Antibody response against koala retrovirus (KoRV) in koalas harboring KoRV-A in the presence or absence of KoRV-B.

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.

Receptors and entry cofactors for retroviruses include single and multiple transmembrane-spanning proteins as well as newly described glycophosphatidylinositol-anchored and secreted proteins.

In the past few years, many retrovirus receptors, coreceptors, and cofactors have been identified. These molecules are important for some aspects of viral entry, although in some cases it remains to be determined whether they are required for binding or postbinding stages in entry, such as fusion. There are certain common features to the molecules that many retroviruses use to gain entry into the cell. For example, the receptors for most mammalian oncoretroviruses are multiple membrane-spanning transport proteins. However, avian retroviruses use single-pass membrane proteins, and a sheep retrovirus uses a glycosylphosphatidylinositol-anchored molecule as its receptor. For some retroviruses, particularly the lentiviruses, two cell surface molecules are required for efficient entry. More recently, a soluble protein that is required for viral entry has been identified for a feline oncoretrovirus. In this review, we will focus on the various strategies used by mammalian retroviruses to gain entry into the cell. The choice of receptors will also be discussed in light of pressures that drive viral evolution and persistence.

Suppression of the chemically transformed phenotype of BHK cells by a human cDNA.

Transformation of the baby hamster kidney cell line BHK SN-10 by chemical carcinogens such as nitrosylmethylurea (NMU) is mediated by the loss of a gene product critical for the suppression of malignant transformation. Somatic cell hybrids between chemically transformed BHK SN-10 cells and either normal hamster kidney or human fibroblast cells are nontransformed; therefore, a recessive mechanism underlies the malignant transformation of BHK SN-10 cells after chemical carcinogenesis (A. Stoler and N. P. Bouck, Proc. Natl. Acad. Sci. USA 82:570-574, 1985). A human fibroblast cDNA library was constructed and introduced into NMU-transformed BHK SN-10 cells (NMU 34m) in order to identify a human cDNA capable of suppressing cellular transformation. NMU-transformed BHK cells were analyzed for reversion to an anchorage-dependent normal cellular phenotype after transfection with human cDNA. The human cDNA capable of inducing stable reversion of NMU 34m cells encodes the intermediate filament protein vimentin, which is apparently required for maintenance of the normal phenotype in BHK SN-10 cells.

Quantitative micro P30 and reverse transcriptase assays for Moloney murine leukemia virus.

An anti-P30 immunohistochemical and a reverse transcriptase assay for Moloney murine leukemia virus (MoMLV) are adapted to 96-well plates. The assay results are shown to be directly proportional to the number of infectious particles, and can therefore be used to estimate the infective titers of a virus preparation. The micro P30 assay yields a direct estimate of infectious centers, and the reverse transcriptase assay quantitates progeny from a single cycle of replication. The semi-automated nature of these assays is well suited to the analysis of a large number of samples and therefore permits the examination of the efficiency of the process of retroviral/MoMLV infection under varied times or conditions.

Viral and cellular factors governing hamster cell infection by murine and gibbon ape leukemia viruses.

Hamster cells are resistant to infection by most retroviruses, including Moloney murine leukemia virus (MoMLV) and gibbon ape leukemia viruses (GaLVs). We have constructed MoMLV-GaLV hybrid virions to identify viral and cellular determinants responsible for the inability of GaLV and MoMLV to infect hamster cells. The substitution of MoMLV core components for GaLV core components circumvents the resistance of hamster cells to infection by GaLV, demonstrating that hamster cells have receptors for GaLV but are not efficiently infected by this primate retrovirus because of a postpenetration block. In contrast, hamster cells are apparently resistant to MoMLV infection because although they bear a receptor for MoMLV, the receptor is nonfunctional. Treatment of CHO K1 or BHK 21 hamster cells with the glycosylation inhibitor tunicamycin allows the cells to be infected by MoMLV. The construction of MoMLV-GaLV hybrid virions that can efficiently infect resistant cells has allowed the identification of viral and cellular factors responsible for restricting infection of hamster cells by MoMLV and GaLV.

