Ecotropic Virus Research Articles

Naturally Occurring Polymorphisms of the Mouse Gammaretrovirus Receptors CAT-1 and XPR1 Alter Virus Tropism and Pathogenicity

Gammaretroviruses of several different host range subgroups have been isolated from laboratory mice. The ecotropic viruses infect mouse cells and rely on the host CAT-1 receptor. The xenotropic/polytropic viruses, and the related human-derived XMRV, can infect cells of other mammalian species and use the XPR1 receptor for entry. The coevolution of these viruses and their receptors in infected mouse populations provides a good example of how genetic conflicts can drive diversifying selection. Genetic and epigenetic variations in the virus envelope glycoproteins can result in altered host range and pathogenicity, and changes in the virus binding sites of the receptors are responsible for host restrictions that reduce virus entry or block it altogether. These battleground regions are marked by mutational changes that have produced 2 functionally distinct variants of the CAT-1 receptor and 5 variants of the XPR1 receptor in mice, as well as a diverse set of infectious viruses, and several endogenous retroviruses coopted by the host to interfere with entry.

EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2

Bakground:The EVI1(ecotropic virus integration site 1) gene codes for a zinc-finger transcription factor, whose transcriptional activation leads to a particularly aggressive form of acute myeloid leukaemia (AML). Although, EVI1 interactions with key proteins in hematopoiesis have been previously described, the precise role of this transcription factor in promoting leukaemic transformation is not completely understood. Recent works have identified specific microRNA (miRNA) signatures in different AML subgroups. However, there is no analysis of miRNAs profiles associated with EVI1 overexpression in humans.Methods:We performed QT-RT–PCR to assess the expression of 250 miRNAs in cell lines with or without EVI1 overexpression and in patient samples. We used ChIP assays to evaluated the possible binding of EVI1 binding to the putative miRNA promoter. Proliferation of the different cell lines transfected with the anti- or pre-miRs was quantified by MTT.Results:Our data showed that EVI1 expression was significantly correlated with the expression of miR-1-2 and miR-133-a-1 in established cell lines and in patient samples. ChIP assays confirmed that EVI1 binds directly to the promoter of these two miRNAs. However, only miR-1-2 was involved in abnormal proliferation in EVI1 expressing cell lines.Conclusions:Our data showed that EVI1 controls proliferation in AML through modulation of miR-1-2. This study contributes to further understand the transcriptional networks involving transcription factors and miRNAs in AML.

EVI1 overexpression in distinct subtypes of pediatric acute myeloid leukemia

Overexpression of the ecotropic virus integration-1 (EVI1) gene (EVI1+), localized at chromosome 3q26, is associated with adverse outcome in adult acute myeloid leukemia (AML). In pediatric AML, 3q26 abnormalities are rare, and the role of EVI1 is unknown. We studied 228 pediatric AML samples for EVI1+ using gene expression profiling and RQ-PCR. EVI1+ was found in 20/213 (9%) of children with de novo AML, and in 4/8 with secondary AML. It was predominantly found in MLL-rearranged AML (13/47), monosomy 7 (2/3), or FAB M6/7 (6/10), and mutually exclusive with core-binding factor AML, t(15;17), and NPM1 mutations. Fluorescent in situ hybridization (FISH) was performed to detect cryptic 3q26 abnormalities. However, none of the EVI1+ patients harbored structural 3q26 alterations. Although significant differences in 4 years pEFS for EVI1+ and EVI1- pediatric AML were observed (28%+/-11 vs 44%+/-4, P=0.04), multivariate analysis did not identify EVI1+ as an independent prognostic factor. We conclude that EVI1+ can be found in approximately 10% of pediatric AML. Although EVI1+ was not an independent prognostic factor, it was predominantly found in subtypes of pediatric AML that are related with an intermediate to unfavorable prognosis. Further research should explain the role of EVI1+ in disease biology in these cases. Remarkably, no 3q26 abnormalities were identified in EVI1+ pediatric AML.

