scholarly journals Incorporation of Fowl Plague Virus Hemagglutinin into Murine Leukemia Virus Particles and Analysis of the Infectivity of the Pseudotyped Retroviruses

1998 ◽  
Vol 72 (6) ◽  
pp. 5313-5317 ◽  
Author(s):  
Theodora Hatziioannou ◽  
Sandrine Valsesia-Wittmann ◽  
Stephen J. Russell ◽  
François-Loïc Cosset
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Retroviral Vector ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
Cell Surface Molecule ◽  
Virus Attachment ◽  
Virus Particles ◽  
Fusion Ability ◽  
Murine Leukemia

ABSTRACT We describe retrovirus particles carrying the fowl plague virus (FPV) hemagglutinin (HA). When expressed in cells providing Moloney murine leukemia virus (MoMLV) Gag and Pol proteins and alacZ retroviral vector, FPV HA was found to be efficiently expressed, correctly processed, and stably incorporated into retroviral particles. HA-bearing retroviruses were infectious with a wide host range and were only 10-fold less infectious than retroviruses carrying wild-type MLV retroviral envelopes. We also coexpressed HA proteins in retroviral particles with chimeric MoMLV-derived envelope glycoproteins that efficiently retarget virus attachment but are only weakly fusogenic. Our results suggest that HA can in some cases enhance the fusion ability of these retroviral particles, depending on the cell surface molecule that is used as a receptor.

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 Receptor-Mediated Moloney Murine Leukemia Virus Entry Can Occur Independently of the Clathrin-Coated-Pit-Mediated Endocytic Pathway

1999 ◽  
Vol 73 (7) ◽  
pp. 5994-6005 ◽  
Author(s):  
Sunyoung Lee ◽  
Yi Zhao ◽  
W. French Anderson
Keyword(s):  
Hela Cells ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
293 Cells ◽  
Immunofluorescence Labeling ◽  
Murine Leukemia

ABSTRACT To investigate receptor-mediated Moloney murine leukemia virus (MoMuLV) entry, the green fluorescent protein (GFP)-tagged ecotropic receptor designated murine cationic amino acid transporter (MCAT-1) (MCAT-1-GFP) was constructed and expressed in 293 cells (293/MCAT-1-GFP). 293/MCAT-1-GFP cells displayed green fluorescence primarily at the cell membrane and supported wild-type levels of MoMuLV vector binding and transduction. Using immunofluorescence labeling and confocal microscopy, it was demonstrated that the surface envelope protein (SU) gp70 of MoMuLV virions began to appear inside cells 5 min after virus binding and was colocalized with MCAT-1-GFP. However, clathrin was not colocalized with MCAT-1-GFP, suggesting that MoMuLV entry, mediated by MCAT-1, does not involve clathrin. Double immunofluorescence labeling of SU and clathrin in 293 cells expressing untagged receptor (293/MCAT-1) gave the same results, i.e., SU and clathrin did not colocalize. In addition, we examined the transduction ability of MoMuLV vector on HeLa cells overexpressing the dominant-negative GTPase mutant of dynamin (K44A). HeLa cells overexpressing mutant dynamin have a severe block in endocytosis by the clathrin-coated-pit pathway. No significant titer difference was observed when MoMuLV vector was tranduced into HeLa cells overexpressing either wild-type or mutant dynamin, while the transduction ability of vesicular stomatitis virus glycoprotein pseudotyped vector into HeLa cells overexpressing mutant dynamin was decreased significantly. Taken together, these data suggest that MoMuLV entry does not occur through the clathrin-coated-pit-mediated endocytic pathway.

scholarly journals Structure-Based Moloney Murine Leukemia Virus Reverse Transcriptase Mutants with Altered Intracellular Direct-Repeat Deletion Frequencies

2000 ◽  
Vol 74 (20) ◽  
pp. 9629-9636 ◽  
Author(s):  
Julie K. Pfeiffer ◽  
Millie M. Georgiadis ◽  
Alice Telesnitsky
Keyword(s):  
Reverse Transcriptase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Direct Repeat ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type Virus ◽  
Template Switching ◽  
Wild Type ◽  
Catalytic Core ◽  
Murine Leukemia

ABSTRACT Template switching rates of Moloney murine leukemia virus reverse transcriptase mutants were tested using a retroviral vector-based direct-repeat deletion assay. The reverse transcriptase mutants contained alterations in residues that modeling of substrates into the catalytic core had suggested might affect interactions with primer and/or template strands. As assessed by the frequency of functionallacZ gene generation from vectors in which lacZwas disrupted by insertion of a sequence duplication, the frequency of template switching varied more than threefold among fully replication-competent mutants. Some mutants displayed deletion rates that were lower and others displayed rates that were higher than that of wild-type virus. Replication for the mutants with the most significant alterations in template switching frequencies was similar to that of the wild type. These data suggest that reverse transcriptase template switching rates can be altered significantly without destroying normal replication functions.

