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1
Seliger, B., R. Kollek, C. Stocking, T. Franz, and W. Ostertag. "Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene." Molecular and Cellular Biology 6, no. 1 (January 1986): 286–93. http://dx.doi.org/10.1128/mcb.6.1.286.
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A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.
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Onodera, Masafumi, David M. Nelson, Akihiro Yachie, G. Jayashree Jagadeesh, Bruce A. Bunnell, Richard A. Morgan, and R. Michael Blaese. "Development of Improved Adenosine Deaminase Retroviral Vectors." Journal of Virology 72, no. 3 (March 1, 1998): 1769–74. http://dx.doi.org/10.1128/jvi.72.3.1769-1774.1998.
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ABSTRACT A series of adenosine deaminase (ADA) retroviral vectors were designed and constructed with the goal of improved performance over the PA317/LASN vector currently used in clinical trials. First, the bacterial selectable-marker neomycin phosphotransferase (neo) gene was removed to create a “simplified” vector. Second, the Moloney murine leukemia virus long terminal repeat (LTR) promoter used for ADA expression was replaced with either the myeloproliferative sarcoma virus (MPSV) or SL3-3 LTR. Supernatant from each ADA vector was used to transduce ADA-deficient (ADA−) B- and T-cell lines as well as primary peripheral blood mononuclear cells (PBMC) from an ADA− severe combined immunodeficiency patient. Total ADA enzyme activity and ADA activity per integrant in the transduced cells demonstrated that the MPSV LTR splicing vector design provided the highest level of ADA expression per cell. This ADA(MPSV) vector was then tested in packaging cell lines containing either the gibbon ape leukemia virus envelope (PG13 cells), the murine amphotropic envelope (FLYA13 cells), or the feline endogenous virus RD114 envelope (FLYRD18 cells). The results indicate that FLYRD18/ADA(MPSV), a simplified ADA retroviral vector with the MPSV LTR, provides a 17-fold-higher level of ADA expression in human lymphohematopoietic cells than the PA317/LASN vector currently in use.
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Seliger, B., R. Kollek, C. Stocking, T. Franz, and W. Ostertag. "Viral transfer, transcription, and rescue of a selectable myeloproliferative sarcoma virus in embryonal cell lines: expression of the mos oncogene." Molecular and Cellular Biology 6, no. 1 (January 1986): 286–93. http://dx.doi.org/10.1128/mcb.6.1.286-293.1986.
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A derivative of the myeloproliferative sarcoma virus (Neor-MPSV) carrying the mos oncogene and dominant selection marker for neomycin resistance (Neor) was introduced into embryonal carcinoma and embryo-derived cell lines by transfection and infection using pseudotypes with Friend helper virus (Friend murine leukemia virus [F-MuLV]). Cells resistant to G418 (a neomycin analog) were cloned and expanded. Transductants retained an undifferentiated phenotype as judged by morphology, tumorigenicity, and cell-surface antigen analyses. Nucleic acid analysis of infectants revealed both Neor-MPSV and F-MuLV proviruses, although no virus was released. G418-resistant transductants remained nonpermissive for the expression of other proviruses and for subsequent superinfection. Northern analysis showed expression of full-length Neor-MPSV, as well as mos-specific subgenomic RNA. mos sequences were deleted from Neor-MPSV (Neor mos-1), and pseudotypes were used to infect embryonal carcinoma cells. No morphological differences were observed in either mos+ or mos- transductants as compared with parental cell lines. However, mos+ transductants showed an enhanced anchorage-independent growth compared with that of mos- transductants in agar cloning. PCC4 transductants were induced to differentiate with retinoic acid and superinfected with F-MuLV. Infection with viral supernatant in fibroblasts and in mice confirmed the rescue of biologically active Neor-MPSV.
4
Le Bousse-Kerdiles, MC, M. Souyri, F. Smadja-Joffe, V. Praloran, C. Jasmin, and HJ Ziltener. "Enhanced hematopoietic growth factor production in an experimental myeloproliferative syndrome." Blood 79, no. 12 (June 15, 1992): 3179–87. http://dx.doi.org/10.1182/blood.v79.12.3179.3179.
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Abstract The murine myeloproliferative syndrome induced by the myeloproliferative sarcoma virus (MPSV) has numerous similarities to human primary myelofibrosis. We have shown that medium conditioned by spleen cells of MPSV-infected mice has the capacity to support the growth of primitive blast cell colonies. The detection of this activity associated with MPSV infection stimulated us to characterize the hematopoietins responsible for this activity. Northern blot analysis showed a large increase, or induction, of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage- CSF (CSF-1), and granulocyte-CSF (G-CSF) transcripts in the hematopoietic organs of MPSV-infected mice; however, no IL-3 transcript could be detected in either MPSV-infected or normal mice. Significant levels of IL-1 alpha, IL-6, G-CSF, and CSF-1 bioactivities were found in the serum of MPSV-infected mice, but not in controls. Additionally, analysis of medium conditioned by spleen cells of MPSV-infected mice showed the presence of tumor necrosis factor alpha bioactivity. The increased production of cytokines that are able to stimulate pluripotent hematopoietic stem cells corroborates the hypothesis of a possible involvement of hematopoietic growth factors in the development of some myeloproliferative disorders.
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Le Bousse-Kerdiles, MC, M. Souyri, F. Smadja-Joffe, V. Praloran, C. Jasmin, and HJ Ziltener. "Enhanced hematopoietic growth factor production in an experimental myeloproliferative syndrome." Blood 79, no. 12 (June 15, 1992): 3179–87. http://dx.doi.org/10.1182/blood.v79.12.3179.bloodjournal79123179.
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The murine myeloproliferative syndrome induced by the myeloproliferative sarcoma virus (MPSV) has numerous similarities to human primary myelofibrosis. We have shown that medium conditioned by spleen cells of MPSV-infected mice has the capacity to support the growth of primitive blast cell colonies. The detection of this activity associated with MPSV infection stimulated us to characterize the hematopoietins responsible for this activity. Northern blot analysis showed a large increase, or induction, of interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage- CSF (CSF-1), and granulocyte-CSF (G-CSF) transcripts in the hematopoietic organs of MPSV-infected mice; however, no IL-3 transcript could be detected in either MPSV-infected or normal mice. Significant levels of IL-1 alpha, IL-6, G-CSF, and CSF-1 bioactivities were found in the serum of MPSV-infected mice, but not in controls. Additionally, analysis of medium conditioned by spleen cells of MPSV-infected mice showed the presence of tumor necrosis factor alpha bioactivity. The increased production of cytokines that are able to stimulate pluripotent hematopoietic stem cells corroborates the hypothesis of a possible involvement of hematopoietic growth factors in the development of some myeloproliferative disorders.
6
Laker, C., G. Gräning, A. Kelso, C. Stocking, and W. Ostertag. "Abrogation of the requirement for feeder cell interaction and T cell receptor stimulation of lymphocytes infected with retroviral vectors." Journal of Experimental Medicine 172, no. 2 (August 1, 1990): 447–56. http://dx.doi.org/10.1084/jem.172.2.447.
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Infection of sensitive adult mice with myeloproliferative sarcoma virus (MPSV) results in a myeloproliferative syndrome. Two components of the viral genome are required to induce this unique pathology: the mos oncogene and sequences within the U3 region of the long terminal repeat (LTR). In studies designed to identify the target cell of MPSV and thus better understand the mechanism by which a myeloproliferative syndrome is induced, we have infected a series of T cell lines with MPSV-based vectors. The results presented here show that infection with neoR MPSV abrogates the requirement for an antigen-specific or feeder cell-dependent stimulation, without altering the requirement for interleukin 2. Significantly, this response is not dependent on the mos oncogene, but requires sequences within the U3 region of the MPSV LTR. No alteration in the constitutive or induced levels of lymphokines released by these cells was observed. These results suggest a model in which T cells acquire a proliferative advantage by uncoupling the proliferative response from the lymphokine synthesis that is induced by activation of the T cell receptor. These cells are thus poised for antigen stimulation and secretion of cytokines that stimulate myelopoiesis.
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Allay, JA, LL Dumenco, ON Koc, L. Liu, and SL Gerson. "Retroviral transduction and expression of the human alkyltransferase cDNA provides nitrosourea resistance to hematopoietic cells." Blood 85, no. 11 (June 1, 1995): 3342–51. http://dx.doi.org/10.1182/blood.v85.11.3342.bloodjournal85113342.
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Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy. This toxicity predominantly involves modification of the O6 position of guanine with an alkyl moiety. The enzyme responsible for repair of O6-alkylguanine adducts, O6-alkylguanine-DNA alkyltransferase (alkyltransferase), is expressed at low levels in bone marrow (BM) cells. High alkyltransferase expression prevents the cytotoxicity and carcinogenicity of nitrosoureas in several transgenic and in vitro gene transfer models. We used gene transfer using a novel myeloproliferative sarcoma virus (MPSV) based retrovirus (vM5MGMT) to express the human alkyltransferase cDNA (MGMT) in human and murine hematopoietic cells. Transduced K562 cells had very high levels of alkyltransferase expression and significantly increased resistance to 1,3-bis (2-chloroethyl) nitrosourea (BCNU) as compared with untransduced K562 cells. Primary murine BM progenitors showed a high transduction efficiency with vM5MGMT and have increased BCNU resistance in vitro. After BM transplantation with vM5MGMT-transduced BM cells and BCNU treatment of these mice, BM, spleen and thymus had a 10- to 40-fold increase in alkyltransferase expression that persisted for at least 23 weeks posttransplantation. Progenitor cells procured from mice expressing high levels of alkyltransferase also had increased resistance to BCNU. Thus, an MPSV-based retroviral vector transduces mouse and human hematopoietic cells at high efficiency and results in high levels of gene expression both in vitro and in vivo. Overexpression of the alkyltransferase protein may protect hematopoietic progenitors from nitrosourea-induced myelosuppression.
