Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging.

Apolipoprotein B editing enzyme, catalytic polypeptide 3 (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to retrovirus infection. Complex retroviruses like human immunodeficiency virus type 1 (HIV-1) encode the viral infectivity factor (Vif) protein to counteract APOBEC3 proteins. Vif induces degradation of APOBEC3G and other APOBEC3 proteins and thereby prevents their packaging into virions. It is not known if murine leukemia virus (MLV) encodes a Vif-like protein. Here, we show that the MLV P50 protein, produced from an alternatively spliced gag RNA, interacts with the C terminus of mouse APOBEC3 and prevents its packaging without causing its degradation. By infecting APOBEC3 knockout (KO) and wild-type (WT) mice with Friend or Moloney MLV P50-deficient viruses, we found that APOBEC3 restricts the mutant viruses more than WT viruses in vivo Replication of P50-mutant viruses in an APOBEC3-expressing stable cell line was also much slower than that of WT viruses, and overexpressing P50 in this cell line enhanced mutant virus replication. Thus, MLV encodes a protein, P50, that overcomes APOBEC3 restriction by preventing its packaging into virions.IMPORTANCE MLV has existed in mice for at least a million years, in spite of the existence of host restriction factors that block infection. Although MLV is considered a simple retrovirus compared to lentiviruses, it does encode proteins generated from alternatively spliced RNAs. Here, we show that P50, generated from an alternatively spliced RNA encoded in gag, counteracts APOBEC3 by blocking its packaging. MLV also encodes a protein, glycoGag, that increases capsid stability and limits APOBEC3 access to the reverse transcription complex (RTC). Thus, MLV has evolved multiple means of preventing APOBEC3 from blocking infection, explaining its survival as an infectious pathogen in mice.

Sequential activation of the three protomers in the Moloney murine leukemia virus Env.

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3 → (SU-TM)2TM → (SU-TM)TM2 → TM3 This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+ from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.

Mouse Siglec-1 Mediates trans-Infection of Surface-bound Murine Leukemia Virus in a Sialic Acid N-Acyl Side Chain-dependent Manner.

Siglec-1 (sialoadhesin, CD169) is a surface receptor on human cells that mediates trans-enhancement of HIV-1 infection through recognition of sialic acid moieties in virus membrane gangliosides. Here, we demonstrate that mouse Siglec-1, expressed on the surface of primary macrophages in an interferon-α-responsive manner, captures murine leukemia virus (MLV) particles and mediates their transfer to proliferating lymphocytes. The MLV infection of primary B-cells was markedly more efficient than that of primary T-cells. The major structural protein of MLV particles, Gag, frequently co-localized with Siglec-1, and trans-infection, primarily of surface-bound MLV particles, efficiently occurred. To explore the role of sialic acid for MLV trans-infection at a submolecular level, we analyzed the potential of six sialic acid precursor analogs to modulate the sialylated ganglioside-dependent interaction of MLV particles with Siglec-1. Biosynthetically engineered sialic acids were detected in both the glycolipid and glycoprotein fractions of MLV producer cells. MLV released from cells carrying N-acyl-modified sialic acids displayed strikingly different capacities for Siglec-1-mediated capture and trans-infection; N-butanoyl, N-isobutanoyl, N-glycolyl, or N-pentanoyl side chain modifications resulted in up to 92 and 80% reduction of virus particle capture and trans-infection, respectively, whereas N-propanoyl or N-cyclopropylcarbamyl side chains had no effect. In agreement with these functional analyses, molecular modeling indicated reduced binding affinities for non-functional N-acyl modifications. Thus, Siglec-1 is a key receptor for macrophage/lymphocyte trans-infection of surface-bound virions, and the N-acyl side chain of sialic acid is a critical determinant for the Siglec-1/MLV interaction.

Incorporation of mouse APOBEC3 into murine leukemia virus virions decreases the activity and fidelity of reverse transcriptase.

APOBEC3 proteins are restriction factors that induce G→A hypermutation in retroviruses during replication as a result of cytidine deamination of minus-strand DNA transcripts. However, the mechanism of APOBEC inhibition of murine leukemia viruses (MuLVs) does not appear to be G→A hypermutation and is unclear. In this report, the incorporation of mA3 in virions resulted in a loss in virion reverse transcriptase (RT) activity and RT fidelity that correlated with the loss of virion-specific infectivity.

Haploinsufficiency of SAMD9L, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy 7.