Mutational analysis of the proposed gibbon ape leukemia virus binding site in Pit1 suggests that other regions are important for infection.

Region A of Pit1 (residues 550 to 558 in domain IV) and related receptors has remained the only sequence implicated in gibbon ape leukemia virus (GALV) infection, and an acidic residue at the first position appeared indispensable. The region has also been proposed to be the GALV binding site, but this lacks empirical support. Whether an acidic residue at the first position in this sequence is a definitive requirement for GALV infection has also remained unclear; certain receptors retain function even in the absence of this acidic residue. We report here that in Pit1 an acidic residue is dispensable not only at position 550 but also at 553 alone and at both positions. Further, the virus requires no specific residue at either position. Mutations generated a collection of region A sequences, often with fundamentally different physicochemical properties (overall hydrophobicity or hydrophilicity and net charge of -1, or 0, or +1), and yet Pit1 remained an efficient GALV receptor. A comparison of these sequences and a few previously published ones from highly efficient GALV receptors revealed that every position in region A can vary without affecting GALV entry. Even Pit2 is nonfunctional for GALV only because it has lysine at the first position in its region A, which is otherwise highly diverse from region A of Pit1. We propose that region A itself is not the GALV binding motif and that other sequences are required for virus entry. Indeed, certain Pit1/Pit2 chimeras revealed that sequences outside domain IV are specifically important for GALV infection.

Transplanted CNS stem cells form functional synapses in vivo.

An understanding of developmental mechanisms and new cell therapies can be achieved by transplantation into the nervous system. Multipotential stem cells have been isolated from the foetal and adult central nervous system (CNS). Immortalized and primary precursor cells integrate into the developing brain generating both neurons and glia as defined by immunological and morphological criteria. Here we show for the first time that in vitro-expanded CNS precursors, upon transplantation into the brains of rats, form electrically active and functionally connected neurons. These neurons exhibit spontaneous and evoked postsynaptic events and respond to focal glutamate application. Donor cells were grafted into the foetal hippocampus, and the amplitude and frequency of spontaneous synaptic events were monitored in the grafted cells in area CA1 for the first month of postnatal life. The formation of synapses onto grafted neurons indicates that grafted CNS stem cells can be used to study synaptic development in vivo and has important implications for clinical cell replacement therapies.

The requirements for viral entry differ from those for virally induced syncytium formation in NIH 3T3/DTras cells exposed to Moloney murine leukemia virus.

Moloney murine leukemia virus (Mo-MuLV) has the unique ability to infect different cells via either a low-pH-dependent or a pH-independent entry pathway. Only the pH-independent mechanism of Mo-MuLV entry has been associated with Mo-MuLV-induced syncytium formation. We have now identified a transformed cell line (NIH 3T3/DTras) which efficiently forms syncytia when exposed to Mo-MuLV, yet is low pH dependent for Mo-MuLV entry. Treatment of NIH 3T3/DTras cells with chloroquine, an agent which raises endosomal pH, blocks Mo-MuLV entry, but not Mo-MuLV-induced syncytium formation. This demonstrates that fusion which accompanies viral entry and fusion which is responsible for syncytium formation occur as independent processes in these cells. In addition, we determined that neither inherent differences in the Mo-MuLV receptor nor reduced affinity for Mo-MuLV gp70 can account for resistance of NIH 3T3 cells to Mo-MuLV-induced syncytium formation.

Properties of a unique form of the murine amphotropic leukemia virus receptor expressed on hamster cells.