Cathepsin L is required for ecotropic murine leukemia virus infection in NIH3T3 cells

Recently it has been reported that a cathepsin B inhibitor, CA-074Me, attenuates ecotropic murine leukemia virus (Eco-MLV) infection in NIH3T3 cells, suggesting that cathepsin B is required for the Eco-MLV infection. However, cathepsin B activity was negative or extremely low in NIH3T3 cells. How did CA-074Me attenuate the Eco-MLV infection? The CA-074Me treatment of NIH3T3 cells inhibited cathepsin L activity, and a cathepsin L specific inhibitor, CLIK148, attenuated the Eco-MLV vector infection. These results indicate that the suppression of cathepsin L activity by CA-074Me induces the inhibition of Eco-MLV infection, suggesting that cathepsin L is required for the Eco-MLV infection in NIH3T3 cells. The CA-074Me treatment inhibited the Eco-MLV infection in human cells expressing the exogenous mouse ecotropic receptor and endogenous cathepsins B and L, but the CLIK148 treatment did not, showing that only the cathepsin L suppression by CLIK148 is not enough to prevent the Eco-MLV infection in cells expressing both of cathepsins B and L, and CA-074Me inhibits the Eco-MLV infection by suppressing both of cathepsins B and L. These results suggest that either cathepsin B or L is sufficient for the Eco-MLV infection.

Visualizing fusion of pseudotyped HIV-1 particles in real time by live cell microscopy

BackgroundMost retroviruses enter their host cells by fusing the viral envelope with the plasma membrane. Although the protein machinery promoting fusion has been characterized extensively, the dynamics of the process are largely unknown.ResultsWe generated human immunodeficiency virus-1 (HIV-1) particles pseudotyped with the envelope (Env) protein of ecotropic murine leukemia virus eMLV to study retrovirus entry at the plasma membrane using live-cell microscopy. This Env protein mediates highly efficient pH independent fusion at the cell surface and can be functionally tagged with a fluorescent protein. To detect fusion events, double labeled particles carrying one fluorophor in Env and the other in the matrix (MA) domain of Gag were generated and characterized. Fusion events were defined as loss of Env signal after virus-cell contact. Single particle tracking of >20,000 individual traces in two color channels recorded 28 events of color separation, where particles lost the Env protein, with the MA layer remaining stable at least for a short period. Fourty-five events were detected where both colors were lost simultaneously. Importantly, the first type of event was never observed when particles were pseudotyped with a non-fusogenic Env.ConclusionThese results reveal rapid retroviral fusion at the plasma membrane and permit studies of the immediate post-fusion events.

Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIH 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.

Novel HIV‐1 clade B candidate vaccines designed for HLA‐B*5101+ patients protected mice against chimaeric ecotropic HIV‐1 challenge

Novel candidate HIV-1 vaccines have been constructed, which are tailor-designed for HLA-B*5101(+) patients infected with HIV-1 clade B. These vaccines employ novel immunogen HIVB-B*5101 derived from consensus HIV-1 clade B Gag p17 and p24 regions coupled to two Pol-derived B*5101-restricted epitopes, which are together with a third B*5101 epitope in Gag dominant in HIV-1-infected long-term non-progressing patients. Both plasmid DNA and modified vaccinia virus Ankara (MVA) vectors supported high expression levels of the HIVB-B*5101 immunogen in cultured cells. Heterologous DNA prime-recombinant MVA boost regimen induced efficiently HIV-1-specific CD8(+) T-cell responses in BALB/c mice. These vaccine-elicited T cells were multifunctional, killed efficiently target cells in vivo, and protected mice against challenge with ecotropic HIV-1/NL4-3 and ecotropic HIV-1/NDK chimaeric viruses with HIV-1 clade B or D backbones, respectively, and ecotropic murine leukemia virus gp80 envelope, and therefore did so in the absence of anti-HIV-1 gp120 antibodies. These results support further development of HIVB-B*5101 vaccines in combined heterologous-modality regimens. The use of allele-specific vaccines in humans is discussed in the context of other developments in the HIV-1 field.

Evidence that Ecotropic Murine Leukemia Virus Contamination in TZM-bl Cells Does Not Affect the Outcome of Neutralizing Antibody Assays with Human Immunodeficiency Virus Type 1

The TZM-bl cell line that is commonly used to assess neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was recently reported to be contaminated with an ecotropic murine leukemia virus (MLV) (Y. Takeuchi, M. O. McClure, and M. Pizzato, J. Virol. 82:12585-12588, 2008), raising questions about the validity of results obtained with this cell line. Here we confirm this observation and show that HIV-1 neutralization assays performed with a variety of serologic reagents in a similar cell line that does not harbor MLV yield results that are equivalent to those obtained in TZM-bl cells. We conclude that MLV contamination has no measurable effect on HIV-1 neutralization when TZM-bl cells are used as targets for infection.