Monosomy 7 and interstitial deletion of 7q (-7/7q-) are well-recognized nonrandom chromosomal abnormalities frequently found among patients with myelodysplastic syndromes (MDSs) and myeloid leukemias. We previously identified candidate myeloid tumor suppressor genes (SAMD9, SAMD9-like = SAMD9L, and Miki) in the 7q21.3 subband. We established SAMD9L-deficient mice and found that SAMD9L(+/-) mice as well as SAMD9L(-/-) mice develop myeloid diseases resembling human diseases associated with -7/7q-. SAMD9L-deficient hematopoietic stem cells showed enhanced colony formation potential and in vivo reconstitution ability. SAMD9L localizes in early endosomes. SAMD9L-deficient cells showed delays in homotypic endosome fusion, resulting in persistence of ligand-bound cytokine receptors. These findings suggest that haploinsufficiency of SAMD9L and/or SAMD9 gene(s) contributes to myeloid transformation.

Relationship between B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas.

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase IIIß (topoIIIß) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIIIß, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA-1 (p= 0.003), H2AX (p=0.024) and topoIIIß (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

Second site mutation in the virus envelope expands the host range of a cytopathic variant of Moloney murine leukemia virus.

Spl574 MLV (murine leukemia virus) is a variant of Moloney ecotropic MLV (MoMLV) that is cytopathic in Mus dunni cells and restricted by other mouse cells. Its host range and cytopathicity are due to a mutation, S82F, at a site critical for binding to the CAT-1 receptor. To identify residues that affect affinity for receptor variants, virus with S82F was passed in restrictive cells. The env genes of the adapted viruses contained 18 novel mutations, including one, E114G, present in 6 of 30 sequenced envs. MoMLV-E114G efficiently infected all mouse cells as well as ecotropic MLV resistant Chinese hamster cells. Virus with E114G and S82F induced large multinucleated syncytia in NIH 3T3 and SC-1 cells as well as M. dunni cells. Inoculation of Mo-S82F,E114G into mice produced lymphomas typical of MoMLV. Residues at env position 114 are thus important determinants of host range, and E114G suppresses host range restriction due to S82F, but does not affect S82F-governed cytopathicity.

Retrovirus-induced lymphomagenesis: a correlation between disease pathogenesis and flow cytometric analysis.

Perinatal infection with a temperature-sensitive mutant (ts-1) of Moloney murine leukemia virus (MoMuLV) results in massive splenomegaly and thymomegaly in mice and development of lymphoma in >55 % of infected pups. Previous flow cytometry studies showed a decrease in CD4(+) cells in perinatally infected pups, but cell population changes in infected animals with lymphoma compared with infected animals without lymphoma has not yet been reported. In the current study, BALB/c mice were infected with ts-1 through breast milk transmission and observed until development of clinical signs and symptoms of lymphoma and/or symptomatic ts-1 infection. Flow cytometry studies were performed on blood, spleen and thymus samples and correlated with gross morphology and histological changes, resulting from the development of lymphoma. Infected animals with lymphoma had significant decreases in CD4(+) and CD8(+) cell counts in blood and spleen compared with controls. The spleens of infected animals without lymphoma showed a decrease in CD4(+) and CD8(+) cell counts, but this was not significant compared with controls. In the thymus, CD4(+) and CD8(+) cell counts also decreased, but this was not significant in infected animals with and without lymphoma compared with controls. Markers of myeloid cell dysfunction increased in the thymus of animals with infection with and without lymphoma compared with controls. Thus, immunosuppression and CD4(+)/CD8(+) cell decreases in the spleen and thymus are associated with malignant transformation and development of lymphoma in this animal model.

Persistent retroviral infection with MoMuLV influences neuropathological signature and phenotype of prion disease.

A fundamental step in pathophysiology of prion diseases is the conversion of the host encoded prion protein (PrP(C)) into a misfolded isoform (PrP(Sc)) that accumulates mainly in neuronal but also non-neuronal tissues. Prion diseases are transmissible within and between species. In a subset of prion diseases, peripheral prion uptake and subsequent transport to the central nervous system are key to disease initiation. The involvement of retroviruses in this process has been postulated based on the findings that retroviral infections enhance the spread of prion infectivity and PrP(Sc) from cell to cell in vitro. To study whether retroviral infection influences the phenotype of prion disease or the spread of prion infectivity and PrP(Sc) in vivo, we developed a murine model with persistent Moloney murine leukemia retrovirus (MoMuLV) infection with and without additional prion infection. We investigated the pathophysiology of prion disease in MoMuLV and prion-infected mice, monitoring temporal kinetics of PrP(Sc) spread and prion infectivity, as well as clinical presentation. Unexpectedly, infection of MoMuLV challenged mice with prions did not change incubation time to clinical prion disease. However, clinical presentation of prion disease was altered in mice infected with both pathogens. This was paralleled by remarkably enhanced astrogliosis and pathognomonic astrocyte morphology in the brain of these mice. Therefore, we conclude that persistent viral infection might act as a disease modifier in prion disease.