Identification and cloning of the receptors for amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV) have both enabled the determination of the normal function of these virus receptors in cells and initiated experimental examination of how these receptors interact with their respective viruses. GaLV and A-MuLV have distinct host ranges and use different receptors to infect human cells. It was therefore surprising to find that the human GaLV and A-MuLV receptors were not only structurally similar but performed similar cellular functions (B. O'Hara, S. V. Johann, H. P. Klinger, D. G. Blair, H. Rubinson, K. J. Dunn, P. Sass, S. M. Vitek, and T. Robbins, Cell Growth Differ. 1:119-127, 1990; M. van Zeijl, S. V. Johann, E. Closs, J. Cunningham, R. Eddy, T. B. Shows, and B. O'Hara, Proc. Natl. Acad. Sci. USA 91:1168-1172, 1994; M. P. Kavanaugh, D. G. Miller, W. Zhang, W. Law, S. L. Kozak, D. Kabat, and A. D. Miller, Proc. Natl. Acad. Sci. USA 91:7071-7075, 1994; and Z. Olah, C. Lehel, W. B. Anderson, M. V. Eiden, and C. A. Wilson, J. Biol. Chem., in press). We have now determined that the murine retrovirus 10A1 can use both the human GaLV receptor and the human A-MuLV receptor to infect cells. Furthermore, we have cloned and functionally characterized a unique form of the amphotropic receptor homolog expressed in E36 hamster cells. This receptor (EAR) can serve as both a GaLV receptor and an A-MuLV receptor, and it therefore differs from the receptors expressed in human cells, which function exclusively as either GaLV or A-MuLV receptors.

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.

Substitution of a single amino acid residue is sufficient to allow the human amphotropic murine leukemia virus receptor to also function as a gibbon ape leukemia virus receptor.

We have previously reported the unique properties of a receptor for amphotropic murine leukemia viruses (A-MuLVs) expressed on Chinese hamster E36 cells (C.A. Wilson, K.B. Farrell, and M.V. Eiden, J. Virol. 68:7697-7703, 1994). This receptor, HaPiT2 (formerly designated EAR), in contrast to the human form of the A-MuLV receptor (PiT2), functions as a receptor not only for A-MuLVs but also for gibbon ape leukemia virus (GALV). Comparison of the deduced amino acid sequences of the HaPiT2 and PiT2 proteins suggested that differences in the amino acid composition of the extracellular region(s) of the hamster and human proteins account for their functional differences. We substituted extracellular regions of HaPiT2 for those of PiT2 to map the region of the HaPiT2 protein required for GALV receptor function. Only those PiT2-HaPiT2 chimeric receptors containing the fourth and fifth extracellular regions of HaPiT2 functioned as GALV receptors. We have now determined that the substitution of a single amino acid residue, glutamic acid, for the lysine residue at position 522 in the fourth extracellular region of the PiT2 protein is sufficient to render PiT2 functional as a GALV receptor.

Glycosylation-dependent inactivation of the ecotropic murine leukemia virus receptor.

The ecotropic murine leukemia virus (E-MuLV) receptor expressed on Mus dunni tail fibroblast (MDTF) cells is a receptor for all E-MuLVs with the notable of Moloney murine leukemia virus (Mo-MuLV). Substitution of isoleucine for valine at position 214 in the third extracellular region (the putative E-MuLV binding site) of the MDTF receptor molecule allows this molecule to function as a Mo-MuLV receptor (M.V. Eiden, K. Farrell, J. Warsowe, L. A. Mahan, and C. A. Wilson, J. Virol. 67:4056-4061, 1993). We have now determined that treating MDTF cells with tunicamycin, an inhibitor of N-linked glycosylation, also renders them susceptible to Mo-MuLV infection. Two potential N-linked glycosylation sites are present in the third extracellular regions of both the NIH 3T3 and MDTF ecotropic receptors. The glycosylation site at position 229 of the MDTF receptor cDNA was eliminated by substituting a threonine codon for the asparagine codon. Mo-MuLV-resistant human HOS cells, expressing this form of the receptor, are susceptible to Mo-MuLV infection. Thus, our studies suggest that without a glycan moiety at position 229, the valine residue at 214 is no longer restrictive for Mo-MuLV infection. BHK-21 and CHO K1 hamster cells also express glycosylation-inactivated forms of the ecotropic receptor. Sequence analysis of these receptors together with our analysis of MDTF receptor function suggests that a single asparagine-linked glycosylation site is responsible for glycosylation inactivation of these receptors.