8
Hayashi, Y., T. Tange, Y. Urano, F. Smadja-Joffe, M. C. Le Bousse-Kerdiles, and C. Jasmin. "Histopathologic Studies on Myeloproliferative Sarcoma Virus (MPSV) Induced Leukemias and Hemangiosarcoma in Jar-2 Rats." Pathology - Research and Practice 183, no. 3 (June 1988): 314–20. http://dx.doi.org/10.1016/s0344-0338(88)80128-6.
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Chinnasamy, Dhanalakshmi, Nachimuthu Chinnasamy, Melissa J. Enriquez, Makoto Otsu, Richard A. Morgan, and Fabio Candotti. "Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins." Blood 96, no. 4 (August 15, 2000): 1309–16. http://dx.doi.org/10.1182/blood.v96.4.1309.
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Abstract Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)–based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1–based vectors requires the presence of viral accessory proteins.
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Chinnasamy, Dhanalakshmi, Nachimuthu Chinnasamy, Melissa J. Enriquez, Makoto Otsu, Richard A. Morgan, and Fabio Candotti. "Lentiviral-mediated gene transfer into human lymphocytes: role of HIV-1 accessory proteins." Blood 96, no. 4 (August 15, 2000): 1309–16. http://dx.doi.org/10.1182/blood.v96.4.1309.h8001309_1309_1316.
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Resting lymphocytes are refractory to gene transfer using Moloney murine leukemia virus (MMLV)-based retroviral vectors because of their quiescent status. Recently, it has been shown that lentiviral vectors are capable of transferring genes into nondividing and terminally differentiated cells. We used human immunodeficiency virus type-1 (HIV-1)–based vectors expressing enhanced green fluorescent protein (EGFP) driven by different promoters (CMV, MPSV, or PGK) and investigated their ability to transduce human T- and B-cell lines, as well as resting or activated primary peripheral and umbilical cord blood lymphocytes. The effects of the presence or the absence of HIV-1 accessory proteins (Vif, Vpr, Vpu, and Nef) in the vector system were also assessed. Flow cytometry analysis showed no differences in the ability of these vectors of transferring the reporter gene into lymphocytic lines and mitogen-stimulated primary lymphocytes in the presence or the absence of HIV-1 accessory proteins (APs). Similarly, viral supernatants generated in the presence of accessory genes could efficiently transduce various subsets of resting lymphocytes and provide long-term expression of the transgene. No significant transduction-induced changes in cell activation or cycling status were observed and Alu-HIV-1 long terminal repeat polymerase chain reaction (LTR PCR) analysis demonstrated integration of the vector sequences at the molecular level. In contrast, in the absence of HIV-1 APs, lentiviral vectors failed to integrate and express the transgene in resting lymphocytes. These results show that transduction of primary resting lymphocytes with HIV-1–based vectors requires the presence of viral accessory proteins.
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Pawliuk, R., R. Kay, P. Lansdorp, and RK Humphries. "Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer." Blood 84, no. 9 (November 1, 1994): 2868–77. http://dx.doi.org/10.1182/blood.v84.9.2868.2868.
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Abstract We have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent- activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony- forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. Our results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and should provide a unique tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells.
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Pawliuk, R., R. Kay, P. Lansdorp, and RK Humphries. "Selection of retrovirally transduced hematopoietic cells using CD24 as a marker of gene transfer." Blood 84, no. 9 (November 1, 1994): 2868–77. http://dx.doi.org/10.1182/blood.v84.9.2868.bloodjournal8492868.
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We have investigated the use of a cell surface antigen as a dominant selectable marker to facilitate the detection and selection of retrovirally infected target cells. The small coding region of the human cell surface antigen CD24 (approximately 240 bp) was introduced into a myeloproliferative sarcoma virus (MPSV)-based retroviral vector, which was then used to infect day 4 5-fluorouracil (5-FU)-treated murine bone marrow cells. Within 48 hours of termination of the infection procedure CD24-expressing cells were selected by fluorescent- activated cell sorting (FACS) with an antibody directed against the CD24 antigen. Functional analysis of these cells showed that they included not only in vitro clonogenic progenitors and day 12 colony- forming unit-spleen but also cells capable of competitive long-term hematopoietic repopulation. Double-antibody labeling studies performed on recipients of retrovirally transduced marrow cells showed that some granulocytes, macrophages, erythrocytes, and, to a lesser extent, B and T lymphocytes still expressed the transduced CD24 gene at high levels 4 months later. No gross abnormalities in hematopoiesis were detected in mice repopulated with CD24-expressing cells. Our results show that the use of the CD24 cell surface antigen as a retrovirally encoded marker enables the rapid, efficient, and nontoxic selection in vitro of infected primary cells, facilitates tracking and phenotyping of their progeny, and should provide a unique tool to identify elements that regulate the expression of transduced genes in the most primitive hematopoietic cells.
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Tegerstedt, K., K. Andreasson, A. Vlastos, K. O. Hedlund, T. Dalianis, and T. Ramqvist. "Murine pneumotropic virus VP1 virus-like particles (VLPs) bind to several cell types independent of sialic acid residues and do not serologically cross react with murine polyomavirus VP1 VLPs." Journal of General Virology 84, no. 12 (December 1, 2003): 3443–52. http://dx.doi.org/10.1099/vir.0.19443-0.
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The ability of murine pneumotropic virus (MPtV) major capsid protein VP1 to form virus-like particles (VLPs) was examined. MPtV-VLPs obtained were used to estimate the potential of MPtV to attach to different cells and to assess some characteristics of the MPtV cell receptor. Furthermore, to evaluate if MPtV-VLPs could potentially complement murine polyomavirus (MPyV) VP1 VLPs (MPyV-VLPs) as vectors for prime–boost gene therapy, the capability of MPtV-VLPs to serologically cross react with MPyV-VLPs and to transduce DNA into cells was examined. MPtV VP1 obtained in a recombinant baculovirus system formed MPtV-VLPs readily. MPtV-VLPs were shown by FACS analysis to bind to different cells, independent of MHC class I antigen expression. In addition, MPtV-VLPs did not cause haemagglutination of red blood cells and MPtV-VLP binding to cells was neuraminidase resistant but mostly trypsin and papain sensitive, indicating that the MPtV receptor lacks sialic acid components. When tested by ELISA and in vivo neutralization assays, MPtV-VLPs did not serologically cross react with MPyV-VLPs, suggesting that MPtV-VLPs and MPyV-VLPs could potentially be interchanged as carriers of DNA in repeated gene therapy. Finally, MPtV-VLPs were shown to transduce foreign DNA in vitro and in vivo. In conclusion, the data suggest that MPtV-VLPs, and possibly also MPtV, bind to several different cell types, that binding is neuraminidase resistant and that MPtV-VLPs should potentially be able to complement MPyV-VLPs for prime–boost gene transfer in vivo.
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Gottwein, Eva, Jochen Bodem, Barbara Müller, Ariane Schmechel, Hanswalter Zentgraf, and Hans-Georg Kräusslich. "The Mason-Pfizer Monkey Virus PPPY and PSAP Motifs Both Contribute to Virus Release." Journal of Virology 77, no. 17 (September 1, 2003): 9474–85. http://dx.doi.org/10.1128/jvi.77.17.9474-9485.2003.
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ABSTRACT Late (L) domains are required for the efficient release of several groups of enveloped viruses. Three amino acid motifs have been shown to provide L-domain function, namely, PPXY, PT/SAP, or YPDL. The retrovirus Mason-Pfizer monkey virus (MPMV) carries closely spaced PPPY and PSAP motifs. Mutation of the PPPY motif results in a complete loss of virus release. Here, we show that the PSAP motif acts as an additional L domain and promotes the efficient release of MPMV but requires an intact PPPY motif to perform its function. Examination of HeLaP4 cells expressing PSAP mutant virus by electron microscopy revealed mostly late budding structures and chains of viruses accumulating at the cell surface with little free virus. In the case of the PPPY mutant virus, budding appeared to be mostly arrested at an earlier stage before induction of membrane curvature. The cellular protein TSG101, which interacts with the human immunodeficiency virus type 1 (HIV-1) PTAP L domain, was packaged into MPMV in a PSAP-dependent manner. Since TSG101 is crucial for HIV-1 release, this result suggests that the Gag-TSG101 interaction is responsible for the virus release function of the MPMV PSAP motif. Nedd4, which has been shown to interact with viral PPPY motifs, was also detected in MPMV particles, albeit at much lower levels. Consistent with a role of VPS4A in the budding of both PPPY and PTAP motif-containing viruses, the overexpression of ATPase-defective GFP-VPS4A fusion proteins blocked both wild-type and PSAP mutant virus release.
