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

2003 ◽  
Vol 77 (21) ◽  
pp. 11651-11660 ◽  
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.

scholarly journals Murine Leukemia Virus Nucleocapsid Mutant Particles Lacking Viral RNA Encapsidate Ribosomes

2002 ◽  
Vol 76 (22) ◽  
pp. 11405-11413 ◽  
Author(s):  
Delphine Muriaux ◽  
Jane Mirro ◽  
Kunio Nagashima ◽  
Demetria Harvin ◽  
Alan Rein
Keyword(s):  
Murine Leukemia Virus ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Leukemia Virus ◽  
Biological Significance ◽  
Viral Rna ◽  
Genomic Rna ◽  
Wild Type ◽  
Particle Assembly ◽  
Murine Leukemia

ABSTRACT A single retroviral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. Gag normally selects the genomic RNA of the virus with high specificity; the nucleocapsid (NC) domain of Gag plays a crucial role in this selection process. However, encapsidation of the viral RNA is completely unnecessary for particle assembly. We previously showed that mutant murine leukemia virus (MuLV) particles that lack viral RNA because of a deletion in the cis-acting packaging signal (“Ψ”) in the genomic RNA compensate for the loss of the viral RNA by incorporating cellular mRNA. The RNA in wild-type and Ψ− particles was also found to be necessary for virion core structure. In the present work, we explored the role of RNA in MuLV particles that lack genomic RNA because of mutations in the NC domain of Gag. Using a fluorescent dye assay, we observed that NC mutant particles contain the same amount of RNA that wild-type virions do. Surprisingly enough, these particles contained large amounts of rRNAs. Furthermore, ribosomal proteins were detected by immunoblotting, and ribosomes were observed inside the particles by electron microscopy. The biological significance of the presence of ribosomes in NC mutant particles lacking genomic RNA is discussed.

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 Mutant murine leukemia virus Gag proteins lacking proline at the N-terminus of the capsid domain block infectivity in virions containing wild-type Gag

Virology ◽  
2006 ◽  
Vol 347 (2) ◽  
pp. 364-371 ◽  
Author(s):  
S.J. Rulli ◽  
D. Muriaux ◽  
K. Nagashima ◽  
J. Mirro ◽  
M. Oshima ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Gag Proteins ◽  
N Terminus ◽  
Domain Block ◽  
Murine Leukemia

scholarly journals Tissue- and Tumor-Specific Targeting of Murine Leukemia Virus-Based Replication-Competent Retroviral Vectors

2006 ◽  
Vol 80 (14) ◽  
pp. 7070-7078 ◽  
Author(s):  
Christian Metzl ◽  
Daniela Mischek ◽  
Brian Salmons ◽  
Walter H. Günzburg ◽  
Matthias Renner ◽  
...  
Keyword(s):  
T Cell ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Genomic Stability ◽  
Tata Box ◽  
Regulatory Sequence ◽  
Wild Type ◽  
T Cell Factor ◽  
Dividing Cells ◽  
Murine Leukemia

ABSTRACT Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human α1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and β-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.

scholarly journals Akv murine leukemia virus enhances lymphomagenesis in myc-kappa transgenic and in wild-type mice

Virology ◽  
1995 ◽  
Vol 206 (1) ◽  
pp. 93-99 ◽  
Author(s):  
Cornelia Speth ◽  
Arne Luz ◽  
P. Günter Strauss ◽  
Susanne Wendel ◽  
Reinhardt Zeidler ◽  
...  
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Murine Leukemia

scholarly journals Cleavage of the Murine Leukemia Virus Transmembrane Env Protein by Human Immunodeficiency Virus Type 1 Protease: Transdominant Inhibition by Matrix Mutations

1998 ◽  
Vol 72 (12) ◽  
pp. 9621-9627 ◽  
Author(s):  
Rosemary E. Kiernan ◽  
Eric O. Freed
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Env Protein ◽  
Hiv 1 ◽  
Murine Leukemia