scholarly journals Moloney Murine Leukemia Virus Integrase Protein Augments Viral DNA Synthesis in Infected Cells

2001 ◽  
Vol 75 (23) ◽  
pp. 11365-11372 ◽  
Author(s):  
Lilin Lai ◽  
Hongmei Liu ◽  
Xiaoyun Wu ◽  
John C. Kappes
Keyword(s):  
Dna Synthesis ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Specific Interaction ◽  
Proteolytic Processing ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
Viral Dna ◽  
Infected Cells ◽  
Murine Leukemia

ABSTRACT Mutations in the IN domain of retroviral DNA may affect multiple steps of the virus life cycle, suggesting that the IN protein may have other functions in addition to its integration function. We previously reported that the human immunodeficiency virus type 1 IN protein is required for efficient viral DNA synthesis and that this function requires specific interaction with other viral components but not enzyme (integration) activity. In this report, we characterized the structure and function of the Moloney murine leukemia virus (MLV) IN protein in viral DNA synthesis. Using an MLV vector containing green fluorescent protein as a sensitive reporter for virus infection, we found that mutations in either the catalytic triad (D184A) or the HHCC motif (H61A) reduced infectivity by approximately 1,000-fold. Mutations that deleted the entire IN (ΔIN) or 34 C-terminal amino acid residues (Δ34) were more severely defective, with infectivity levels consistently reduced by 10,000-fold. Immunoblot analysis indicated that these mutants were similar to wild-type MLV with respect to virion production and proteolytic processing of the Gag and Pol precursor proteins. Using semiquantitative PCR to analyze viral cDNA synthesis in infected cells, we found the Δ34 and ΔIN mutants to be markedly impaired while the D184A and H61A mutants synthesized cDNA at levels similar to the wild type. The DNA synthesis defect was rescued by complementing the Δ34 and ΔIN mutants intrans with either wild-type IN or the D184A mutant IN, provided as a Gag-IN fusion protein. However, the DNA synthesis defect of ΔIN mutant virions could not be complemented with the Δ34 IN mutant. Taken together, these analyses strongly suggested that the MLV IN protein itself is required for efficient viral DNA synthesis and that this function may be conserved among other retroviruses.

Mapping of DNase I-hypersensitive sites in the 5' and 3' long terminal repeats of integrated moloney murine leukemia virus proviral DNA

1985 ◽  
Vol 5 (4) ◽  
pp. 601-609
Author(s):  
T Thompson ◽  
H Fan
Keyword(s):  
Murine Leukemia Virus ◽  
Transcription Initiation ◽  
Leukemia Virus ◽  
Dnase I ◽  
Moloney Murine Leukemia Virus ◽  
Wild Type ◽  
Long Terminal Repeats ◽  
Proviral Dna ◽  
Enhancer Sequences ◽  
Murine Leukemia

The chromatin state of integrated Moloney murine leukemia virus (M-MuLV) proviral DNA was investigated. Nuclei from M-MuLV-infected mouse NIH 3T3 cells were digested with limited amounts of DNase I, and hypersensitive (HS) sites were mapped by the indirect end labeling technique. Particular emphasis was placed on the 5' long terminal repeat (LTR), since viral transcription initiates there. M-MuLV proviral DNA showed two strong DNase I-HS sites in the 5' LTR, one coincident with the transcription initiation (cap) site and the other with the transcriptional enhancers. Two weaker DNase I-HS sites were also detected in internal proviral DNA. The 3' LTR also showed a strong HS site in the region of the enhancers, but an HS site at the cap site of the 3' LTR was not detected. Thus, the chromatin configurations of the 5' and 3' LTRs of integrated M-MuLV proviruses appear to be different. The chromatin configuration of M-MuLV proviruses which contain LTR insertions of polyomavirus enhancer sequences was also studied. The 5' LTR of M-MuLV proviruses containing polyoma enhancer sequences substituted for the M-MuLV enhancers showed two strong HS sites, one in the polyoma sequences and one at the cap site. The 5' LTR of M-MuLV proviruses containing polyoma enhancer sequences inserted into the wild-type M-MuLV LTR between the cap site and the M-MuLV enhancers showed three HS sites. Two HS sites corresponded to those of the wild-type M-MuLV LTR, whereas the third mapped to the inserted polyoma sequences. The HS site associated with the inserted polyoma sequences was considerably stronger than the M-MuLV-associated HS sites.

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