Gibbon ape leukemia virus and the amphotropic murine leukemia virus 4070A exhibit an unusual interference pattern on E36 Chinese hamster cells.

The gibbon ape leukemia virus (GaLV), the amphotropic mouse leukemia virus (A-MLV) 4070A, and the xenotropic mouse leukemia virus (X-MLV) exhibit wide but not identical species host ranges. However, most Chinese hamster cells resist infection by all three viruses. We have now determined that the Chinese hamster cell line E36 differs from other Chinese hamster cell lines in that it is susceptible to infection by wild-type GaLV, A-MLV, and X-MLV. Surprisingly, analysis of the interference pattern of GaLV and A-MLV in E36 cells indicated that GaLV and A-MLV interfere in a nonreciprocal fashion. E36 cells productively infected with GaLV were resistant to superinfection by both GaLV and amphotropically packaged recombinant retroviral vectors. In contrast, E36 cells infected with A-MLV were resistant to superinfection with an amphotropic vector but could still be infected by a GaLV vector. These results imply the existence of a receptor on E36 cells that interacts with both GaLV and A-MLV.

Characterization of a naturally occurring ecotropic receptor that does not facilitate entry of all ecotropic murine retroviruses.

A fibroblast cell line (MDTF) derived from the feral mouse Mus dunni is resistant to infection by Moloney murine leukemia virus (Mo-MuLV), an ecotropic murine leukemia virus (E-MuLV) (M. R. Lander and S. K. Chattopadadhyay, J. Virol. 52:695-698, 1984). MDTF cells can be infected by other E-MuLVs such as Friend MuLV and Rauscher MuLV, which have been demonstrated to use the same receptor as Mo-MuLV in NIH 3T3 cells (A. Rein and A. Schultz, Virology 136:144-152, 1984). We have now shown that the block to Mo-MuLV infection of MDTF cells occurs at the level of the envelope-receptor interaction. We have cloned the ecotropic receptor cDNA from MDTF cells (dRec) and compared its sequence with that of the NIH 3T3 cell receptor (mRec). Although the deduced dRec and mRec proteins differ at only four amino acid residues, we demonstrate that these changes account for the resistance of MDTF cells to Mo-MuLV infection. Our findings suggest that retroviruses in the same receptor class can exhibit different host ranges due to single amino acid differences in their cellular receptor.

The cellular receptor for gibbon ape leukemia virus is a novel high affinity sodium-dependent phosphate transporter.

The primate type C retrovirus gibbon ape leukemia virus (GaLV) has been shown to use a widely expressed, multiple membrane-spanning protein of unknown function as its cell surface receptor on human cells (GLVR1) (Johann, S. V., Gibbons, J. J., and O'Hara, B. (1992) J. Virol. 66, 1635-1640; O'Hara, B., Johann, S. V., Klinger, H. P., Blair, D. G., Rubinson, H., Dunni, K.J., Sass, P., Vitek, S. M., and Robins, T. (1990) Cell Growth Diff. 1, 119-127). Here we present evidence that the receptor for GaLV (GLVR1) functions as a sodium-dependent transporter of inorganic phosphate. GLVR1 is shown to have approximately 3-4-fold higher affinity for phosphate than other mammalian phosphate transporters described to date. Productive infection of GLVR1-expressing cells by GaLV, but not other retroviruses, results in the complete blockade of GLVR1-specific uptake of inorganic phosphate. Since productive infection of cells with GaLV is generally not cytotoxic, it is likely that more than one phosphate transporter exists on the cell surface. Our data suggest that GLVR1 represents a sodium-dependent phosphate transporter that differs from other mammalian phosphate transporters in structure, affinity for phosphate, and function.