SUMO-interacting motifs of human TRIM5α are important for antiviral activity.

Human TRIM5α potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5α in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5α shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5α revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5α consensus SUMO conjugation site did not affect the antiviral activity of TRIM5α in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5α antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5α restriction of N-MLV. Our data suggest a novel aspect of TRIM5α-mediated restriction, in which the presence of intact SIMs in TRIM5α, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA.

Monosodium luminol upregulates the expression of Bcl-2 and VEGF in retrovirus-infected mice through downregulation of corresponding miRNAs.

The retrovirus ts1 is a mutant of Moloney murine leukemia virus (MoMuLV) that causes neurodegeneration (ND) in susceptible mice. Our previous studies showed that the antioxidant drug monosodium luminol (GVT) prevented the development of ND in ts1-infected mice. In this study, we analyzed effect of GVT on the expression of B-cell lymphoma-2 protein (Bcl-2) and vascular endothelial growth factor (VEGF) in central nervous system (CNS) tissues of these animals. Our data showed that GVT treatment of ts1-infected mice significantly increased their expression of Bcl-2 and VEGF in brainstem compared with ts1-infected untreated mice. We also studied the expression of specific microRNAs (miRNAs) such as miRNA-15 and -16 (targeting Bcl-2), and miRNA-20 (targeting VEGF). We found that the expression of miRNAs inversely correlated with the upregulation of their target proteins in ts1-infected untreated as well as in GVT-treated-ts1-infected mice. The data showed that GVT treatment prevented ts1-induced ND at least in part by upregulating Bcl-2 and VEGF expression, what likely occurred as a consequence of downregulation of their corresponding miRNAs.

Enhanced replication and pathogenesis of Moloney murine leukemia virus in mice defective in the murine APOBEC3 gene.

Human APOBEC3G (hA3G), a member of the AID/APOBEC family of deaminases, is a restriction factor for human immunodeficiency virus (HIV). In the absence of the viral Vif protein hA3G is packaged into virions and during reverse transcription in a recipient cell it deaminates cytosines, leading to G-->A hypermutation and inactivation of the viral DNA. Unlike humans, who carry seven APOBEC3 genes, mice only carry one, mA3. Thus the role of mA3 in restriction of retroviral infection could be studied in mA3 -/- knockout mice, where the gene is inactivated. M-MuLV-infected mA3 -/- mice showed substantially higher levels of infection at very early times compared to wild-type mice (ca. 2 logs at 0-10 days), particularly in the bone marrow and spleen. Restriction of M-MuLV infection was studied ex vivo in primary bone marrow-derived dendritic cells (BMDCs) that express or lack mA3, using an M-MuLV-based retroviral vector expressing enhanced jellyfish green fluorescent protein (EGFP). The results indicated that mA3 within the virions as well as mA3 in the recipient cell contribute to resistance to infection in BMDCs. Finally, M-MuLV-infected mA3 +/+ mice developed leukemia more slowly compared to animals lacking one or both copies of mA3 although the resulting disease was similar (T-lymphoma). These studies indicate that mA3 restricts replication and pathogenesis of M-MuLV in vivo.

The incorporation of APOBEC3 proteins into murine leukemia viruses.

APOBEC3 proteins represent a group of potent intrinsic inhibitors of retroviral replication. Murine APOBEC3 (mA3) is able to inhibit HIV-1, whereas it is inactive against Moloney murine leukemia virus (MLV). In this work, we present evidence showing that mA3, compared to hA3G, is incorporated inefficiently into MLV, while the abilities of mA3 and hA3G to be packaged into HIV-1 are similar. The nucleocapsid (NC) domain of HIV-1 is capable of facilitating the interaction of mA3 with HIV-1 Gag, and thereby the incorporation of mA3 into HIV-1. Swapping studies of the NC domains in HIV-1 and MLV indicate that MLV NC domain is responsible for viral exclusion of mA3, due to its inability to facilitate the mA3/Gag interaction. The interaction between mA3 and HIV-1 Gag is mediated by the linker region between two zinc coordination motifs in mA3, similar to what has been found for the incorporation of hA3G into both HIV-1 and MLV. These results suggest that the interaction between NC domains and the linker regions might represent a common mechanism for viral incorporation of APOBEC3 proteins, and the inefficient incorporation of endogenous mA3 appears to be a strategy by which MLV escapes the inhibitory effect of mA3.

