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

2005 ◽  
Vol 79 (18) ◽  
pp. 11618-11626 ◽  
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.

scholarly journals Development of an In Vivo Assay To Identify Structural Determinants in Murine Leukemia Virus Reverse Transcriptase Important for Fidelity

2000 ◽  
Vol 74 (1) ◽  
pp. 312-319 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Gene Transfection ◽  
Leukemia Virus ◽  
Rnase H ◽  
Wild Type ◽  
Structural Determinants ◽  
In Vivo Assay ◽  
Murine Leukemia

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.

scholarly journals Zinc Finger Domain of Murine Leukemia Virus Nucleocapsid Protein Enhances the Rate of Viral DNA Synthesis in Vivo

2002 ◽  
Vol 76 (15) ◽  
pp. 7473-7484 ◽  
Author(s):  
Wen-hui Zhang ◽  
Carey K. Hwang ◽  
Wei-Shau Hu ◽  
Robert J. Gorelick ◽  
Vinay K. Pathak
Keyword(s):  
Secondary Structure ◽  
Dna Synthesis ◽  
Zinc Finger ◽  
Rna Secondary Structure ◽  
Leukemia Virus ◽  
Template Switching ◽  
Zinc Finger Domain ◽  
Wild Type ◽  
Murine Leukemia

ABSTRACT In vitro studies have indicated that retroviral nucleocapsid (NC) protein facilitates both DNA synthesis by reverse transcriptase (RT) and annealing of the nascent DNA with acceptor template. Increasing the rate of DNA synthesis is expected to reduce the frequency of RT template switching, whereas annealing the nascent DNA with acceptor template promotes template switching. We performed a mutational analysis of the murine leukemia virus (MLV) NC zinc finger domain to study its effect on RT template switching in vivo and to explore the role of NC during reverse transcription. The effects of NC mutations on RT template switching were determined by using a previously described in vivo direct-repeat deletion assay. A trans-complementation assay was also developed in which replication-defective NC mutants were rescued by coexpression of replication-defective RT mutants that provided wild-type NC in trans. We found that mutations in the MLV NC zinc finger domain increased the frequency of template switching approximately twofold. When a predicted stem-loop RNA secondary structure was introduced into the template RNA, the template-switching frequency increased 5-fold for wild-type NC and further increased up to an additional 6-fold for NC zinc finger domain mutants, resulting in an overall increase of as much as 30-fold. Thus, wild-type NC increased the efficiency with which RT was able to reverse transcribe through regions of RNA secondary structure that might serve as RT pause sites. These results provide the first in vivo evidence that NC enhances the rate of DNA synthesis by RT in regions of the template possessing stable RNA secondary structure.

scholarly journals Mutation in the Glycosylated Gag Protein of Murine Leukemia Virus Results in Reduced In Vivo Infectivity and a Novel Defect in Viral Budding or Release

2007 ◽  
Vol 81 (8) ◽  
pp. 3685-3692 ◽  
Author(s):  
Audrey Low ◽  
Shoibal Datta ◽  
Yurii Kuznetsov ◽  
Sohail Jahid ◽  
Nayantara Kothari ◽  
...  
Keyword(s):  
Leukemia Virus ◽  
Spherical Particles ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Viral Budding ◽  
Leukemia Viruses ◽  
Murine Leukemia

ABSTRACT All gammaretroviruses, including murine leukemia viruses (MuLVs), feline leukemia viruses, and gibbon-ape leukemia virus, encode an alternate, glycosylated form of Gag polyprotein (glyco-Gag or gPr80 gag ) in addition to the polyprotein precursor of the viral capsid proteins (Pr65 gag ). gPr80 gag is translated from an upstream in-frame CUG initiation codon, in contrast to the AUG codon used for Pr65 gag . The role of glyco-Gag in MuLV replication has been unclear, since gPr80 gag -negative Moloney MuLV (M-MuLV) mutants are replication competent in vitro and pathogenic in vivo. However, reversion to the wild type is frequently observed in vivo. In these experiments, in vivo inoculation of a gPr80 gag mutant, Ab-X-M-MuLV, showed substantially lower (2 log) initial infectivity in newborn NIH Swiss mice than that of wild-type virus, and revertants to the wild type could be detected by PCR cloning and DNA sequencing as early as 15 days postinfection. Atomic force microscopy of Ab-X-M-MuLV-infected producer cells or of the PA317 amphotropic MuLV-based vector packaging line (also gPr80 gag negative) revealed the presence of tube-like viral structures on the cell surface. In contrast, wild-type virus-infected cells showed the typical spherical, 145-nm particles observed previously. Expression of gPr80 gag in PA317 cells converted the tube-like structures to typical spherical particles. PA317 cells expressing gPr80 gag produced 5- to 10-fold more infectious vector or viral particles as well. Metabolic labeling studies indicated that this reflected enhanced virus particle release rather than increased viral protein synthesis. These results indicate that gPr80 gag is important for M-MuLV replication in vivo and in vitro and that the protein may be involved in a late step in viral budding or release.

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

1999 ◽  
Vol 73 (3) ◽  
pp. 2509-2516 ◽  
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.

scholarly journals Antisense Transcription in Gammaretroviruses as a Mechanism of Insertional Activation of Host Genes

2010 ◽  
Vol 84 (8) ◽  
pp. 3780-3788 ◽  
Author(s):  
Mads Heilskov Rasmussen ◽  
Borja Ballarín-González ◽  
Jinghua Liu ◽  
Louise Berkhoudt Lassen ◽  
Annette Füchtbauer ◽  
...  
Keyword(s):  
B Cell ◽  
Leukemia Virus ◽  
Negative Polarity ◽  
Antisense Transcription ◽  
B Cell Lymphomas ◽  
Eukaryotic Transcription ◽  
R Region ◽  
Rapid Amplification ◽  
Host Genes

ABSTRACT Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5′ long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5′ rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5′ LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5′ LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.

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

1999 ◽  
Vol 73 (3) ◽  
pp. 2434-2441 ◽  
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.

scholarly journals Selection of Optimal Polypurine Tract Region Sequences during Moloney Murine Leukemia Virus Replication

2000 ◽  
Vol 74 (22) ◽  
pp. 10293-10303 ◽  
Author(s):  
Nicole D. Robson ◽  
Alice Telesnitsky
Keyword(s):  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Upstream Sequence ◽  
Wild Type ◽  
The Core ◽  
Polypurine Tract ◽  
Efficient Replication ◽  
Marked Preference ◽  
Leukemia Viruses ◽  
Murine Leukemia

ABSTRACT Retrovirus plus-strand synthesis is primed by a cleavage remnant of the polypurine tract (PPT) region of viral RNA. In this study, we tested replication properties for Moloney murine leukemia viruses with targeted mutations in the PPT and in conserved sequences upstream, as well as for pools of mutants with randomized sequences in these regions. The importance of maintaining some purine residues within the PPT was indicated both by examining the evolution of random PPT pools and from the replication properties of targeted mutants. Although many different PPT sequences could support efficient replication and one mutant that contained two differences in the core PPT was found to replicate as well as the wild type, some sequences in the core PPT clearly conferred advantages over others. Contributions of sequences upstream of the core PPT were examined with deletion mutants. A conserved T-stretch within the upstream sequence was examined in detail and found to be unimportant to helper functions. Evolution of virus pools containing randomized T-stretch sequences demonstrated marked preference for the wild-type sequence in six of its eight positions. These findings demonstrate that maintenance of the T-rich element is more important to viral replication than is maintenance of the core PPT.

Sign in / Sign up

Export Citation Format

Share Document