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Rumlová, Michaela, Ivana Křížová, Romana Hadravová, Michal Doležal, Karolína Strohalmová, Alena Keprová, Iva Pichová, and Tomáš Ruml. "Breast cancer-associated protein – a novel binding partner of Mason-Pfizer monkey virus protease." Journal of General Virology 95, no. 6 (June 1, 2014): 1383–89. http://dx.doi.org/10.1099/vir.0.064345-0.
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We identified breast cancer-associated protein (BCA3) as a novel binding partner of Mason-Pfizer monkey virus (MPMV) protease (PR). The interaction was confirmed by co-immunoprecipitation and immunocolocalization of MPMV PR and BCA3. Full-length but not C-terminally truncated BCA3 was incorporated into MPMV virions. We ruled out the potential role of the G-patch domain, a glycine-rich domain located at the C terminus of MPMV PR, in BCA3 interaction and virion incorporation. Expression of BCA3 did not affect MPMV particle release and proteolytic processing; however, it slightly increased MPMV infectivity.
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Cowled, Christopher. "A new arbovirus in northern Australia." Microbiology Australia 30, no. 4 (2009): 131. http://dx.doi.org/10.1071/ma09131.
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Routine arbovirus surveillance has unearthed a number of novel viruses circulating in domestic and wild animals in northern Australia. One of these is a new virus named Middle Point orbivirus (MPOV). While its disease potential remains unknown, evidence suggests that this virus emerged quite recently in Australia and it has now become the single most commonly isolated animal virus in the Northern Territory. The discovery of MPOV highlights the importance of obtaining prototype data on novel Australian viruses.
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Tsutsumi, Kazuhito, Toshiro Takafuta, Michiko Kida, Tatsuji Mino, Miki Kido, Masayuki Shimojima, Masayuki Saijo, and Takuo Ito. "Effects of Plasma Exchange (PE) Therapy in a Severe Fever with Thrombocytopenia Syndrome (SFTS) Patient with High Virus Quantities." Blood 128, no. 22 (December 2, 2016): 4900. http://dx.doi.org/10.1182/blood.v128.22.4900.4900.
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Abstract Background: Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by the SFTS bunyavirus, which is carried by ticks, and presents various symptoms such as fever, fatigue, digestive symptoms, and hemophagocytic lymphhistiocytosis (HLH). Since the first case was reported from China in 2011, there has been a rapid increase of newly diagnosed patients in Japan. Because the mortality rate has remained high (10-30%), there is an urgent need to establish a management strategy for SFTS. Method: We retrospectively reviewed two newly-diagnosed cases of severe SFTS in our hospital from April 2015 to July 2015. Diagnosis of SFTS was confirmed rapidly using a conventional one-step RT-PCR (cvPCR) method, and the quantity of SFTS virus in each serum was determined with a quantitative one-step RT-PCR (qPCR) method described by Yoshikawa et al. (JCM2014, 52, 3325-33). Virus titer is indicated in units of log10 copies/ml. Results: Case 1 was an 87-year-old female and Case 2 was an 81-year-old male. They showed similar severe clinical symptoms including disturbance of consciousness and a variety of abnormal laboratory findings. Both cases were quickly confirmed as SFTS using cvPCR on the next day after blood sampling. Bone marrow (BM) aspiration was performed at diagnosis, and confirmed HLH in both cases. [Case 1] Although methylprednisolone (mPSL) 250 mg was administrated to control HLH on day 3, the data indicated that HLH had become worse on day 4. To control HLH, mPSL pulse therapy (1000 mg/day) was administrated from day four to day six. However, multiple organ failure proceeded rapidly, and she expired on day eight. Serum virus quantities increased markedly from 6.99 (day 3) to 9.05 (day 6). In this case, the virus level at diagnosis was extremely high compared to previously reported cases and it was possible that the high-dose mPSL therapy suppressed anti-virus immunity. [Case 2] Although mPSL 500 mg was administrated from day two, organ damage progressed on day three. In this case, instead of increasing the dosage of mPSL, plasma exchange (PE) was performed from day four to day six, and organ damage was relieved and the patient recovered in good condition. The serum virus quantities decreased slowly from 6.62 (day 4) to 6.48 (day 7) after PE. Discussion: Yoshikawa et al. reported that virus level at diagnosis correlated to the outcomes of SFTS patients and that in most fatal cases virus quantities at diagnosis were over 5.0. In our cases, the patients had higher virus quantities at diagnosis and presented with severe symptoms. In the first case of fatality, the virus quantities remarkably increased shortly after mPSL therapy. As most SFTS patients are over the age of 50 and the mortality rate is higher in the elderly, lowered immunity in elderly patients might be the cause of SFTS progression. Moreover, immunosuppression therapy, including high dose mPSL therapy for HLH, might enhance the accelerated proliferation of SFTS virus. Although mPSL therapy is effective in some cases, immunosuppressive therapy for a SFTS patient is still controversial. In the second case, after PE therapy the virus level didn't increase and symptoms were improved. PE therapy does not suppress humoral and cellular immunity, which is different from mPSL therapy. As such, PE therapy might be effective in serious cases of SFTS with high virus quantities. In these cases, treatment plans were developed without information on virus quantities, and we obtained qPCR results afterwards. In the future, if qPCR can be performed as quickly as cvPCR, then data on the virus level at diagnosis may be the most important information for planning treatment. Disclosures No relevant conflicts of interest to declare.
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Tsujimura, Akane, Koichi Miyamura, Yachiyo Kuwatsuka, Yoshihiro Inamoto, Taku Oba, Seitaro Terakura, and Yoshihisa Kodera. "Steroid Therapy Increase Only Adenovirus Infection but Not Cytomegalovirus Infection in Dose Dependent Manner after Hematopoietic Stem Cell Transplantation." Blood 108, no. 11 (November 16, 2006): 5292. http://dx.doi.org/10.1182/blood.v108.11.5292.5292.
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Abstract Steroid is the key drug for acute GVHD, however, steroid therapy often increases the incidence of virus infections. But the direct evidence of steroid therapy and various virus infections after transplantation has not been reported yet. Here we evaluated the relationship between the amount of steroid therapy and incidence of virus infections during the first 100 days post transplantation. Two hundred eight patients transplanted between 1997 and 2003 were retrospectively analyzed. Methylpredonisone (mPSL) (1–2mg/kg) was used for the treatment of acute GVHD. Between 1997 and 1999, we used mPSL pulse therapy (500mg–1g/day) for the secondary treatment of GVHD. Most of these patients died of progressive gut GVHD. However, autopsy revealed that cause of death was transplantation related microangiopathy. (Nishida et. al. BMT33, 1143) Thus, since 2001, mPSL pulse therapy has not been used for steroid resistant GVHD. In addition, for the initial treatment of GVHD, we prefer to use 1mg/kg of mPSL for the same reason. Thus, in our institute, amount of steroid was decreased after 2001. Patients were divided into 2 groups according to the initial dose of methylpredonisone (mPSL) used before day 100; Group A (0.5–1.0 mg/kg), Group B (≥2 mg/kg). Interestingly, the incidence of adenovirus infection was well correlated with the percentage of patients in Group B. (Table and Figure) In 1997, 1998 and 1999, percentage of patients in Group B (≥2mg/kg of mPSL) was 32.1, 22.2 and 18.6%, respectively, and incidence of adenovirus infection was 21.4, 14.8 and 17.9%, respectively. Whereas in 2001, 2002 and 2003, percentage of patients in Group B was 12.5%, 3.4% and 11.8%, respectively and incidence of adenovirus infection was 3.1, 3.4 and 5.9%, respectively. On the contrary, incidence of CMV, HSV and HZV infections was not correlated with the steroid therapy, suggesting that each virus has its own threshold of immunosuppression for reactivation. Reactivation of CMV (antigenemia) generally occurred after transplantation independent on the amount of steroid used. However, reactivation of adenovirus may require additional immunosuppression such as middle to high dose of mPSL. In our previous study, steroid therapy was a risk factor of adenovirus infection. (Miyamura et. al. BMT4, 535, BMT25, 545) There is no relationship was observed steroid therapy and HSV and HZV infections. They are commonly observed in a host whose immunological status is normal, suggesting that immuno-suppression is not crucial for these herpes virus infection. Three patients who received ATG developed EBV infection in this population. Reactivation of EBV may need greater immunosuppression than reactivation of adenovirus needs. These differences may reflect the precursor frequencies of CTL against each virus. percentage of patients who received >2mg mPSL and Incidence of virus infection 1997 1998 1999 2000 2001 2002 2003 mPSL ≥2mg/kg 32.1 22.2 18.6 17.2 12.5 3.4 11.8 ADV 21.4 14.8 17.9 6.9 3.1 3.4 5.9 CMV 42.9 55.6 60.7 34.5 56.3 51.7 55.9 VZV 3.6 14.8 7.1 17.2 9.4 17.2 5.9 HSV 25.0 3.7 7.1 0.0 3.1 3.4 5.9 Figure Figure
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Attoui, Houssam, Fauziah Mohd Jaafar, Mourad Belhouchet, Philippe de Micco, Xavier de Lamballerie, and Corina P. D. Brussaard. "Micromonas pusilla reovirus: a new member of the family Reoviridae assigned to a novel proposed genus (Mimoreovirus)." Journal of General Virology 87, no. 5 (May 1, 2006): 1375–83. http://dx.doi.org/10.1099/vir.0.81584-0.
