certain GPCR ligands that stimulated cAMP production. Loss of Rapgef2 inhibited cAMP- and ERK-dependent neuritogenesis, suggesting that Rapgef2 connects these two pathways in neuronal and endocrine cells. G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR)–mediated increases in the second messenger cyclic adenosine monophosphate (cAMP) activate the mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase (ERK), and in neuroendocrine cells, this pathway leads to cAMP-dependent neuritogenesis mediated through Rap1 and B-Raf. We found that the Rap guanine nucleotide exchange factor Rapgef2 was enriched from primary bovine neuroendocrine cells by cAMP-agarose affinity chromatography and that it was specifically eluted by cAMP. With loss-of-function experiments in the rat neuronal cell line Neuroscreen-1 (NS-1) and gain-of-function experiments in human embryonic kidney 293T cells, we demonstrated that Rapgef2 connected GPCR-dependent activation of adenylate cyclase and increased cAMP concentration with the activation of ERK in neurons and endocrine cells. Furthermore, knockdown of Rapgef2 blocked cAMP- and ERK-dependent neuritogenesis. Our data are consistent with a pathway involving the cAMP-mediated activation of Rapgef2, which then stimulates Rap1, leading to increases in B-Raf, MEK, and ERK activity.