scholarly journals Characterization of Moloney Murine Leukemia Virus p12 Mutants Blocked during Early Events of Infection

2002 ◽  
Vol 76 (21) ◽  
pp. 10801-10810 ◽  
Author(s):  
Bing Yuan ◽  
Ariberto Fassati ◽  
Andrew Yueh ◽  
Stephen P. Goff
Keyword(s):  
Life Cycle ◽  
Murine Leukemia Virus ◽  
Early Phase ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Gag Protein ◽  
Viral Dnas ◽  
Murine Leukemia

ABSTRACT Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3′ ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.

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 Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes

2002 ◽  
Vol 76 (23) ◽  
pp. 12376-12380 ◽  
Author(s):  
Youichi Suzuki ◽  
Robert Craigie
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Preintegration Complex ◽  
Retroviral Integration ◽  
Target Dna ◽  
Initial Salt ◽  
Murine Leukemia

ABSTRACT Retroviral integration is mediated by a preintegration complex (PIC) which contains the viral DNA made by reverse transcription together with associated protein factors. Prior to association with target DNA, the PIC must avoid suicidal intramolecular integration of its viral DNA (autointegration). We have demonstrated that barrier-to-autointegration factor (BAF) blocks the autointegration of Moloney murine leukemia virus (MoMLV) PICs in vitro. In this study, we show that BAF is an authentic component of MoMLV. Analysis of the sedimentation properties of initial, salt-stripped, and BAF-reconstituted PICs reveals that the viral DNA within the PIC is reversibly compacted by BAF, consistent with the functional role of BAF in protecting the viral DNA from autointegration. Furthermore, we find that BAF can promote the association of PICs with target DNA. Thus, our data suggest that BAF plays critical roles in promoting preferential intermolecular integration by both blocking autointegration and stimulating the capture of target DNA.

scholarly journals Assembly and Catalysis of Concerted Two-End Integration Events by Moloney Murine Leukemia Virus Integrase

2001 ◽  
Vol 75 (20) ◽  
pp. 9561-9570 ◽  
Author(s):  
Fan Yang ◽  
Monica J. Roth
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Strand Transfer ◽  
Viral Dna ◽  
Retroviral Integration ◽  
Target Dna ◽  
The Individual ◽  
Murine Leukemia

ABSTRACT Retroviral integration results in the stable and coordinated insertion of the two termini of the linear viral DNA into the host genome. An in vitro concerted two-end integration reaction catalyzed by the Moloney murine leukemia virus (M-MuLV) integrase (IN) was used to investigate the binding and coordination of the two viral DNA ends. Comparison of the two-end integration and strand transfer assays indicates that zinc is required for efficient concerted integration utilizing plasmid DNA as target. Complementation assays using a pair of nonoverlapping integrase domains, consisting of the HHCC domain and the core/C-terminal region, yielded products containing the correct 4-base target site duplication. The efficiency of the coordinated two-end integration varied depending on the order of addition of the individual protein and DNA components in the complementation assay. Two-end integration was most efficient when the long terminal repeat (LTR) was premixed with either the target DNA or the HHCC domain. The preference for two-end integration through preincubation of the HHCC finger with the viral DNA supports the role of this domain in the recognition and/or positioning of the LTR.

scholarly journals Moloney murine leukemia virus p12 is required for histone loading onto retroviral DNAs

2021 ◽  
Author(s):  
Gary Z. Wang ◽  
Stephen P. Goff
Keyword(s):  
Rna Polymerase Ii ◽  
Murine Leukemia Virus ◽  
Viral Protein ◽  
De Novo ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Dna ◽  
Viral Dnas ◽  
Host Chromatin ◽  
Murine Leukemia

During retrovirus infection, a histone-free DNA copy of the viral RNA genome is synthesized and rapidly loaded with nucleosomes de novo upon nuclear entry. The potential role of viral accessory proteins in histone loading onto retroviral DNAs has not been extensively investigated. The p12 protein of Moloney murine leukemia virus (MMLV) is a virion protein critical for tethering the incoming viral DNA to host chromatin in the early stages of infection. Infection by virions containing a mutant p12 (PM14) defective in chromatin tethering results in the formation of viral DNAs that do not accumulate in the nucleus. In this report, we show that viral DNAs of these mutants are not loaded with histones. Moreover, the DNA genomes delivered by mutant p12 show prolonged association with viral structural proteins nucleocapsid (NC) and capsid (CA). The histone-poor viral DNA genomes do not become associated with the host RNA polymerase II machinery. These findings provide insights into fundamental aspects of retroviral biology, indicating that tethering to host chromatin by p12 and retention in the nucleus are required to allow loading of histones onto the viral DNA. Importance: Incoming retroviral DNAs are rapidly loaded with nucleosomal histones upon entry into the nucleus and before integration into the host genome. The entry of murine leukemia virus DNA into the nucleus only occurs upon dissolution of the nuclear membrane in mitosis, and retention in the nucleus requires the action of a viral protein, p12, which tethers the DNA to host chromatin. Data presented here show that the tethering activity of p12 is required for the loading of histones onto the viral DNA. p12 mutants lacking tethering activity fail to acquire histones, retain capsid and nucleocapsid proteins, and are poorly transcribed. The work defines a new requirement for a viral protein to allow chromatinization of viral DNA.