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Micromonas pusilla reovirus (MpRV) is an 11-segmented, double-stranded RNA virus isolated from the marine protist Micromonas pusilla. Sequence analysis (including conserved termini and presence of core motifs of reovirus polymerase), morphology and physicochemical properties confirmed the status of MpRV as a member of the family Reoviridae. Electron microscopy showed that intact virus particles are unusually larger (90–95 nm) than the known size of particles of viruses belonging to the family Reoviridae. Particles that were purified on caesium chloride gradients had a mean size of 75 nm (a size similar to the size of intact particles of members of the family Reoviridae), indicating that they lost outer-coat components. The subcore particles had a mean size of 50 nm and a smooth surface, indicating that MpRV belongs to the non-turreted Reoviridae. The maximum amino acid identity with other reovirus proteins was 21 %, which is compatible with values existing between distinct genera. Based on morphological and sequence findings, this virus should be classified as the representative of a novel genus within the family Reoviridae, designated Mimoreovirus (from Micromonas pusilla reovirus). The topology of the phylogenetic tree built with putative polymerase sequences of the family Reoviridae suggested that the branch of MpRV could be ancestral. Further analysis showed that segment 1 of MpRV was much longer (5792 bp) than any other reovirus segment and encoded a protein of 200 kDa (VP1). This protein exhibited significant similarities to O-glycosylated proteins, including viral envelope proteins, and is likely to represent the additional outer coat of MpRV.
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Hull, Stacey, and Kathleen Boris-Lawrie. "RU5 of Mason-Pfizer Monkey Virus 5′ Long Terminal Repeat Enhances Cytoplasmic Expression of Human Immunodeficiency Virus Type 1 gag-pol and Nonviral Reporter RNA." Journal of Virology 76, no. 20 (October 15, 2002): 10211–18. http://dx.doi.org/10.1128/jvi.76.20.10211-10218.2002.
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ABSTRACT Retroviruses utilize an unspliced version of their primary transcription product as an RNA template for synthesis of viral Gag and Pol structural and enzymatic proteins. Cytoplasmic expression of the gag-pol RNA is achieved despite the lack of intron removal and the presence of a long and highly structured 5′ untranslated region that inhibits efficient ribosome scanning. In this study, we have identified for the first time that the 5′ long terminal repeat (LTR) of Mason-Pfizer monkey virus (MPMV) facilitates Rev/Rev-responsive element-independent expression of HIV-1 gag-pol reporter RNA. The MPMV RU5 region of the LTR is necessary and directs functional interaction with cellular posttranscriptional modulators present in human 293 and monkey COS cells but not in quail QT-6 cells and does not require any viral protein. Deletion of MPMV RU5 decreases the abundance of spliced mRNA but has little effect on cytoplasmic accumulation of unspliced gag-pol RNA despite complete elimination of detectable Gag protein production. MPMV RU5 also exerts a positive effect on the cytoplasmic expression of intronless luc RNA, and ribosomal profile analysis demonstrates that MPMV RU5 directs subcellular localization of the luc transcript to polyribosomes. Our findings have a number of similarities with those of reports on 5′ terminal posttranscriptional control elements in spleen necrosis virus and human foamy virus RNA and support the model that divergent retroviruses share 5′ terminal RNA elements that interact with host proteins to program retroviral RNA for productive cytoplasmic expression.
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Doehle, Brian P., Hal P. Bogerd, Heather L. Wiegand, Nolwenn Jouvenet, Paul D. Bieniasz, Eric Hunter, and Bryan R. Cullen. "The Betaretrovirus Mason-Pfizer Monkey Virus Selectively Excludes Simian APOBEC3G from Virion Particles." Journal of Virology 80, no. 24 (October 11, 2006): 12102–8. http://dx.doi.org/10.1128/jvi.01600-06.
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ABSTRACT The APOBEC3 protein family can constitute a potent barrier to the successful infection of mammalian species by retroviruses. Therefore, any retrovirus that has evolved the ability to replicate in a given animal must have developed mechanisms that allow it to avoid or inhibit the APOBEC3 proteins expressed in that animal. Here, we demonstrate that Mason-Pfizer monkey virus (MPMV) is resistant to inhibition by the APOBEC3G protein expressed in its normal host, the rhesus macaque, but highly susceptible to inhibition by murine APOBEC3 (mA3). MPMV virion particles fail to package rhesus APOBEC3G (rA3G), and MPMV Gag binds rA3G poorly in coexpressing cells. In contrast, MPMV virions package mA3 efficiently and MPMV Gag-mA3 complexes are readily detected. Moreover, mA3, but not rA3G, partially colocalizes with MPMV Gag in the cytoplasm of coexpressing cells. Previously, we have demonstrated that murine leukemia virus also escapes inhibition by APOBEC3 proteins by avoiding virion incorporation of its cognate APOBEC3 protein, mA3, yet is inhibited by primate APOBEC3G proteins, which it packages effectively (B. P. Doehle, A. Schäfer, H. L. Wiegand, H. P. Bogerd, and B. R. Cullen, J. Virol. 79:8201-8207, 2005). The finding that two essentially unrelated beta- and gammaretroviruses use similar mechanisms to escape inhibition by the APOBEC3 proteins found in their normal host species suggests that the selective exclusion of APOBEC3 proteins from virion particles may be a general mechanism used by simple mammalian retroviruses.
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Hagmaier, K., N. Stock, B. Precious, K. Childs, L. F. Wang, S. Goodbourn, and R. E. Randall. "Mapuera virus, a rubulavirus that inhibits interferon signalling in a wide variety of mammalian cells without degrading STATs." Journal of General Virology 88, no. 3 (March 1, 2007): 956–66. http://dx.doi.org/10.1099/vir.0.82579-0.
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Mapuera virus (MPRV) is a paramyxovirus that was originally isolated from bats, but its host range remains unknown. It was classified as a member of the genus Rubulavirus on the basis of structural and genetic features. Like other rubulaviruses it encodes a V protein (MPRV/V) that functions as an interferon (IFN) antagonist. Here we show that MPRV/V differs from the IFN antagonists of other rubulaviruses in that it does not induce the proteasomal degradation of STAT proteins, key factors in the IFN signalling cascade. Rather, MPRV/V prevents the nuclear translocation of STATs in response to IFN stimulation and inhibits the formation of the transcription factor complex ISGF3. We also show that MPRV/V blocks IFN signalling in cells from diverse mammalian species and discuss the IFN response as a barrier to cross-species infections.
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Cowled, Chris, Lorna Melville, Richard Weir, Susan Walsh, Alex Hyatt, Rosey Van Driel, Steven Davis, Aneta Gubala, and David Boyle. "Genetic and epidemiological characterization of Middle Point orbivirus, a novel virus isolated from sentinel cattle in northern Australia." Journal of General Virology 88, no. 12 (December 1, 2007): 3413–22. http://dx.doi.org/10.1099/vir.0.83231-0.
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Middle Point orbivirus (MPOV) was isolated in 1998 from a healthy cow pastured at Beatrice Hill farm, Middle Point (formerly Coastal Plains Research Station), 50 km east of Darwin in Australia's Northern Territory. The isolate could not be identified by using conventional serological tests, and electron microscopy indicated that it belongs to the family Reoviridae, genus Orbivirus. Genetic sequencing of segments 2 and 3 revealed that this virus is related to Yunnan orbivirus, an orbivirus known only from China and not previously associated with a vertebrate host. A real-time RT-PCR test was developed to study the epidemiology of this virus in the field. Over 150 previously unidentified viruses isolated from cattle between 1994 and 2006 were positively identified as isolates of MPOV. Serology was used to demonstrate the development of antibody responses to MPOV in cattle from multiple locations across the Northern Territory.
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Otero, Glen C., Matthew E. Harris, John E. Donello, and Thomas J. Hope. "Leptomycin B Inhibits Equine Infectious Anemia Virus Rev and Feline Immunodeficiency Virus Rev Function but Not the Function of the Hepatitis B Virus Posttranscriptional Regulatory Element." Journal of Virology 72, no. 9 (September 1, 1998): 7593–97. http://dx.doi.org/10.1128/jvi.72.9.7593-7597.1998.
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ABSTRACT Human immunodeficiency virus type 1 Rev export depends upon the presence of the nuclear export signal (NES), a leucine-rich stretch of hydrophobic amino acids. Recently, the nuclear NES-binding receptor has been identified as CRM1 or exportin 1. Rev export has been shown to be CRM1 dependent. The function of the atypical NES-containing Rev-like proteins of equine infectious anemia virus (EIAV) and feline immunodeficiency virus (FIV) is inhibited by leptomycin B, a drug that specifically blocks NES-CRM1 interactions. These data suggest that the function of atypical NES-containing proteins is CRM1 dependent. In contrast to the inhibition of EIAV Rev and FIV Rev, the cytoplasmic accumulation of hepatitis B virus (HBV) posttranscriptional regulatory element (PRE)-containing and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-containing RNAs is not inhibited by leptomycin B treatment. We conclude that the HBV PRE, like the MPMV CTE, functions independently of an NES receptor-exportin 1 interaction.