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.

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 epsilon epitope- and green fluorescent protein (GFP)-tagged human Pit-2 proteins in mammalian cells. Overexpression of epsilon-epitope-tagged Pit-2 transporters in NIH 3T3 cells resulted in a marked increase in sodium-dependent P(i) uptake. This increase in P(i) uptake 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 P(i) transporter function. The overexpressed epitope-tagged transporters were detected in cell lysates, by Western blot analysis using both epsilon-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.

Formation of infectious hybrid virions with gibbon ape leukemia virus and human T-cell leukemia virus retroviral envelope glycoproteins and the gag and pol proteins of Moloney murine leukemia virus.

The gibbon ape leukemia virus, SEATO strain, and human T-cell leukemia virus type I envelope glycoproteins can be functionally assembled with a Moloney murine leukemia virus core into infectious particles. The envelope-host cell receptor interaction is the major determinant of the host cell specificity for these hybrid virions.

Entry of amphotropic murine leukemia virus is influenced by residues in the putative second extracellular domain of its receptor, Pit2.

Human cells express distinct but related receptors for the gibbon ape leukemia virus (GALV) and the amphotropic murine leukemia virus (A-MuLV), termed Pit1 and Pit2, respectively. Pit1 is not able to function as a receptor for A-MuLV infection, while Pit2 does not confer susceptibility to GALV. Previous studies of chimeric receptors constructed by interchanging regions of Pit1 and Pit2 failed to clarify the determinants unique to Pit2 which correlate with A-MuLV receptor function. In order to identify which regions of Pit2 are involved in A-MuLV receptor function, we exchanged the putative second and third extracellular domains of Pit1, either individually or together, with the corresponding regions of Pit2. Our functional characterization of these receptors indicates a role for the putative second extracellular domain (domain II) in A-MuLV infection. We further investigated the influence of domain II with respect to A-MuLV receptor function by performing site-specific mutagenesis within this region of Pit2. Many of the mutations had little or no effect on receptor function. However, the substitution of serine for methionine at position 138 (S138M) in a Pit1 chimera containing domain II of Pit2 resulted in a 1,000-fold reduction in A-MuLV receptor function. Additional mutations made within domain II of the nonfunctional S138M mutant restored receptor function to nearly wild-type efficiency. The high degree of tolerance for mutations as well as the compensatory effect of particular substitutions observed within domain II suggests that an element of secondary structure within this region plays a critical role in the interaction of the receptor with A-MuLV.

Evaluation of retroviral vectors based on the gibbon ape leukemia virus.

The gibbon ape leukemia viruses (GaLVs) are primate-derived C-type retroviruses with a broad host range. Using an infectious, full-length clone of the GaLV SEATO strain, we have determined that this virus replicates efficiently in 13 of 17 human cell lines tested. In fact, the SB lymphoblast cell line, while resistant to infection by wild-type amphotropic mouse leukemia virus (A-MLV), was infected by GaLV-SEATO. We constructed vectors containing GaLV components and compared the performance of genomes containing an enhancer and promoter derived either from the SEATO or SF strains of GaLV. The GaLV vector genomes were packaged in a Moloney (Mo)MLV core with either an A-MLV or GaLV SEATO envelope. We found that, in some cases, the vector genome appeared to be critical in obtaining optimal infection. For example, vectors with a GaLV SF-based genome infected the human HL60 cell line, whereas vectors with a GaLV SEATO-based genome did not. We also found that most, but not all, of the human cell lines tested were more susceptible to vectors packaged with the GaLV SEATO than A-MLV envelope. The source of the viral core was also important, in that some human cells appeared susceptible to infection only with GaLV genomes packaged in particles composed of a GaLV core and envelope. Our results show that GaLV-based packageable genomes can be expressed in target cells not efficiently infected by vectors containing MoMLV-based genomes. These results suggest that judicious combinations of retroviral genomes and structural components can significantly improve gene transfer into human cells.