ABSTRACT We have identified mutations in the human immunodeficiency virus type 1 (HIV-1) matrix protein (MA) which block infectivity of virions pseudotyped with murine leukemia virus (MuLV) envelope (Env) glycoproteins without affecting infectivity conferred by HIV-1 Env or vesicular stomatitis virus G glycoproteins. This inhibition is very potent and displays a strong transdominant effect; infectivity is reduced more than 100-fold when wild-type and mutant molecular clones are cotransfected at a 1:1 ratio. This phenomenon is observed with both ecotropic and amphotropic MuLV Env. The MA mutations do not affect the incorporation of MuLV Env into virions. We demonstrate that in HIV-1 virions pseudotyped with MuLV Env, the HIV-1 protease (PR) efficiently catalyzes the cleavage of the p15(E) transmembrane (TM) protein to p12(E). Immunoprecipitation analysis of pseudotyped virions reveals that the mutant MA blocks this HIV-1 PR-mediated cleavage of MuLV TM. Furthermore, the transdominant inhibition exerted by the mutant MA on wild-type infectivity correlates with the relative level of p15(E) cleavage. Consistent with the hypothesis that abrogation of infectivity imposed by the mutant MA is due to inhibition of p15(E) cleavage, mutant virions are significantly more infectious when pseudotyped with a truncated p12(E) form of MuLV Env. These results indicate that HIV-1 Gag sequences can influence the viral PR-mediated processing of the MuLV TM Env protein p15(E). These findings have implications for the development of HIV-1-based retroviral vectors pseudotyped with MuLV Env, since p15(E) cleavage is essential for activating membrane fusion and virus infectivity.

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 Increased Induction of Osteopetrosis, but Unaltered Lymphomagenicity, by Murine Leukemia Virus SL3-3 after Mutation of a Nuclear Factor 1 Site in the Enhancer

1999 ◽  
Vol 73 (12) ◽  
pp. 10406-10415 ◽  
Author(s):  
Steen Ethelberg ◽  
Barbara D. Tzschaschel ◽  
Arne Luz ◽  
Salvador J. Diaz-Cano ◽  
Finn Skou Pedersen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Transcription Factor Binding Sites ◽  
Nuclear Factor ◽  
Wild Type ◽  
Factor Binding ◽  
Nuclear Factor 1 ◽  
Murine Leukemia

ABSTRACT SL3-3 is a murine leukemia virus which is only weakly bone pathogenic but highly T-cell lymphomagenic. A major pathogenic determinant is the transcriptional enhancer comprising several transcription factor binding sites, among which are three identical sites for nuclear factor 1 (NF1). We have investigated the pathogenic properties of NF1 site enhancer mutants of SL3-3. Two different mutants carrying a 3-bp mutation either in all three NF1 sites or in the central site alone were constructed and assayed in inbred NMRI mice. The wild type and both mutants induced lymphomas in all mice, with a mean latency period of 9 weeks. However, there was a considerable difference in osteopetrosis induction. Wild-type SL3-3 induced osteopetrosis in 11% of the mice (2 of 19), and the triple NF1 site mutant induced osteopetrosis in none of the mice (0 of 19), whereas the single NF1 site mutant induced osteopetrosis in 56% (10 of 18) of the mice, as determined by X-ray analysis. A detailed histological examination of the femurs of the mice was carried out and found to support this diagnosis. Thus, the NF1 sites of SL3-3 are major determinants of osteopetrosis induction, without determining lymphomagenesis. This conclusion was further supported by evaluation of the bone pathogenicity of other SL3-3 enhancer variants, the lymphomagenicity of which had been examined previously. This evaluation furthermore strongly indicated that the core sites, a second group of transcription factor binding sites in the viral enhancer, are necessary for the osteopetrosis induction potential of SL3-3.

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.

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