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Ernst, R. K., M. Bray, D. Rekosh, and M. L. Hammarskjöld. "A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA." Molecular and Cellular Biology 17, no. 1 (January 1997): 135–44. http://dx.doi.org/10.1128/mcb.17.1.135.
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A common feature of gene expression in all retroviruses is that unspliced, intron-containing RNA is exported to the cytoplasm despite the fact that cellular RNAs which contain introns are usually restricted to the nucleus. In complex retroviruses, the export of intron-containing RNA is mediated by specific viral regulatory proteins (e.g., human immunodeficiency virus type 1 [HIV-1] Rev) that bind to elements in the viral RNA. However, simpler retroviruses do not encode such regulatory proteins. Here we show that the genome of the simpler retrovirus Mason-Pfizer monkey virus (MPMV) contains an element that serves as an autonomous nuclear export signal for intron-containing RNA. This element is essential for MPMV replication; however, its function can be complemented by HIV-1 Rev and the Rev-responsive element. The element can also facilitate the export of cellular intron-containing RNA. These results suggest that the MPMV element mimics cellular RNA transport signals and mediates RNA export through interaction with endogenous cellular factors.
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Wendling, F., JF Penciolelli, M. Charon, and P. Tambourin. "Factor-independent erythropoietic progenitor cells in leukemia induced by the myeloproliferative leukemia virus." Blood 73, no. 5 (April 1, 1989): 1161–67. http://dx.doi.org/10.1182/blood.v73.5.1161.1161.
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Abstract The myeloproliferative leukemia virus (MPLV), a novel murine retroviral complex that does not transform fibroblasts, has been shown to cause an acute leukemia in adult mice accompanied by a progressive polycythemia. The present study demonstrates that, on in vivo inoculation, MPLV induces a rapid suppression of growth factor requirement for in vitro colony formation by both the late and the primitive erythroid progenitor cells. CFU-e-derived erythrocytic colonies developed and differentiated in semi-solid medium without the addition of erythropoietin (Epo). In addition, the formation of CFU-e colonies was not altered by the presence of specific neutralizing Epo antibodies. In the spleen, the CFU-e pool size increased rapidly up to 30-fold. By day 6 postinfection, 100% of these progenitor cells were Epo-independent. The in vivo effects of MPLV-infection on early erythroid progenitor cell compartments were examined in cultures grown for seven days. The concentration of erythroid progenitor cells was twofold elevated in spleen from MPLV-infected mice. As early as day 4 postinfection, 50% of these progenitors produced fully hemoglobinized colonies in serum-free cultures without the addition of interleukin-3 (IL-3) and Epo. Most spontaneous colonies were large and contained up to 10(5) cells per colony. They were composed of either erythroblasts only (16%) or erythroblasts and megakaryocytes (70%); few of them were multipotential (14%). In the marrow, the total number of BFU-e was reduced and only few factor-independent bursts were observed, suggesting a rapid migration of infected progenitors from marrow to spleen. Furthermore, the data show that abnormal erythropoiesis was due to the replication defective MPLV information and was not influenced by the Fv-2 locus.
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Wendling, F., JF Penciolelli, M. Charon, and P. Tambourin. "Factor-independent erythropoietic progenitor cells in leukemia induced by the myeloproliferative leukemia virus." Blood 73, no. 5 (April 1, 1989): 1161–67. http://dx.doi.org/10.1182/blood.v73.5.1161.bloodjournal7351161.
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The myeloproliferative leukemia virus (MPLV), a novel murine retroviral complex that does not transform fibroblasts, has been shown to cause an acute leukemia in adult mice accompanied by a progressive polycythemia. The present study demonstrates that, on in vivo inoculation, MPLV induces a rapid suppression of growth factor requirement for in vitro colony formation by both the late and the primitive erythroid progenitor cells. CFU-e-derived erythrocytic colonies developed and differentiated in semi-solid medium without the addition of erythropoietin (Epo). In addition, the formation of CFU-e colonies was not altered by the presence of specific neutralizing Epo antibodies. In the spleen, the CFU-e pool size increased rapidly up to 30-fold. By day 6 postinfection, 100% of these progenitor cells were Epo-independent. The in vivo effects of MPLV-infection on early erythroid progenitor cell compartments were examined in cultures grown for seven days. The concentration of erythroid progenitor cells was twofold elevated in spleen from MPLV-infected mice. As early as day 4 postinfection, 50% of these progenitors produced fully hemoglobinized colonies in serum-free cultures without the addition of interleukin-3 (IL-3) and Epo. Most spontaneous colonies were large and contained up to 10(5) cells per colony. They were composed of either erythroblasts only (16%) or erythroblasts and megakaryocytes (70%); few of them were multipotential (14%). In the marrow, the total number of BFU-e was reduced and only few factor-independent bursts were observed, suggesting a rapid migration of infected progenitors from marrow to spleen. Furthermore, the data show that abnormal erythropoiesis was due to the replication defective MPLV information and was not influenced by the Fv-2 locus.
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Krönig, Holger, Kathrin Hofer, Daniel Sommermeyer, Christian Peschel, Wolfgang Uckert, and Helga Bernhard. "Generating of Human CD4+ and CD8+ T Lymphocytes toward NY-ESO-1 by T Cell Receptor (TCR) Modification and Transduction for Adoptive TCR Gene-Transfer." Blood 112, no. 11 (November 16, 2008): 3533. http://dx.doi.org/10.1182/blood.v112.11.3533.3533.
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Abstract The Cancer Testis (CT) antigen NY-ESO-1 is one of the most immunogenic cancer antigens eliciting strong humoral and cellular immune responses in tumor patients and therefore it is a promising candidate antigen for successful adoptive T cell transfer. The aim of our studies is the transfer of autologous T cells re-directed towards CT antigens by T cell receptor (TCR) gene transfer. The first precondition for genetic transfer of CT-Ag-specific TCRs is the availability of tumor-reactive CD4+ and CD8+ T cell clones that express a CT-Ag-specific TCR. Therefore, we generated the autologous CD8+ T cell clone ThP2 through stimulating HLA-A2.1− PBMCs with autologous HLA-A2+DCs loaded with synthetic NY-ESO-1157–165. After two restimulations, FACS-sorting and cloning, the T cell line specifically recognized the NY-ESO-1157–165 peptide and also specifically lysed NY-ESO-1157–165 expressing tumor cells. In addition, we generated NY-ESO-1 specific T helper1 clones from HLA-DR1+ and HLA-DR4+ healthy donors by stimulation of CD4+ T cells with autologous dendritic cells (DC) pulsed with the NY-ESO-187–111 peptide. The specificity of CD4+ T helper cell clones was determined by proliferation assays and IFN gamma ELISPOT through screening with the NY-ESO-187–111 peptide. By limiting dilution of the NYESO- 1-specific T cell populations we succeeded to isolate CD4+ T cell clones, which recognized NY-ESO-1-pulsed target cells and DCs pulsed with NY-ESO-1 protein. The second precondition for TCR gene transfer is the availability of efficient vector systems. Using vectors based upon mouse myelo-proliferative sarcoma virus (MPSV), it was possible to achieve a high transgene expression in the TCR-transduced T cells. Therefore, we cloned the TCR of the HL-A2-restricted NY-ESO-1-specific CTL clone ThP2 in the retroviral vector and documented the correct expression of the TCR-chains using peptide/HLA-multimers following retroviral transduction of peripheral PBMCs. Moreover, the NY-ESO-1 specific lysis of HLA-A2+ NY-ESO-1+ tumor cell lines after transduction in primary T cells was as well effective as the primary T cell clone. Because the expression of naive transgenic T cell receptors in recipient human T cells is often insufficient to achieve highly reactive T cell bulks we modified the TCR of the ThP2 CTL clone by, murinisation, codon optimalization or by introducing cysteins into the constant regions. Afterwards we compared the expression efficiency of the three different modifications on naive T cells by tetramer-staining. We were able to show that codon optimalization leads to an increase in the expression levels of the transgenic TCRs in human CD8+ T cells. The next step is the development of T cell transfer regiments, which are based on class-II-restricted TCR-transduced T cells.
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Choi, Gyu, Sunyoung Park, Bongkun Choi, Suntaek Hong, Jiyeon Lee, Eric Hunter, and Sung S. Rhee. "Identification of a Cytoplasmic Targeting/Retention Signal in a Retroviral Gag Polyprotein." Journal of Virology 73, no. 7 (July 1, 1999): 5431–37. http://dx.doi.org/10.1128/jvi.73.7.5431-5437.1999.
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ABSTRACT Retroviral capsid assembly can occur by either of two distinct morphogenic processes: in type C viruses, the capsid assembles and buds at the plasma membrane, while in type B and D viruses, the capsid assembles within the cytoplasm and is then transported to the plasma membrane for budding. We have previously reported that a single-amino-acid substitution of a tryptophan for an arginine in the matrix protein (MA) of Mason-Pfizer monkey virus (MPMV) converts its capsid assembly from that of a type D retrovirus to that of the type C viruses (S. S. Rhee and E. Hunter, Cell 63:77–86, 1990). Here we identify a region of 18 amino acids within the MA of MPMV that is responsible for type D-specific morphogenesis. Insertion of these 18 amino acids into the MA of type C Moloney murine leukemia virus causes it to assemble an immature capsid in the cytoplasm. Furthermore, fusion of the MPMV MA to the green fluorescent protein resulted in altered intracellular targeting and a punctate accumulation of the fusion protein in the cytoplasm. These 18 amino acids, which are necessary and sufficient to target retroviral Gag polyproteins to defined sites in the cytoplasm, appear to define a novel mammalian cytoplasmic targeting/retention signal.