A retrovirus uses a cationic amino acid transporter as a cell surface receptor.

New studies have indicated that the cell membrane receptor for the ecotropic murine leukemia virus is the classic membrane amino acid transporter y+, the principal transporter of cationic L-acids in mammalian cells. This finding has been hailed as a landmark in cell physiology which may reveal new mechanisms of viral pathogenesis. These studies represent the first amino acid transporter to be cloned, as well as the first example of a virus subverting a transmembrane protein as a receptor.

Mobilization of Endogenous Retroviruses in Mice after Infection with an Exogenous Retrovirus

Mammalian genomes harbor a large number of retroviral elements acquired as germ line insertions during evolution. Although many of the endogenous retroviruses are defective, several contain one or more intact viral genes that are expressed under certain physiological or pathological conditions. This is true of the endogenous polytropic retroviruses that generate recombinant polytropic murine leukemia viruses (MuLVs). In these recombinants the env gene sequences of exogenous ecotropic MuLVs are replaced with env gene sequences from an endogenous polytropic retrovirus. Although replication-competent endogenous polytropic retroviruses have not been observed, the recombinant polytropic viruses are capable of replicating in numerous species. Recombination occurs during reverse transcription of a virion RNA heterodimer comprised of an RNA transcript from an endogenous polytropic virus and an RNA transcript from an exogenous ecotropic MuLV RNA. It is possible that homodimers corresponding to two full-length endogenous RNA genomes are also packaged. Thus, infection by an exogenous virus may result not only in recombination with endogenous sequences, but also in the mobilization of complete endogenous retrovirus genomes via pseudotyping within exogenous retroviral virions. We report that the infection of mice with an ecotropic virus results in pseudotyping of intact endogenous viruses that have not undergone recombination. The endogenous retroviruses infect and are integrated into target cell genomes and subsequently replicate and spread as pseudotyped viruses. The mobilization of endogenous retroviruses upon infection with an exogenous retrovirus may represent a major interaction of exogenous retroviruses with endogenous retroviruses and may have profound effects on the pathogenicity of retroviral infections.

Effects of the ligand sequence modifications on the retargeted transduction by the retroviral vector having a ligand-chimeric Env protein

There have been various attempts to redirect the cell entry receptor tropism of the murine leukemia virus vectors. We have recently reported the successful retargeting of the ecotropic Moloney murine leukemia virus vector. This vector (S3-D84K) contains a viral envelope (Env) protein into which a full-length (68 aa) stromal cell-derived factor-1alpha (SDF-1alpha) was inserted at Pro-79. The S3-D84K vector transduces a certain human cell line through the CXC chemokine receptor 4 (CXCR4) at a titre of about 10(4) c.f.u. ml(-1). Here, the S3-D84K vector was found to transduce another human cell line through CXCR4 with a titre close to 10(6) c.f.u. ml(-1). The SDF-1alpha ligand of the S3-D84K Env protein was modified in different ways. In one, C-terminal truncations (by 3-51 aa) with or without a Cys-to-Gly change were performed, and in the other, Cys-to-Ala changes of the disulfide-forming cysteines without truncation were made. Seven truncation and three alanine mutant chimeric Env proteins were examined for virion incorporation, and the retroviral vectors displaying the mutant protein were examined for CXCR4 binding and retargeted transduction. Two mutant vectors showed transduction through CXCR4 with titres not higher than those of the S3-D84K vector, while the other mutant vectors minimally transduced cells through CXCR4 either due to a defect in virion incorporation of the chimeric Env protein or an inability to bind to CXCR4. These results suggest that a full-length sequence that may fold into a distinct domain within the chimeric Env protein is preferable as a targeting ligand.

EVI1 Overexpression in Pediatric Acute Myeloid Leukemia Associated with Unfavorable Subtypes.

