scholarly journals R-Peptide Cleavage Potentiates Fusion-Controlling Isomerization of the Intersubunit Disulfide in Moloney Murine Leukemia Virus Env

2007 ◽  
Vol 82 (5) ◽  
pp. 2594-2597 ◽  
Author(s):  
Robin Löving ◽  
Kejun Li ◽  
Michael Wallin ◽  
Mathilda Sjöberg ◽  
Henrik Garoff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Isomerization Reaction ◽  
Peptide Cleavage ◽  
In Vivo Assays ◽  
Env Protein ◽  
Murine Leukemia

ABSTRACT Fusion of the membrane of the Moloney murine leukemia virus (Mo-MLV) Env protein is facilitated by cleavage of the R peptide from the cytoplasmic tail of its TM subunit, but the mechanism for this effect has remained obscure. The fusion is also controlled by the isomerization of the intersubunit disulfide of the Env SU-TM complex. In the present study, we used several R-peptide-cleavage-inhibited virus mutants to show that the R peptide suppresses the isomerization reaction in both in vitro and in vivo assays. Thus, the R peptide affects early steps in the activation pathway of murine leukemia virus Env.

scholarly journals Expression of Moloney Murine Leukemia Virus RNase H Rescues the Growth Defect of an Escherichia coliMutant

2001 ◽  
Vol 75 (13) ◽  
pp. 6212-6217 ◽  
Author(s):  
Andrew G. Campbell
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
In Vitro Assays ◽  
Growth Defect ◽  
Protein Variant ◽  
Murine Leukemia

ABSTRACT A 157-amino-acid fragment of Moloney murine leukemia virus reverse transcriptase encoding RNase H is shown to rescue the growth-defective phenotype of an Escherichia coli mutant. In vitro assays of the recombinant wild-type protein purified from the conditionally defective mutant confirm that it is catalytically active. Mutagenesis of one of the presumptive RNase H-catalytic residues results in production of a protein variant incapable of rescue and which lacks activity in vitro. Analyses of additional active site mutants demonstrate that their encoded variant proteins lack robust activity yet are able to rescue the bacterial mutant. These results suggest that genetic complementation may be useful for in vivo screening of mutant viral RNase H gene fragments and in evaluating their function under conditions that more closely mimic physiological conditions. The rescue system may also be useful in verifying the functional outcomes of mutations based on protein structural predictions and modeling.

scholarly journals Mutations of the RNase H C Helix of the Moloney Murine Leukemia Virus Reverse Transcriptase Reveal Defects in Polypurine Tract Recognition

2002 ◽  
Vol 76 (16) ◽  
pp. 8360-8373 ◽  
Author(s):  
David Lim ◽  
Marianna Orlova ◽  
Stephen P. Goff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Rnase H ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Polypurine Tract ◽  
Dna Ends ◽  
Murine Leukemia

ABSTRACT Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and Escherichia coli RNase H possess a positively charged α-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (ΔC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5′ and 3′ viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5′ end.

scholarly journals Diminished Potential for B-Lymphoid Differentiation after Murine Leukemia Virus Infection In Vivo and in EML Hematopoietic Progenitor Cells

2007 ◽  
Vol 81 (13) ◽  
pp. 7274-7279 ◽  
Author(s):  
Samantha L. Finstad ◽  
Naomi Rosenberg ◽  
Laura S. Levy
Keyword(s):  
Bone Marrow ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Selective Advantage ◽  
Moloney Murine Leukemia Virus ◽  
Vitro Model ◽  
B Lymphoid ◽  
Murine Leukemia

ABSTRACT Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.

scholarly journals Stabilization of TM Trimer Interactions during Activation of Moloney Murine Leukemia Virus Env

2007 ◽  
Vol 82 (5) ◽  
pp. 2358-2366 ◽  
Author(s):  
Mathilda Sjöberg ◽  
Birgitta Lindqvist ◽  
Henrik Garoff
Keyword(s):  
Cell Membrane ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Moloney Murine Leukemia Virus ◽  
Structural Conversion ◽  
Murine Leukemia

