An SL3-3 murine leukemia virus enhancer variant more pathogenic than the wild type obtained by assisted molecular evolution in vivo.

ABSTRACT Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations. Structural features of retroviral RTs that are important for accuracy of DNA synthesis in vivo are not known. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE P also contains an MLV-based retroviral vector (GA-1) which encodes a wild-type bacterial β-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild-type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal), and the frequencies of inactivating mutations in lacZ were quantified. Three mutations in the YVDD motif (V223M, V223S, and V223A) and two mutations in the RNase H domain (S526A and R657S) exhibited frequencies of lacZ inactivation 1.2- to 2.3-fold higher than that for the wild-type MLV RT (P < 0.005). Two mutations (V223I and Y598V) did not affect the frequency oflacZ inactivation. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.

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Moloney Murine Leukemia Virus Infects Cells of the Developing Hair Follicle after Neonatal Subcutaneous Inoculation in Mice

Journal of Virology ◽

10.1128/jvi.73.3.2509-2516.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2509-2516 ◽

Cited By ~ 3

Author(s):

Michael A. Okimoto ◽

Hung Fan

Keyword(s):

Bone Marrow ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Hair Follicles ◽

Moloney Murine Leukemia Virus ◽

Wild Type ◽

Outer Root Sheath ◽

Murine Leukemia

ABSTRACT The nature of Moloney murine leukemia virus (M-MuLV) infection after a subcutaneous (s.c.) inoculation was studied. We have previously shown that an enhancer variant of M-MuLV, Mo+PyF101 M-MuLV, is poorly leukemogenic when used to inoculate mice s.c., but not when inoculated intraperitoneally. This attenuation of leukemogenesis correlated with an inability of Mo+PyF101 M-MuLV to establish infection in the bone marrow of mice at early times postinfection. These results suggested that a cell type(s) is infected in the skin by wild-type but not Mo+PyF101 M-MuLV after s.c. inoculation and that this infection is important for the delivery of infection to the bone marrow, as well as for efficient leukemogenesis. To determine the nature of the cell types infected by M-MuLV and Mo+PyF101 M-MuLV in the skin after a s.c. inoculation, immunohistochemistry with an anti-M-MuLV CA antibody was performed. Cells of developing hair follicles, specifically cells of the outer root sheath (ORS), were extensively infected by M-MuLV after s.c. inoculation. The Mo+PyF101 M-MuLV variant also infected cells of the ORS but the level of infection was lower. By Western blot analysis, the level of infection in skin by Mo+PyF101 M-MuLV was approximately 4- to 10-fold less than that of wild-type M-MuLV. Similar results were seen when a mouse keratinocyte line was infected in vitro with both viruses. Cells of the ORS are a primary target of infection in vivo, since a replication defective M-MuLV-based vector expressing β-galactosidase also infected these cells after a s.c. inoculation.

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Effects of nonleukemogenic and wild-type Moloney murine leukemia virus on lymphoid cells in vivo: identification of a preleukemic shift in thymocyte subpopulations.

Journal of Virology ◽

10.1128/jvi.60.2.423-430.1986 ◽

1986 ◽

Vol 60 (2) ◽

pp. 423-430 ◽

Cited By ~ 10

Author(s):

B R Davis ◽

K G Chandy ◽

B K Brightman ◽

S Gupta ◽

H Fan

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Lymphoid Cells ◽

Moloney Murine Leukemia Virus ◽

Wild Type ◽

Murine Leukemia

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Decreased Virus Population Diversity in p53-Null Mice Infected with Weakly Oncogenic Abelson Virus

Journal of Virology ◽

10.1128/jvi.79.18.11618-11626.2005 ◽

2005 ◽

Vol 79 (18) ◽

pp. 11618-11626 ◽

Cited By ~ 2

Author(s):

Erica Marchlik ◽

Richard Kalman ◽

Naomi Rosenberg

Keyword(s):

Tumor Suppressor ◽

Recombination Event ◽

Leukemia Virus ◽

Population Diversity ◽

Virus Population ◽

Wild Type ◽

Emerging Viruses ◽

Host Genes ◽

Murine Leukemia

ABSTRACT The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53 +/+ and p53 − / − tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.

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Moloney Murine Leukemia Virus-Induced Preleukemic Thymic Atrophy and Enhanced Thymocyte Apoptosis Correlate with Disease Pathogenicity

Journal of Virology ◽

10.1128/jvi.73.3.2434-2441.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2434-2441 ◽

Cited By ~ 19

Author(s):