The EVI1 (ecotropic virus integration-1) gene plays an important role in hematopoiesis especially in megakaryocyte development. The MDS1 gene is located upstream of EVI1, and its function is currently unknown. Normally the MDS1/EVI1 intergenic splice variant is co-expressed with EVI1. In adult acute myeloid leukemia (AML) overexpression of EVI1 (EVI1+) can be found in patients with chromosome 3q26-rearrangements. Often, these patients do not co-express MDS1/EVI1. Recently high EVI1 expression was also discovered in a separate subgroup of patients that did not have 3q26-rearrangements. Occasionally, they did not show overexpression of MDS1/EVI1. In these patients cryptic inversions of chromosome 3 were identified with fluorescence in situ hybridization (FISH). Of interest, EVI1+ was found to be an independent poor prognostic marker in adult AML (Lugthart et al, Blood 2008). In pediatric AML, 3q26-rearrangements are rare and the role of EVI1 is unknown. In this study, we investigated the frequency and clinical relevance of EVI1+ in pediatric AML. EVI1 expression was analyzed in 233 pediatric AML patients, of whom microarray gene expression profiling data were available. EVI1+ was found in 25 pediatric AML patients (11%), and confirmed with real-time quantitative PCR. This included 13/49 (26%) patients with MLL-rearranged AML: 5/22 (23%) cases with t(9;11); and all (n=4) cases with t(6;11). Moreover, EVI1+ was found in 4/7 (57%) cases with AML M7; in 2/3 (66%) cases with AML M6; in both cases with monosomy 7; in 1/43 (2%) cases with normal cytogenetics; in 2 patients with random cytogenetics, and in 1 patient with a cytogenetic failure. EVI1+ was not found in the t(8;21), inv(16) and t(15;17) subgroups. 3/25 EVI1+ patients lacked the MDS/EVI1 transcript, but no cryptic 3q26-rearrangements were detected with FISH. Molecular analysis showed that one patient had a CEBPα mutation; one patient had an FLT3-ITD; and 3 patients showed a mutation in the RAS oncogene. EVI+ was not correlated with sex or white blood cell count. However, the frequency in children younger than 10 years old was twice as high when compared to older children (14% vs 7%, p=0.12). Survival analysis was restricted to the subset of patients who were treated using uniform DCOG and BFM treatment protocols (n=204). In this cohort, EVI1+ patients had a worse 5-years event-free survival (pEFS) compared to patients without EVI1+ (30 vs. 43%, p=0.02). However, multivariate analysis, including cytogenetics (favorable [t(8;21, inv(16), t(15;17)] vs. other), FLT3-ITD, age and WBC, showed that EVI1+ was not an independent prognostic factor for survival. Moreover, within the unfavorable/normal cytogenetic subgroup, there was no difference in outcome between patients with and without EVI1+. We conclude that EVI1+ is found in ~10% of pediatric AML, and highly correlated with specific unfavorable cytogenetic (MLL-rearrangements) and morphologic (FAB M6/7) subtypes. In contrast to adult AML, no 3q26-rearrangements or cryptic inversions were found, and EVI1+ was not an independent prognostic factor. This difference in prognostic relevance may be due to differences in treatment. Alternatively, these results may indicate that EVI1 plays a different role in disease biology between adult and pediatric AML. This is at least suggested by the lack of 3q26 aberrations in pediatric AML.

Identification of Gammaretroviruses Constitutively Released from Cell Lines Used for Human Immunodeficiency Virus Research

Three human cell lines used in human immunodeficiency virus research were found to be contaminated with previously undetected retroviruses. On the bases of partial nucleotide sequence, capsid protein antigenicity, vector mobilization, and receptor usage studies, these contaminants were shown to be replication competent and to belong to the Gammaretrovirus genus. While the TZM-bl cells harbor ecotropic murine leukemia virus (MLV), Jurkat J6 cells were found to release xenotropic MLV and the A3.01/F7 cells to produce gibbon ape leukemia virus. These findings highlight the importance of routine testing of cell lines for retrovirus contamination to prevent potential experimental artifacts and allow correct biohazard assessment.

The significance of the pre-challenge immune status of mice for development of retrovirus-induced immunodeficiency syndrome (MAIDS).

The effects of vaccination with RNA-free viral pseudoparticles, preinfection with non-pathogenic ecotropic virus, and induction of tolerance to viral proteins in newborns on the outcome of murine immunodeficiency syndrome (MAIDS) were studied. The parameters used to follow disease progression were: lymphopenia, circulating B and T8 cells, serum IgG and IgM levels, lymphoproliferation and skin graft rejection. Immunization with RNA-free viral pseudoparticles had no effect on any of these parameters. Preinfection of adults with ecotropic virus and the induction of tolerance in newborns to virus antigens both attenuated the early symptoms of viral infection and delayed the onset of immunodeficiency and lymphoproliferation in some mice, but did not significantly alter the number of deaths due to MAIDS. Failure of immune-based therapy to produce successful protection against MAIDS suggests that immune destruction caused by the persistent virus rather than hyperimmune activity is the main pathogenic factor in this disease.