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Adu-Gyamfi, Emmanuel, Lori S. Kim, Theodore S. Jardetzky, and Robert A. Lamb. "Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity." Journal of Virology 90, no. 17 (June 22, 2016): 7778–88. http://dx.doi.org/10.1128/jvi.00896-16.
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ABSTRACTParamyxoviridaeconsist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering.IMPORTANCEParamyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.
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Grange, Marie-Pierre, Vincent Blot, Lelia Delamarre, Isabelle Bouchaert, Anna Rocca, Alice Dautry-Varsat, and Marie-Christine Dokhélar. "Identification of Two Intracellular Mechanisms Leading to Reduced Expression of Oncoretrovirus Envelope Glycoproteins at the Cell Surface." Journal of Virology 74, no. 24 (December 15, 2000): 11734–43. http://dx.doi.org/10.1128/jvi.74.24.11734-11743.2000.
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ABSTRACT All retrovirus glycoproteins have a cytoplasmic domain that plays several roles in virus replication. We have determined whether and how the cytoplasmic domains of oncoretrovirus glycoproteins modulate their intracellular trafficking, by using chimeric proteins that combined the α-chain of the interleukin-2 receptor with the glycoprotein cytoplasmic domains of five oncoretroviruses: human T-cell leukemia virus type 1 (HTLV-1), Rous sarcoma virus (RSV), bovine leukemia virus (BLV), murine leukemia virus (MuLV), and Mason-Pfizer monkey virus (MPMV). All of these proteins were synthesized and matured in the same way as a control protein with no retrovirus cytoplasmic domain. However, the amounts of all chimeric proteins at the cell surface were smaller than that of the control protein. The protein appearing at and leaving the cell surface and endocytosis were measured in stable transfectants expressing the chimera. We identified two groups of proteins which followed distinct intracellular pathways. Group 1 included chimeric proteins that reached the cell surface normally but were rapidly endocytosed afterwards. This group included the chimeric proteins with HTLV-1, RSV, and BLV cytoplasmic domains. Group 2 included chimeric proteins that were not detected at the cell surface, despite normal intracellular concentrations, and were accumulated in the Golgi complex. This group included the chimeric proteins with MuLV and MPMV cytoplasmic domains. Finally, we verified that the MuLV envelope glycoproteins behaved in the same way as the corresponding chimeras. These results indicate that retroviruses have evolved two distinct mechanisms to ensure a similar biological feature: low concentrations of their glycoproteins at the cell surface.
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Ward, Alex M., David Rekosh, and Marie-Louise Hammarskjold. "Trafficking through the Rev/RRE Pathway Is Essential for Efficient Inhibition of Human Immunodeficiency Virus Type 1 by an Antisense RNA Derived from the Envelope Gene." Journal of Virology 83, no. 2 (October 29, 2008): 940–52. http://dx.doi.org/10.1128/jvi.01520-08.
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ABSTRACT A human immunodeficiency virus type 1 (HIV-1)-based vector expressing an antisense RNA directed against HIV-1 is currently in clinical trials. This vector has shown a remarkable ability to inhibit HIV-1 replication, in spite of the fact that therapeutic use of unmodified antisense RNAs has generally been disappointing. To further analyze the basis for this, we examined the effects of different plasmid-based HIV-1 long-terminal-repeat-driven constructs expressing antisense RNA to the same target region in HIV-1 but containing different export elements. Two of these vectors were designed to express antisense RNA containing either a Rev response element (RRE) or a Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE). In the third vector, no specific transport element was provided. Efficient inhibition of HIV-1 virus production was obtained with the RRE-driven antisense RNA. This construct also efficiently inhibited p24 production from a pNL4-3 provirus that used the MPMV CTE for RNA export. In contrast, little inhibition was observed with the constructs lacking an RRE. Furthermore, when the RRE-driven antisense RNA was redirected to the Tap/Nxf1 pathway, utilized by the MPMV CTE, through the expression of a RevM10-Tap fusion protein, the efficiency of antisense inhibition was greatly reduced. These results indicate that efficient inhibition requires trafficking of the antisense RNA through the Rev/RRE pathway. Mechanistic studies indicated that the Rev/RRE-mediated inhibition did not involve either nuclear retention or degradation of target mRNA, since target RNA was found to export and associate normally with polyribosomes. However, protein levels were significantly reduced. Taken together, our results suggest a new mechanism for antisense inhibition of HIV mediated by Rev/RRE.
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Browning, Matthew T., Russell D. Schmidt, Kathy A. Lew, and Tahir A. Rizvi. "Primate and Feline Lentivirus Vector RNA Packaging and Propagation by Heterologous Lentivirus Virions." Journal of Virology 75, no. 11 (June 1, 2001): 5129–40. http://dx.doi.org/10.1128/jvi.75.11.5129-5140.2001.
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ABSTRACT Development of safe and effective gene transfer systems is critical to the success of gene therapy protocols for human diseases. Currently, several primate lentivirus-based gene transfer systems, such as those based on human and simian immunodeficiency viruses (HIV/SIV), are being tested; however, their use in humans raises safety concerns, such as the generation of replication-competent viruses through recombination with related endogenous retroviruses or retrovirus-like elements. Due to the greater phylogenetic distance from primate lentiviruses, feline immunodeficiency virus (FIV) is becoming the lentivirus of choice for human gene transfer systems. However, the safety of FIV-based vector systems has not been tested experimentally. Since lentiviruses such as HIV-1 and SIV have been shown to cross-package their RNA genomes, we tested the ability of FIV RNA to get cross-packaged into primate lentivirus particles such as HIV-1 and SIV, as well as a nonlentiviral retrovirus such as Mason-Pfizer monkey virus (MPMV), and vice versa. Our results reveal that FIV RNA can be cross-packaged by primate lentivirus particles such as HIV-1 and SIV and vice versa; however, a nonlentivirus particle such as MPMV is unable to package FIV RNA. Interestingly, FIV particles can package MPMV RNA but cannot propagate the vector RNA further for other steps of the retrovirus life cycle. These findings reveal that diverse retroviruses are functionally more similar than originally thought and suggest that upon coinfection of the same host, cross- or copackaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential.
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Pérez-Losada, Marcos, Ryan G. Christensen, David A. McClellan, Byron J. Adams, Raphael P. Viscidi, James C. Demma, and Keith A. Crandall. "Comparing Phylogenetic Codivergence between Polyomaviruses and Their Hosts." Journal of Virology 80, no. 12 (June 15, 2006): 5663–69. http://dx.doi.org/10.1128/jvi.00056-06.
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ABSTRACT Seventy-two full genomes corresponding to nine mammalian (67 strains) and two avian (5 strains) polyomavirus species were analyzed using maximum likelihood and Bayesian methods of phylogenetic inference. Our fully resolved and well-supported (bootstrap proportions > 90%; posterior probabilities = 1.0) trees separate the bird polyomaviruses (avian polyomavirus and goose hemorrhagic polyomavirus) from the mammalian polyomaviruses, which supports the idea of spitting the genus into two subgenera. Such a split is also consistent with the different viral life strategies of each group. Simian (simian virus 40, simian agent 12 [Sa12], and lymphotropic polyomavirus) and rodent (hamster polyomavirus, mouse polyomavirus, and murine pneumotropic polyomavirus [MPtV]) polyomaviruses did not form monophyletic groups. Using our best hypothesis of polyomavirus evolutionary relationships and established host phylogenies, we performed a cophylogenetic reconciliation analysis of codivergence. Our analyses generated six optimal cophylogenetic scenarios of coevolution, including 12 codivergence events (P< 0.01), suggesting that Polyomaviridae coevolved with their avian and mammal hosts. As individual lineages, our analyses showed evidence of host switching in four terminal branches leading to MPtV, bovine polyomavirus, Sa12, and BK virus, suggesting a combination of vertical and horizontal transfer in the evolutionary history of the polyomaviruses.
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Bogerd, Hal P., Asier Echarri, Ted M. Ross, and Bryan R. Cullen. "Inhibition of Human Immunodeficiency Virus Rev and Human T-Cell Leukemia Virus Rex Function, but Not Mason-Pfizer Monkey Virus Constitutive Transport Element Activity, by a Mutant Human Nucleoporin Targeted to Crm1." Journal of Virology 72, no. 11 (November 1, 1998): 8627–35. http://dx.doi.org/10.1128/jvi.72.11.8627-8635.1998.
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ABSTRACT The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran · GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed ΔCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, ΔCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, ΔCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.
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Fitch, Walter M. "The Variety of Human Virus Evolution." Molecular Phylogenetics and Evolution 5, no. 1 (February 1996): 247–58. http://dx.doi.org/10.1006/mpev.1996.0018.
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Frankel, W. N., J. P. Stoye, B. A. Taylor, and J. M. Coffin. "A linkage map of endogenous murine leukemia proviruses." Genetics 124, no. 2 (February 1, 1990): 221–36. http://dx.doi.org/10.1093/genetics/124.2.221.