Down-regulation of Jab1, HIF-1alpha, and VEGF by Moloney murine leukemia virus-ts1 infection: a possible cause of neurodegeneration.

Moloney murine leukemia virus-temperature sensitive (MoMuLV-ts1)-mediated neuronal death is a result of both loss of glial support and release of cytokines and neurotoxins from ts1-infected glial cells. Here the authors propose vascular endothelial growth factor (VEGF) down-regulation as another contributory factor in neuronal degeneration induced by ts1 infection. To determine how ts1 affects VEGF expression in ts1-infected brain, the authors examined the expression of several proteins that are important in regulating the expression of VEGF. The authors found significant decreases in Jun-activating domain-binding protein 1 (Jab1), hypoxia-inducible factor (HIF)-1alpha, and VEGF levels and increases in p53 protein levels in ts1-infected brains compared to noninfected control brains. The authors suggest that a decrease Jab1 expression in ts1 infection leads to accumulation of p53, which binds to HIF-1alpha to accelerate its degradation. A rapid degradation of HIF-1alpha leads to decreased VEGF production and secretion. Considering that endothelial cells are the most conspicuous in virus replication and production in ts1 infection, but are not killed by the infection, the authors examined the expression of these proteins using infected and noninfected mouse cerebrovascular endothelial (CVE) cells. The ts1- infected CVE cells showed decreased Jab1, HIF-1alpha, and VEGF mRNA and protein levels and increased p53 protein levels compared with noninfected cells, consistent with the results found in vivo. These results confirm that ts1 infection results in insufficient secretion of VEGF from endothelial cells and may result in decreased neuroprotection. This study suggested that ts1-mediated neuropathology in mice may result from changes in expression and activity of Jab1, p53, and HIF-1alpha, with a final target on VEGF expression and neuronal degeneration.

APRIL facilitates viral-induced erythroleukemia but is dispensable for T cell immunity and lymphomagenesis.

The TNF family member, a proliferation-inducing ligand (APRIL), has been suggested to act as a costimulatory molecule in T cell responses. However, studies addressing this role in vivo are largely lacking. Here, we evaluated the effects of APRIL on physiological T cell responses in vivo. Although receptors for APRIL are expressed on a subset of T cells, neither TCR transgenic (Tg) T cell responses nor endogenous TCR responses were affected by Tg APRIL expression in vivo. Moreover, APRIL did not significantly enhance the induction of T cell lymphomas upon Moloney murine leukemia virus (MLV) infection. This clearly contrasts current belief and indicates that APRIL does not serve a major role in T cell immunity or lymphomagenesis. However, we did observe a strong increase in erythroleukemia formation after MLV inoculation of APRIL Tg mice. Strikingly, this erythroleukemia-facilitating property of APRIL was confirmed using the erythroleukemogenic Friend-MLV. Erythroleukemia in APRIL Tg mice was characterized by low hematocrits and grossly enlarged spleens with an increased percentage of erythroid precursors. Altogether, these results unveil new proerythroleukemogenic properties of APRIL.

Retroviral gene insertion in breast milk mediated lymphomagenesis.

We have demonstrated breast milk transmitted MoMuLV-ts1 retrovirus infection and subsequent lymphoma development in offspring of uninfected mothers suckled by infected surrogate mothers. Additionally, we have shown that the lymphoma development occurs as a result of viral gene integration into host genome. A total of 146 pups from Balb/C mice were divided into 5 groups; one control and 4 experimental. All offspring suckled from surrogate infected or control mothers, except one group of infected pups left with their biological mothers. Thirteen of 91 infected pups developed lymphoma. Inverse-PCR, DNA cloning, and quantitative real-time PCR (qRT-PCR) were used to study the virus integration sites (VIS) and alterations in gene expression. VIS were randomly distributed throughout the genome. The majority of insertion sites were found in chromosomes 10, 12 and 13. A total of 209 proviral genomic insertion sites were located with 52 intragenic and 157 intergenic sites. We have identified 29 target genes. Four genes including Tacc3, Aurka, Gfi1 and Ahi1 showed the maximum upregulation of mRNA expression. These four genes can be considered as candidate genes based on their association with cancer. Upregulation of these genes may be involved in this type of lymphoma development. This model provides an important opportunity to gain insight into the relationship of viral gene insertion into host genome and development of lymphoma via natural transmission route such as breast milk.

Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line.

The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.

Role of receptor polymorphism and glycosylation in syncytium induction and host range variation of ecotropic mouse gammaretroviruses.

BACKGROUND: We previously identified unusual variants of Moloney and Friend ecotropic mouse gammaretroviruses that have altered host range and are cytopathic in cells of the wild mouse species Mus dunni. Cytopathicity was attributed to different amino acid substitutions at the same critical env residue involved in receptor interaction: S82F in the Moloney variant Spl574, and S84A in the Friend mouse leukemia virus F-S MLV. Because M. dunni cells carry a variant CAT-1 cell surface virus receptor (dCAT-1), we examined the role of this receptor variant in cytopathicity and host range. RESULTS: We expressed dCAT-1 or mCAT-1 of NIH 3T3 origin in cells that are not normally infectible with ecotropic MLVs and evaluated the transfectants for susceptibility to virus infection and to virus-induced syncytium formation. The dCAT-1 transfectants, but not the mCAT-1 transfectants, were susceptible to virus-induced cytopathicity, and this cytopathic response was accompanied by the accumulation of unintegrated viral DNA. The dCAT-1 transfectants, however, did not also reproduce the relative resistance of M. dunni cells to Moloney MLV, and the mCAT-1 transfectants did not show the relative resistance of NIH 3T3 cells to Spl574. Western analysis, use of glycosylation inhibitors and mutagenesis to remove receptor glycosylation sites identified a possible role for cell-specific glycosylation in the modulation of virus entry. CONCLUSION: Virus entry and virus-induced syncytium formation using the CAT-1 receptor are mediated by a small number of critical amino acid residues in receptor and virus Env. Virus entry is modulated by glycosylation of cellular proteins, and this effect is cell and virus-specific.

A mechanism misregulating p27 in tumors discovered in a functional genomic screen.

The cyclin-dependent kinase inhibitor p27(KIP1) is a tumor suppressor gene in mice, and loss of p27 protein is a negative prognostic indicator in human cancers. Unlike other tumor suppressors, the p27 gene is rarely mutated in tumors. Therefore misregulation of p27, rather than loss of the gene, is responsible for tumor-associated decreases in p27 protein levels. We performed a functional genomic screen in p27(+/-) mice to identify genes that regulate p27 during lymphomagenesis. This study demonstrated that decreased p27 expression in tumors resulted from altered transcription of the p27 gene, and the retroviral tagging strategy enabled us to pinpoint relevant transcription factors. inhibitor of DNA binding 3 (Id3) was isolated and validated as a transcriptional repressor of p27. We further demonstrated that p27 was a downstream target of Id3 in src-family kinase Lck-driven thymic lymphomagenesis and that p27 was an essential regulator of Lck-dependent thymic maturation during normal T-cell development. Thus, we have identified and characterized transcriptional repression of p27 by Id3 as a new mechanism decreasing p27 protein in tumors.

Characterization of R peptide of murine leukemia virus envelope glycoproteins in syncytium formation and entry.

The C-terminal R peptide of ecotropic murine leukemia virus (MLV) envelope protein (Env) negatively controls membrane fusion activity. The R peptide cleavage during virion maturation activates its fusogenicity and is required for viral entry. We analyzed fusogenicity and transduction efficiency of mutant Env proteins of ecotropic, amphotropic, polytropic, and xenotropic MLVs. As the result, we found that the hydrophobic amino acid residues around the R peptide cleavage site are important for membrane fusion inhibition by the R peptide. In addition, we found that Env complexes with R peptide-truncated and -containing Env proteins have lower fusogenicity and transduction efficiency than those with the R-peptide-truncated Env alone, suggesting that efficient R peptide cleavage is required for efficient MLV vector transduction. The role of R peptide cleavage in amphotropic, polytropic, and xenotropic MLV infection has not been investigated. We found in this study that the R peptide cleavage is required for amphotropic, xenotropic, and polytropic MLV vector transduction, like with ecotropic MLV. The R-peptide-truncated Env proteins of the xenotropic and polytropic MLVs, however, had much lower fusogenicity than those of the ecotropic and amphotropic MLVs. These results provide valuable information for construction of efficient MLV vectors and for understanding the retroviral entry mechanism.