Recombinant porcine endogenous retroviruses (PERV-A/C): a new risk for xenotransplantation?

PERVs are integrated in the genome of all pigs. Some of them infect human cells and represent therefore a potential risk for xenotransplantation using pig cells or organs. Three replication-competent subtypes have been described, PERV-A, PERV-B and PERV-C. Whereas PERV-A and PERV-B are polytropic viruses and infect, among others, human cells, PERV-C is an ecotropic virus, infecting only pig cells. Recombinant PERV-A/C are able to infect human cells, they are characterised by high-titre replication and their proviruses have been found de novo integrated in the genome of somatic pig cells, but not in the germ line. This review compares recombinant PERVs with other recombinant retroviruses in order to evaluate their potential pathogenicity.

Optimized Lentiviral Transduction of Mouse Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have attracted much attention as potential platforms for transgene delivery and cell-based therapy for human disease. MSCs have the capability to self-renew and retain multipotency after extensive expansion in vitro, making them attractive targets for ex vivo modification and autologous transplantation. Viral vectors, including lentiviral vectors, provide an efficient means for transgene delivery into human MSCs. In contrast, mouse MSCs have proven more difficult to transduce with lentiviral vectors than their human counterparts, and because many studies use mouse models of human disease, an improved method of transduction would facilitate studies using ex vivo-modified mouse MSCs. We have worked toward improving the production of human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors and optimizing transduction conditions for mouse MSCs using lentivirus vectors pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G), the ecotropic murine leukemia virus envelope glycoprotein (MLV-E), and the glycoproteins derived from the Armstrong and WE strains of lymphocytic choriomeningitis virus (LCMV-Arm, LCMV-WE). Mouse MSCs were readily transduced following overnight incubation using a multiplicity of infection of at least 40. Alternatively, mouse MSCs in suspension were readily transduced after a 1-h exposure to lentiviral pseudotypes immediately following trypsin treatment or retrieval from storage in liquid nitrogen. LCMV-WE pseudotypes resulted in efficient transduction of mouse MSCs with less toxicity than VSV-G pseudotypes. In conclusion, our improved production and transduction conditions for lentiviral vectors resulted in efficient transduction of mouse MSCs, and these improvements should facilitate the application of such cells in the context of mouse models of human disease.

High EVI1 levels predict adverse outcome in acute myeloid leukemia: prevalence of EVI1 overexpression and chromosome 3q26 abnormalities underestimated

AbstractInappropriate expression of EVI1 (ecotropic virus integration-1), in particular splice form EVI1-1D, through chromosome 3q26 lesions or other mechanisms has been implicated in the development of high-risk acute myeloid leukemia (AML). To validate the clinical relevance of EVI1-1D, as well as of the other EVI1 splice forms and the related MDS1/EVI1 (ME) gene, real-time quantitative polymerase chain reaction was performed in 534 untreated adults with de novo AML. EVI1-1D was highly expressed in 6% of cases (n = 32), whereas 7.8% were EVI1+ (n = 41) when all splice variants were taken into account. High EVI1 predicted a distinctly worse event-free survival (HR = 1.9; P = .002) and disease-free survival (HR = 2.1, P = .006) following multivariate analysis. Importantly, we distinguished a subset of EVI1+ cases that lacked expression of ME (EVI1+ME−; n = 17) from cases that were ME+ (EVI1+ME+; n = 24). The atypical EVI1+ME− expression pattern exhibited cytogenetically detectable chromosomal 3q26 breakpoints in 8 cases. Fluorescence in situ hybridization revealed 7 more EVI1+ME− cases that carried cryptic 3q26 breakpoints, which were not found in the EVI1+ME+ group. EVI1+ME− expression predicts an extremely poor prognosis distinguishable from the general EVI1+ AML patients (overall survival [OS]: P < .001 and event-free survival [EFS]: P = .002). We argue that EVI1/ME quantitative expression analysis should be implemented in the molecular diagnostic procedures of AML.