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Abstract Thirty endogenous proviruses belonging to the modified polytropic (Mpmv) class of murine leukemia virus (MLV) were identified by proviral-cellular DNA junction fragment segregation in several sets of recombinant inbred mice. Twenty-six Mpmv loci were mapped to chromosomal regions by matching proviral strain distribution patterns to those of previously assigned genes. Like other endogenous nonecotropic MLVs, Mpmv loci were present on several chromosomes in all strains examined. We pooled recombinant inbred strain linkage data from 110 MLV loci and selected marker genes in order to construct a chromosomal linkage map. Every mouse chromosome was found to harbor at least one proviral insertion, and several regions contained multiple integrations. However, the overall distribution of the 110 mapped proviruses did not deviate significantly from a random distribution. Because of their polymorphism in inbred strains of mice, and the ability to score as many as 57 proviruses per strain using only three hybridization probes, the nonecotropic MLVs mapped in common strains of mice offer a significant advantage over older methods (e.g., biochemical or individual restriction fragment polymorphisms) as genetic markers. These endogenous insertion elements should also be useful for assessing strain purity, and for studying the relatedness of common and not-so-common inbred strains.
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Srinivasakumar, Narasimhachar, and Friedrich Schuening. "Novel Tat-Encoding Bicistronic Human Immunodeficiency Virus Type 1-Based Gene Transfer Vectors for High-Level Transgene Expression." Journal of Virology 74, no. 14 (July 15, 2000): 6659–68. http://dx.doi.org/10.1128/jvi.74.14.6659-6668.2000.
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ABSTRACT We describe bicistronic single-exon Tat (72-amino-acid Tat [Tat72])- and full-length Tat (Tat86)-encoding gene transfer vectors based on human immunodeficiency virus type 1 (HIV-1). We created versions of these vectors that were rendered Rev independent by using the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV). Tat72-encoding vectors performed better than Tat86-expressing vectors in gene transfer experiments. CTE-containing vectors, produced in a Rev-independent packaging system, had gene transfer efficiencies nearly equivalent to those produced using a combination RNA transport (CTE and Rev-Rev response element)-based packaging system. The Tat72-encoding vectors could be efficiently transduced into a variety of cell types, showed higher levels of transgene expression than vectors with the simian cytomegalovirus immediate-early or the simian virus 40 early promoter, and provide an alternative to HIV-1 vectors with internal promoters.
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Cocault, L., D. Bouscary, C. Le Bousse Kerdiles, D. Clay, F. Picard, S. Gisselbrecht, and M. Souyri. "Ectopic expression of murine TPO receptor (c-mpl) in mice is pathogenic and induces erythroblastic proliferation." Blood 88, no. 5 (September 1, 1996): 1656–65. http://dx.doi.org/10.1182/blood.v88.5.1656.1656.
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Abstract c-mpl, the cellular homologue of the v-mpl oncogene transduced in the myeloproliferative leukemia virus (MPLV), encodes the receptor for thrombopoietin, a cytokine involved in the proliferation and differentiation of cells of the megakaryocytic lineage. Here, we show that a retrovirus containing murine c-mpl cDNA (HSFmmpl) is pathogenic in vivo when inoculated in adult mice. All mice developed hepatosplenomegaly and died within 9 to 12 weeks after infection. Histological analysis showed that spleen, liver, and peripheral blood were invaded by erythroblasts at every stage of differentiation. In contrast to the myeloproliferative syndrome induced by MPLV, we did not observe an infiltration of these organs with cells from the granulocytic lineage nor a thrombocytosis. In fact, the platelet count of HSFmmpl mice progressively decreased and a severe thrombocytopenia was observed late in the course of the disease. Further characterization of the target progenitor of HSFmmpl virus in the spleen and bone marrow of diseased animals was accomplished using in vitro clonogenic progenitor cell assays. This analysis indicated that both late and early erythroid compartment (colony-forming unit- erythroid and burst-forming unit-erythroid) were largely increased in the spleens. The colony-forming unit-granulocyte-macrophage compartment was also increased but to a lesser extent. This study shows for the first time that ectopic expression of a member of the cytokine receptor superfamily promotes hematopoietic progenitor cell proliferation and could play a role in leukemogenesis.
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Cocault, L., D. Bouscary, C. Le Bousse Kerdiles, D. Clay, F. Picard, S. Gisselbrecht, and M. Souyri. "Ectopic expression of murine TPO receptor (c-mpl) in mice is pathogenic and induces erythroblastic proliferation." Blood 88, no. 5 (September 1, 1996): 1656–65. http://dx.doi.org/10.1182/blood.v88.5.1656.bloodjournal8851656.
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c-mpl, the cellular homologue of the v-mpl oncogene transduced in the myeloproliferative leukemia virus (MPLV), encodes the receptor for thrombopoietin, a cytokine involved in the proliferation and differentiation of cells of the megakaryocytic lineage. Here, we show that a retrovirus containing murine c-mpl cDNA (HSFmmpl) is pathogenic in vivo when inoculated in adult mice. All mice developed hepatosplenomegaly and died within 9 to 12 weeks after infection. Histological analysis showed that spleen, liver, and peripheral blood were invaded by erythroblasts at every stage of differentiation. In contrast to the myeloproliferative syndrome induced by MPLV, we did not observe an infiltration of these organs with cells from the granulocytic lineage nor a thrombocytosis. In fact, the platelet count of HSFmmpl mice progressively decreased and a severe thrombocytopenia was observed late in the course of the disease. Further characterization of the target progenitor of HSFmmpl virus in the spleen and bone marrow of diseased animals was accomplished using in vitro clonogenic progenitor cell assays. This analysis indicated that both late and early erythroid compartment (colony-forming unit- erythroid and burst-forming unit-erythroid) were largely increased in the spleens. The colony-forming unit-granulocyte-macrophage compartment was also increased but to a lesser extent. This study shows for the first time that ectopic expression of a member of the cytokine receptor superfamily promotes hematopoietic progenitor cell proliferation and could play a role in leukemogenesis.
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Wintermantel, William M., and Laura L. Hladky. "Complete Genome Sequence and Biological Characterization of Moroccan pepper virus (MPV) and Reclassification of Lettuce necrotic stunt virus as MPV." Phytopathology® 103, no. 5 (May 2013): 501–8. http://dx.doi.org/10.1094/phyto-07-12-0166-r.
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Moroccan pepper virus (MPV) and Lettuce necrotic stunt virus (LNSV) have been steadily increasing in prevalence in central Asia and western North America, respectively, over the past decade. Recent sequence analysis of LNSV demonstrated a close relationship between the coat proteins of LNSV and MPV. To determine the full extent of the relationship between LNSV and MPV, the genomes of three MPV isolates were sequenced and compared with that of LNSV. Sequence analysis demonstrated that genomic nucleotide sequences as well as virus-encoded proteins of the three MPV isolates and LNSV shared 97% or greater identity. A full-length clone of a California LNSV isolate was developed and virus derived from infectious transcripts was used to evaluate host plant reactions under controlled conditions. Symptoms of LNSV matched those described previously for MPV on most of a select series of host plants, although some differences were observed. Collectively, these molecular and biological results demonstrate that LNSV should be classified as MPV within the family Tombusviridae, genus Tombusvirus, and confirm the presence of MPV in North America.
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Aebischer, Andrea, Kerstin Wernike, Patricia König, Kati Franzke, Paul J. Wichgers Schreur, Jeroen Kortekaas, Marika Vitikainen, et al. "Development of a Modular Vaccine Platform for Multimeric Antigen Display Using an Orthobunyavirus Model." Vaccines 9, no. 6 (June 15, 2021): 651. http://dx.doi.org/10.3390/vaccines9060651.
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Emerging infectious diseases represent an increasing threat to human and animal health. Therefore, safe and effective vaccines that could be available within a short time frame after an outbreak are required for adequate prevention and control. Here, we developed a robust and versatile self-assembling multimeric protein scaffold particle (MPSP) vaccine platform using lumazine synthase (LS) from Aquifex aeolicus. This scaffold allowed the presentation of peptide epitopes by genetic fusion as well as the presentation of large antigens by bacterial superglue-based conjugation to the pre-assembled particle. Using the orthobunyavirus model Schmallenberg virus (SBV) we designed MPSPs presenting major immunogens of SBV and assessed their efficacy in a mouse model as well as in cattle, a target species of SBV. All prototype vaccines conferred protection from viral challenge infection and the multivalent presentation of the selected antigens on the MPSP markedly improved their immunogenicity compared to the monomeric subunits. Even a single shot vaccination protected about 80% of mice from an otherwise lethal dose of SBV. Most importantly, the MPSPs induced a virtually sterile immunity in cattle. Altogether, LS represents a promising platform for modular and rapid vaccine design.
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Tang, Yao, Ulrike Winkler, Eric O. Freed, Ted A. Torrey, Wankee Kim, Henry Li, Stephen P. Goff, and Herbert C. Morse. "Cellular Motor Protein KIF-4 Associates with Retroviral Gag." Journal of Virology 73, no. 12 (December 1, 1999): 10508–13. http://dx.doi.org/10.1128/jvi.73.12.10508-10513.1999.