ABSTRACT The transmembrane subunit (TM) of the trimeric retrovirus Env complex is thought to direct virus-cell membrane fusion by refolding into a cell membrane-interacting, extended form that subsequently folds back on itself into a very stable trimer of hairpin-like TM polypeptides. However, so far there is only limited evidence for the formation of a stable TM trimer during Env activation. Here we have studied the oligomer composition and stability of an intermediate and the fully activated form of Moloney murine leukemia virus (Mo-MLV) Env. Activation of Mo-MLV Env is controlled by isomerization of its intersubunit disulfide. This results in surface subunit (SU) dissociation and TM refolding. If activation is done in the presence of an alkylator, this will modify the isomerization-active thiol in the SU of Env and arrest Env at an intermediate stage, the isomerization-arrested state (IAS) of its activation pathway. We generated IAS and fully activated Envs in vitro and in vivo and studied their states of oligomerization by two-dimensional blue native polyacrylamide gel electrophoresis (PAGE) and nonreducing sodium dodecyl sulfate (SDS)-PAGE. The IAS Env was composed of trimers of SU-TM complexes, whereas the activated Env consisted of SU monomers and TM trimers. When the oligomers were subjected to mild SDS treatment the TM trimer was found to be 3.5 times more resistant than the IAS oligomer. Thus, this demonstrates that a structural conversion of TM takes place during activation, which results in the formation of a stable TM trimer.

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 Cytoplasmic Tail of Moloney Murine Leukemia Virus Envelope Protein Influences the Conformation of the Extracellular Domain: Implications for Mechanism of Action of the R Peptide

2003 ◽  
Vol 77 (2) ◽  
pp. 1281-1291 ◽  
Author(s):  
Hector C. Aguilar ◽  
W. French Anderson ◽  
Paula M. Cannon
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Cytoplasmic Tail ◽  
Moloney Murine Leukemia Virus ◽  
Virus Envelope ◽  
Peptide Cleavage ◽  
Virus Envelope Protein ◽  
Env Protein ◽  
Membrane Spanning ◽  
Murine Leukemia

ABSTRACT The envelope (Env) protein of Moloney murine leukemia virus (MoMuLV) is a homotrimeric complex whose monomers consist of linked surface (SU) and transmembrane (TM) proteins cleaved from a precursor protein by a cellular protease. In addition, a significant fraction of virion-associated TM is further processed by the viral protease to remove the C-terminal 16 amino acids of the cytoplasmic domain, the R peptide. This cleavage greatly enhances the fusogenicity of the protein and is necessary for the formation of a fully functional Env protein complex. We have previously proposed that R peptide cleavage enhances fusogenicity by altering the conformation of the ectodomain of the protein (Y. Zhao et al., J. Virol. 72:5392-5398, 1998). Using a series of truncation and point mutants of MoMuLV Env, we now provide direct biochemical and immunological evidence that the cytoplasmic tail and the membrane-spanning region of Env can influence the overall structure of the ectodomain of the protein and alter the strength of the SU-TM interaction. The R-peptide-truncated form of the protein, in particular, exhibits a markedly different conformation than the full-length protein.

scholarly journals Synthesis of Virus-Specific High-Mobility DNA after Temperature Upshift of SC-1 Cells Chronically Infected with Moloney Murine Leukemia Virus Mutant ts1

2000 ◽  
Vol 74 (15) ◽  
pp. 7055-7063 ◽  
Author(s):  
Paul F. Szurek ◽  
Benjamin Rix Brooks
Keyword(s):  
Murine Leukemia Virus ◽  
Cultured Cells ◽  
Leukemia Virus ◽  
High Mobility ◽  
Moloney Murine Leukemia Virus ◽  
Permissive Temperature ◽  
Temperature Upshift ◽  
Murine Leukemia

ABSTRACT Premature termination products of reverse transcription that consist physically of viral minus-sense single-stranded DNA that is shorter than one long terminal repeat and partial DNA duplexes are dramatically increased in the central nervous system (CNS) of FVB/N mice that are infected by ts1, a temperature-sensitive mutant of Moloney murine leukemia virus. Due to their migration in agarose gels, these incomplete physical forms of DNA have been designated high-mobility (HM) DNA. In non-CNS tissues, the level of HM DNA is either low or not detectable. In order to determine the conditions that are necessary for the synthesis of HM DNA in vivo, we have characterized the physical forms of HM DNA that were synthesized in vitro in chronically infected SC-1 cells after temperature upshift. At the permissive temperature of 34°C, the chronically infected SC-1 cells did not synthesize HM DNA. After temperature upshift of the cultured cells from 34 to 37°C, the chronically infected SC-1 cells developed extremely high levels of HM DNA. Following temperature downshift of the cultured cells from 37 to 34°C, a decrease in the level of HM DNA and an increase in the level of unintegrated linear proviral DNA occurred simultaneously. These results suggested that the accumulation of HM DNA both in vitro and in vivo may be the result of superinfection.

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