Aqueous extracts from peppermint, sage and lemon balm leaves display potent anti-HIV-1 activity by increasing the virion density

BackgroundAqueous extracts from leaves of well known species of the Lamiaceae family were examined for their potency to inhibit infection by human immunodeficiency virus type 1 (HIV-1).ResultsExtracts from lemon balm (Melissa officinalis L.), peppermint (Mentha × piperita L.), and sage (Salvia officinalis L.) exhibited a high and concentration-dependent activity against the infection of HIV-1 in T-cell lines, primary macrophages, and in ex vivo tonsil histocultures with 50% inhibitory concentrations as low as 0.004%. The aqueous Lamiaceae extracts did not or only at very high concentrations interfere with cell viability. Mechanistically, extract exposure of free virions potently and rapidly inhibited infection, while exposure of surface-bound virions or target cells alone had virtually no antiviral effect. In line with this observation, a virion-fusion assay demonstrated that HIV-1 entry was drastically impaired following treatment of particles with Lamiaceae extracts, and the magnitude of this effect at the early stage of infection correlated with the inhibitory potency on HIV-1 replication. Extracts were active against virions carrying diverse envelopes (X4 and R5 HIV-1, vesicular stomatitis virus, ecotropic murine leukemia virus), but not against a non-enveloped adenovirus. Following exposure to Lamiaceae extracts, the stability of virions as well as virion-associated levels of envelope glycoprotein and processed Gag protein were unaffected, while, surprisingly, sucrose-density equilibrium gradient analyses disclosed a marked increase of virion density.ConclusionAqueous extracts from Lamiaceae can drastically and rapidly reduce the infectivity of HIV-1 virions at non-cytotoxic concentrations. An extract-induced enhancement of the virion's density prior to its surface engagement appears to be the most likely mode of action. By harbouring also a strong activity against herpes simplex virus type 2, these extracts may provide a basis for the development of novel virucidal topical microbicides.

Pathogenic risk of endogenous retrovirus infection in immunodeficient hosts

To investigate the pathogenic risk of endogenous retroviruses (ERVs) infection in immunodeficient hosts, the ERV of N-type ecotropic murine leukemia virus (MuLV) isolated from SL mice, a kind of mice containing considerable infectious ERV particles determined with SC–XC test and developing leukemia spontaneously with average of high frequency of 30% and incubation period of 315 days, was inoculated intraperitoneally into newborn CBA nude mice. The distinct marker of splenomegaly for leukemia was observed in 33% of homozygous ( nu/nu) and 17% of heterozygous ( nu/+) of CBA nude mice with average incubation period of 310 days and 432 days post-inoculation, respectively. Furthermore, the ERV induced leukemia in both the SL mice and CBA nude mice was identified to be B lymphatic, transplantable and with rearrangement of the Evi-1 locus. The higher induction of leukemia and rearrangement of the Evi-1 locus in CBA nude mice are considered to be dependent on the lower immune status of the hosts. These findings indicate that the ERV could present the host immune dependent leukemogenesis in immunodeficient hosts through the Evi-1 gene rearrangement and suggest that screening of ERVs may be necessary in clinical transplantation or transfusion.

Mechanisms underlying glycosylation-mediated loss of ecotropic receptor function in murine MDTF cells and implications for receptor evolution

A Mus dunni tail fibroblast (MDTF) cell line is highly resistant to infection by ecotropic Moloney murine leukemia virus (Mo-MLV). The cationic amino acid transporter type 1 (CAT1) paralogues of murine NIH 3T3 and MDTF cells (mCAT1 and dCAT1, respectively) contain two conserved N-linked glycosylation sites in the third extracellular loop (ECL3, the putative Mo-MLV binding site). Glycosylation of dCAT1 inhibits Mo-MLV infection, but that of mCAT1 does not. Compared with mCAT1, dCAT1 possesses an Ile-to-Val substitution at position 214 and a Gly insertion at position 236 in the ECL3. To determine the residues responsible for the loss of dCAT1 receptor function, mutants of mCAT1 were constructed. The mCAT1/insG receptor (with a Gly residue inserted at mCAT1 position 236) had greatly reduced Mo-MLV receptor function compared with mCAT1. Treatment of mCAT1/insG-expressing cells with tunicamycin, an N-linked glycosylation inhibitor, increased the transduction titre. In addition, the reduced susceptibility to Mo-MLV observed with mCAT1/insG-expressing cells correlated with impaired binding of Mo-MLV. These results show that a single amino acid insertion confers mCAT1 receptor properties on dCAT1 and provide an important insight into the co-evolution of virus-host interactions.