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ABSTRACT Previously we demonstrated that murine retroviral Gag proteins associate with a cellular motor protein, KIF-4. Using the yeast two-hybrid assay, we also found an association of KIF-4 with Gag proteins of Mason-Pfizer monkey virus (MPMV), simian immunodeficiency virus (SIV), and human immunodeficiency virus type 1 (HIV-1). Studies performed with mammalian cell systems confirmed that the HIV-1 Gag protein associates with KIF-4. Soluble cytoplasmic proteins from cells infected with recombinant vaccinia virus expressing the entire Gag-Pol precursor protein of HIV-1 or transfected with HIV-1 molecular clone pNL4-3 were fractionated by sucrose gradient centrifugation and further separated by size-exclusion and anion-exchange chromatographies. KIF-4 and HIV-1 Gag cofractionated in both chromatographic separations. Immunoprecipitation assays have also verified the KIF-4–Gag association. KIF-4 binds mainly to the Gag precursor (Pr55 Gag) and a matrix-capsid processing intermediate (Pr42) but not to other processed Gag products. The binding of Gag is mediated by a domain of KIF-4 proximal to the C terminus. These results, and our previous studies, raise the possibility that KIF-4 may play an important role in retrovirus Gag protein transport.
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Maat, Douwe, Maarten Prins, and Corina Brussaard. "Sediments from Arctic Tide-Water Glaciers Remove Coastal Marine Viruses and Delay Host Infection." Viruses 11, no. 2 (January 30, 2019): 123. http://dx.doi.org/10.3390/v11020123.
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Over the past few decades, the Arctic region has been strongly affected by global warming, leading to increased sea surface temperatures and melting of land and sea ice. Marine terminating (tide-water) glaciers are expected to show higher melting and calving rates, with an increase in the input of fine sediment particles in the coastal marine environment. We experimentally investigated whether marine viruses, which drive microbial interactions and biogeochemical cycling are removed from the water column through adsorption to glacier-delivered fine sediments. Ecologically relevant concentrations of 30, 100 and 200 mg·L−1 sediments were added to filtered lysates of 3 cultured algal viruses and to a natural marine bacterial virus community. Total virus removal increased with sediment concentration whereby the removal rate depended on the virus used (up to 88% for an Arctic algal virus), suggesting a different interaction strength with the sediment. Moreover, we observed that the adsorption of viruses to sediment is a reversible process, and that desorbed viruses are still able to infect their respective hosts. Nonetheless, the addition of sediment to infection experiments with the Arctic prasinovirus MpoV-45T substantially delayed host lysis and the production of progeny viruses. We demonstrate that glacier-derived fine sediments have the potency to alter virus availability and consequently, host population dynamics.
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Vance, Melisa A., Matthew Hirsch, Richard Jude Samulski, Joanne Kurtzberg, Laurie Goodrich, and Llanga Telmo. "AAV Gene Therapy for MPS1 Corneal Clouding and Joint Stiffness." Blood 126, no. 23 (December 3, 2015): 2041. http://dx.doi.org/10.1182/blood.v126.23.2041.2041.
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Abstract Mucopolysaccharidosis type 1 (MPS1), also known as Hurler syndrome, is a genetic lysosomal storage disease that results from the loss-of-function mutations present on the L-iduronidase (IDUA) gene. As a consequence, glycosaminoglycans accumulate aberrantly in lysosomes in multiple organs, leading to hepatosplenomegaly, dwarfism, mental and psychomotor retardation, life-threatening cardiac and pulmonary complications, several skeletal and ocular manifestations and nervous system problems. Untreated pediatric patients often die at 5-10 years of age from progressive heart and lung involvement. Since the early 1980s, successful treatment of MPS1 patients with allogeneic hematopoietic stem cell transplantation after myeloablative chemotherapy has been established. The benefits of this therapy rely on cross-correction of IDUA deficient cells with functional IDUA protein produced by a donor cell circulating through the blood and also from engraftment of donor-derived glial cells in the MPS1 patient brain. As a result, if the stem cell transplantation is performed before two years old, the individual manifests cardiac, liver, pulmonary and neurological improvement as well as his lifetime is significantly prolonged. However, still >30% of the patients have progressive corneal clouding, which leads to blindness. Cornea transplantation is not an option for patients with severe hurler disease and the ones that receive a cornea transplant are at the risk of developing future cloudiness. Recently, we have developed an adeno-associated virus (AAV) capable of delivering idua cDNA to MPS1 patient fibroblasts and human corneas and restoring IDUA protein function. Furthermore, immunohistochemistry analysis of intrastromal IDUA protein in a normal human cornea showed low levels of the protein. Even though the levels of IDUA that are required to correct the loss of sight in MPS1 patients are relatively small, administration of a >50-fold increase in IDUA activity (over wild type levels) with gene therapy does not result in any detectable cellular toxicity. We then investigated AAV serotype tropism by incubating human cornea with different AAV viral capsids carrying AAV-CMV-eGFP. From this experiment, we have identified certain capsids with higher levels of transduction and GFP expression on the human cornea. Moreover, we determined the optimal injection volume required for complete coverage of the corneal center area, which is sufficient for allowing regain of the vision. Considering that: 1) the eye is an easily accessible, immune-privileged organ; 2) intrastromal injections are commonly performed in the clinic to treat fungal keratitis; 3) gene therapy for eye diseases has been performed for nearly two decades, and 4) there are 127 registered clinical trials currently taken place for gene therapy in the eye; we foresee that injection of AAV delivering idua cDNA directly to the cornea will likely reverse the MPS1-associated vision loss. Studies are underway to administrate the therapy to the MPS1 dog model for testing safety and efficacy. Furthermore, due to the success of the developed therapy to reestablish IDUA normal levels in cornea, we are currently focusing on the treatment of joint stiffness. We have identified a novel AAV serotype with the highest levels of transduction of the mouse and equine joint tissue explants as well as in cultured monolayers. We foresee that a combined administration of cord blood stem cells at the Carolinas Cord Blood Bank-Duke University and localized AAV-IDUA cDNA delivery to the human cornea and joint tissue at UNC will result in a significant improvement in quality of life of patients with Hurler disease. Disclosures No relevant conflicts of interest to declare.
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Bowen, Michael D., Clarence J. Peters, and Stuart T. Nichol. "Phylogenetic Analysis of theArenaviridae:Patterns of Virus Evolution and Evidence for Cospeciation between Arenaviruses and Their Rodent Hosts." Molecular Phylogenetics and Evolution 8, no. 3 (December 1997): 301–16. http://dx.doi.org/10.1006/mpev.1997.0436.
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Piedade, Gonçalo, Ella Wesdorp, Elena Montenegro-Borbolla, Douwe Maat, and Corina Brussaard. "Influence of Irradiance and Temperature on the Virus MpoV-45T Infecting the Arctic Picophytoplankter Micromonas polaris." Viruses 10, no. 12 (November 29, 2018): 676. http://dx.doi.org/10.3390/v10120676.
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Arctic marine ecosystems are currently undergoing rapid changes in temperature and light availability. Picophytoplankton, such as Micromonas polaris, are predicted to benefit from such changes. However, little is known about how these environmental changes affect the viruses that exert a strong mortality pressure on these small but omnipresent algae. Here we report on one-step infection experiments, combined with measurements of host physiology and viability, with 2 strains of M. polaris and the virus MpoV-45T under 3 light intensities (5, 60 and 160 μmol quanta m−2 s−1), 2 light period regimes (16:8 and 24:0 h light:dark cycle) and 2 temperatures (3 and 7 °C). Our results show that low light intensity (16:8 h light:dark) delayed the decline in photosynthetic efficiency and cell lysis, while decreasing burst size by 46%. In contrast, continuous light (24:0 h light:dark) shortened the latent period by 5 h for all light intensities, and even increased the maximum virus production rate and burst size under low light (by 157 and 69%, respectively). Higher temperature (7 °C vs 3 °C) led to earlier cell lysis and increased burst size (by 19%), except for the low light conditions. These findings demonstrate the ecological importance of light in combination with temperature as a controlling factor for Arctic phytoplankton host and virus dynamics seasonally, even more so in the light of global warming.
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Tuffy, Kevin M., Rebecca J. Kaddis Maldonado, Jordan Chang, Paul Rosenfeld, Alan Cochrane, and Leslie J. Parent. "HIV-1 Gag Forms Ribonucleoprotein Complexes with Unspliced Viral RNA at Transcription Sites." Viruses 12, no. 11 (November 9, 2020): 1281. http://dx.doi.org/10.3390/v12111281.
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The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.
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Hall, Jeffrey S., Byron Adams, Thomas J. Parsons, Roy French, Leslie C. Lane, and Stanley G. Jensen. "Molecular Cloning, Sequencing, and Phylogenetic Relationships of a New Potyvirus: Sugarcane Streak Mosaic Virus, and a Reevaluation of the Classification of the Potyviridae." Molecular Phylogenetics and Evolution 10, no. 3 (December 1998): 323–32. http://dx.doi.org/10.1006/mpev.1998.0535.
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Hofmann, Wilma, Beate Reichart, Andrea Ewald, Eleonora Müller, Iris Schmitt, Roland H. Stauber, Friedrich Lottspeich, et al. "Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)–And Constitutive Transport Element (Cte)–Containing Retroviral Rnas." Journal of Cell Biology 152, no. 5 (February 26, 2001): 895–910. http://dx.doi.org/10.1083/jcb.152.5.895.
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Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)–mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev–NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).