scholarly journals Sequential activation of the three protomers in the Moloney murine leukemia virus Env

2017 ◽  
Vol 114 (10) ◽  
pp. 2723-2728 ◽  
Author(s):  
Mathilda Sjöberg ◽  
Robin Löving ◽  
Birgitta Lindqvist ◽  
Henrik Garoff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Virus Envelope ◽  
Sequential Activation ◽  
Virus Envelope Protein ◽  
Membrane Fusion Proteins ◽  
Viral Membrane Fusion ◽  
Murine Leukemia

Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney murine leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3→ (SU-TM)2TM → (SU-TM)TM2→ TM3. This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.

Heterogeneity of Murine Leukemia Virus in vitro DNA; Detection of Viral DNA in Mammalian Cells

Science ◽  
1971 ◽  
Vol 172 (3990) ◽  
pp. 1353-1355 ◽  
Author(s):  
L. D. Gelb ◽  
S. A. Aaronson ◽  
M. A. Martin
Keyword(s):  
Murine Leukemia Virus ◽  
Mammalian Cells ◽  
Dna Detection ◽  
Leukemia Virus ◽  
Viral Dna ◽  
Murine Leukemia

Inhibition of the in vitro integration of moloney murine leukemia virus DNA by the DNA minor groove binder netropsin

1994 ◽  
Vol 47 (10) ◽  
pp. 1821-1826 ◽  
Author(s):  
Sandrine Carteau ◽  
Jean Francois Mouscadet ◽  
Hélène Goulaouic ◽  
Frédéric Subra ◽  
Christian Auclair
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Minor Groove ◽  
Moloney Murine Leukemia Virus ◽  
Minor Groove Binder ◽  
Dna Minor Groove ◽  
Murine Leukemia ◽  
Dna Minor Groove Binder

scholarly journals Host Cell Cathepsins Potentiate Moloney Murine Leukemia Virus Infection

2007 ◽  
Vol 81 (19) ◽  
pp. 10506-10514 ◽  
Author(s):  
Pankaj Kumar ◽  
Deepa Nachagari ◽  
Carolyn Fields ◽  
John Franks ◽  
Lorraine M. Albritton
Keyword(s):  
Envelope Protein ◽  
Murine Leukemia Virus ◽  
Cathepsin B ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Dependent Manner ◽  
Virus Binding ◽  
Dose Dependent ◽  
Murine Leukemia

ABSTRACT The roles of cellular proteases in Moloney murine leukemia virus (MLV) infection were investigated using MLV particles pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein as a control for effects on core MLV particles versus effects specific to Moloney MLV envelope protein (Env). The broad-spectrum inhibitors cathepsin inhibitor III and E-64d gave comparable dose-dependent inhibition of Moloney MLV Env and VSV G pseudotypes, suggesting that the decrease did not involve the envelope protein. Whereas, CA-074 Me gave a biphasic response that differentiated between Moloney MLV Env and VSV G at low concentrations, at which the drug is highly selective for cathepsin B, but was similar for both glycoproteins at higher concentrations, at which CA-074 Me inhibits other cathepsins. Moloney MLV infection was lower on cathepsin B knockout fibroblasts than wild-type cells, whereas VSV G infection was not reduced on the B−/− cells. Taken together, these results support the notion that cathepsin B acts at an envelope-dependent step while another cathepsin acts at an envelope-independent step, such as uncoating or viral-DNA synthesis. Virus binding was not affected by CA-074 Me, whereas syncytium induction was inhibited in a dose-dependent manner, consistent with cathepsin B involvement in membrane fusion. Western blot analysis revealed specific cathepsin B cleavage of SU in vitro, while TM and CA remained intact. Infection could be enhanced by preincubation of Moloney MLV with cathepsin B, consistent with SU cleavage potentiating infection. These data suggested that during infection of NIH 3T3 cells, endocytosis brings Moloney MLV to early lysosomes, where the virus encounters cellular proteases, including cathepsin B, that cleave SU.

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