Christine Bonzon ◽

Hung Fan

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Annexin V ◽

Moloney Murine Leukemia Virus ◽

Wild Type ◽

Thymic Atrophy ◽

Thymocyte Apoptosis ◽

Mink Cell ◽

Nih Swiss ◽

Murine Leukemia

ABSTRACT Moloney murine leukemia virus (M-MuLV) is a replication-competent, simple retrovirus that induces T-cell lymphoma with a mean latency of 3 to 4 months. During the preleukemic period (4 to 10 weeks postinoculation) a marked decrease in thymic size is apparent for M-MuLV-inoculated mice in comparison to age-matched uninoculated mice. We were interested in studying whether the thymic regression was due to an increased rate of thymocyte apoptosis in the thymi of M-MuLV-inoculated mice. Neonatal NIH/Swiss mice were inoculated subcutaneously (s.c.) with wild-type M-MuLV (approximately 105 XC PFU). Mice were sacrificed at 4 to 11 weeks postinoculation. Thymic single-cell suspensions were prepared and tested for apoptosis by two-parameter flow cytometry. Indications of apoptosis included changes in cell size and staining with 7-aminoactinomycin D or annexin V. The levels of thymocyte apoptosis were significantly higher in M-MuLV-inoculated mice than in uninoculated control animals, and the levels of apoptosis were correlated with thymic atrophy. To test the relevance of enhanced thymocyte apoptosis to leukemogenesis, mice were inoculated with the Mo+PyF101 enhancer variant of M-MuLV. When inoculated intraperitoneally, a route that results in wild-type M-MuLV leukemogenesis, mice displayed levels of enhanced thymocyte apoptosis comparable to those seen with wild-type M-MuLV. However, in mice inoculated s.c., a route that results in attenuated leukemogenesis, significantly lower levels of apoptosis were observed. This supported a role for higher levels of thymocyte apoptosis in M-MuLV leukemogenesis. To examine the possible role of mink cell focus-forming (MCF) recombinant virus in raising levels of thymocyte apoptosis, MCF-specific focal immunofluorescence assays were performed on thymocytes from preleukemic mice inoculated with M-MuLV and Mo+PyF101 M-MuLV. The results indicated that infection of thymocytes by MCF virus recombinants is not required for the increased level of apoptosis and thymic atrophy.

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Dynamic copy choice: Steady state between murine leukemia virus polymerase and polymerase-dependent RNase H activity determines frequency of in vivo template switching

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.221289898 ◽

2001 ◽

Vol 98 (21) ◽

pp. 12209-12214 ◽

Cited By ~ 61

Author(s):

C. K. Hwang ◽

E. S. Svarovskaia ◽

V. K. Pathak

Keyword(s):

Steady State ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Rnase H ◽

Template Switching ◽

Murine Leukemia

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Strong selection for cells containing new ecotropic recombinant murine leukemia virus provirus after propagation of C57BL/6 radiation-induced thymoma cells in vitro or in vivo

Molecular and Cellular Biology ◽

10.1128/mcb.3.9.1675-1679.1983 ◽

1983 ◽

Vol 3 (9) ◽

pp. 1675-1679

Author(s):

P Jolicoeur ◽

E Rassart ◽

P Sankar-Mistry

Keyword(s):

Cell Lines ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Primary Radiation ◽

Secondary Tumors ◽

Radiation Induced ◽

Viral Sequences ◽

Murine Leukemia

Using the Southern procedure, we have studied the presence of ecotropic-specific murine leukemia viral sequences in genomic DNA isolated from primary X-ray-induced thymomas, from lymphoid cell lines established from them, or from secondary tumors passaged in vivo. We found that primary radiation-induced thymomas and infiltrated spleens do not harbor newly acquired ecotropic provirus. However, additional ecotropic proviruses (which appear recombinant in the gagpol region) could be detected in most of the tumorigenic cell lines established in vitro from them and in tumors arising from subcutaneous transplantation of the primary thymomas. These results suggest that primary radiation-induced thymomas may not be clonal. They also indicate a strong correlation between the presence of ecotropic recombinant proviruses in the genome and the growth ability, both in vitro and in vivo, of specific cells within these thymomas, suggesting a possible mitogenic function for murine leukemia virus.

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Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association

Journal of Virology ◽

10.1128/jvi.77.21.11651-11660.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11651-11660 ◽

Cited By ~ 27

Author(s):

Mariam Andrawiss ◽

Yasuhiro Takeuchi ◽

Lindsay Hewlett ◽

Mary Collins

Keyword(s):

Fluorescence Microscopy ◽

Murine Leukemia Virus ◽

Fluorescent Proteins ◽

Leukemia Virus ◽

Particle Formation ◽

Membrane Association ◽

Wild Type ◽

Particle Assembly ◽

Gag Assembly ◽

Murine Leukemia

ABSTRACT In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.

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Murine Leukemia Virus P50 Protein Counteracts APOBEC3 by Blocking Its Packaging

Journal of Virology ◽

10.1128/jvi.00032-20 ◽

2020 ◽

Vol 94 (18) ◽

Cited By ~ 3

Author(s):

Wenming Zhao ◽

Charbel Akkawi ◽

Marylène Mougel ◽

Susan R. Ross

Keyword(s):

Cell Line ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Stable Cell Line ◽

Host Restriction ◽

C Terminus ◽

Alternatively Spliced ◽

Apobec3 Proteins ◽

Murine Leukemia

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

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