scholarly journals Interaction of Moloney Murine Leukemia Virus Capsid with Ubc9 and PIASy Mediates SUMO-1 Addition Required Early in Infection

2006 ◽  
Vol 80 (1) ◽  
pp. 342-352 ◽  
Author(s):  
Andrew Yueh ◽  
Juliana Leung ◽  
Subarna Bhattacharyya ◽  
Lucy A. Perrone ◽  
Kenia de los Santos ◽  
...  
Keyword(s):  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Viral Gene ◽  
Alanine Scanning Mutagenesis ◽  
Viral Dna ◽  
Murine Leukemia

ABSTRACT Yeast two-hybrid screens led to the identification of Ubc9 and PIASy, the E2 and E3 small ubiquitin-like modifier (SUMO)-conjugating enzymes, as proteins interacting with the capsid (CA) protein of the Moloney murine leukemia virus. The binding site in CA for Ubc9 was mapped by deletion and alanine-scanning mutagenesis to a consensus motif for SUMOylation at residues 202 to 220, and the binding site for PIASy was mapped to residues 114 to 176, directly centered on the major homology region. Expression of CA and a tagged SUMO-1 protein resulted in covalent transfer of SUMO-1 to CA in vivo. Mutations of lysine residues to arginines near the Ubc9 binding site and mutations at the PIASy binding site reduced or eliminated CA SUMOylation. Introduction of these mutations into the complete viral genome blocked virus replication. The mutants exhibited no defects in the late stages of viral gene expression or virion assembly. Upon infection, the mutant viruses were able to carry out reverse transcription to synthesize normal levels of linear viral DNA but were unable to produce the circular viral DNAs or integrated provirus normally found in the nucleus. The results suggest that the SUMOylation of CA mediated by an interaction with Ubc9 and PIASy is required for early events of infection, after reverse transcription and before nuclear entry and viral DNA integration.

scholarly journals Role of Murine Leukemia Virus Reverse Transcriptase Deoxyribonucleoside Triphosphate-Binding Site in Retroviral Replication and In Vivo Fidelity

2000 ◽  
Vol 74 (22) ◽  
pp. 10349-10358 ◽  
Author(s):  
Elias K. Halvas ◽  
Evguenia S. Svarovskaia ◽  
Vinay K. Pathak
Keyword(s):  
Amino Acids ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Binding Site ◽  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Deoxyribonucleoside Triphosphate ◽  
Murine Leukemia

ABSTRACT Retroviral populations exhibit a high evolutionary potential, giving rise to extensive genetic variation. Error-prone DNA synthesis catalyzed by reverse transcriptase (RT) generates variation in retroviral populations. Structural features within RTs are likely to contribute to the high rate of errors that occur during reverse transcription. We sought to determine whether amino acids within murine leukemia virus (MLV) RT that contact the deoxyribonucleoside triphosphate (dNTP) substrate are important for in vivo fidelity of reverse transcription. We utilized the previously described ANGIE P encapsidating cell line, which expresses the amphotropic MLV envelope and a retroviral vector (pGA-1). pGA-1 expresses the bacterial β-galactosidase gene (lacZ), which serves as a reporter of mutations. Extensive mutagenesis was performed on residues likely to interact with the dNTP substrate, and the effects of these mutations on the fidelity of reverse transcription were determined. As expected, most substitution mutations of amino acids that directly interact with the dNTP substrate significantly reduced viral titers (>10,000-fold), indicating that these residues played a critical role in catalysis and viral replication. However, the D153A and A154S substitutions, which are predicted to affect the interactions with the triphosphate, resulted in statistically significant increases in the mutation rate. In addition, the conservative substitution F155W, which may affect interactions with the base and the ribose, increased the mutation rate 2.8-fold. Substitutions of residues in the vicinity of the dNTP-binding site also resulted in statistically significant decreases in fidelity (1.3- to 2.4-fold). These results suggest that mutations of residues that contact the substrate dNTP can affect viral replication as well as alter the fidelity of reverse transcription.

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 Identification of Residues of the Moloney Murine Leukemia Virus Nucleocapsid Critical for Viral DNA Synthesis In Vivo

2001 ◽  
Vol 75 (6) ◽  
pp. 2616-2626 ◽  
Author(s):  
Jason Gonsky ◽  
Eran Bacharach ◽  
Stephen P. Goff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Moloney Murine Leukemia Virus ◽  
Single Amino Acid ◽  
Primer Binding Site ◽  
Nucleic Acid Binding ◽  
Viral Dna ◽  
Murine Leukemia

ABSTRACT The nucleocapsid (NC) protein of retroviruses is a small nucleic acid-binding protein important in virion assembly and in the encapsidation of the viral RNA genome into the virion particle. Multiple single-amino-acid substitutions were introduced into the NC of Moloney murine leukemia virus to examine further its role in viral replication. Two residues were shown to play important roles in the early events of replication. Unlike viruses with previously characterized NC mutations, these viruses showed no impairment in the late events of replication. Viruses containing the substitutions L21A and K30A expressed the normal complement of properly processed viral Gag proteins. Analysis of the RNA content of mutant virions revealed normal levels of unspliced and spliced viral RNA, and the tRNAPro primer was properly annealed to the primer binding site on the viral genome. The virions demonstrated no defect in initiation of reverse transcription using the endogenous tRNA primer or in the synthesis of long viral DNA products in vitro. Nonetheless, viruses possessing these NC mutations demonstrated significant defects in the synthesis and accumulation of viral DNA products in vivo.

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 Retrovirus transduction: generation of infectious retroviruses expressing dominant and selectable genes is associated with in vivo recombination and deletion events.

1983 ◽  
Vol 3 (12) ◽  
pp. 2180-2190 ◽  
Author(s):  
A L Joyner ◽  
A Bernstein
Keyword(s):  
Long Terminal Repeat ◽  
Thymidine Kinase ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Terminal Repeat ◽  
Moloney Murine Leukemia Virus ◽  
Murine Leukemia

We describe the generation of infectious retroviruses containing foreign genes by an in vivo recombination-deletion mechanism. Cotransfection into mouse cells of chimeric plasmids carrying a murine retrovirus 5' long terminal repeat and either the thymidine kinase (tk) gene of herpesvirus or the dominant selectable bacterial gene for neomycin resistance (neo), along with a clone of Moloney murine leukemia virus, results in the generation of infectious thymidine kinase or neomycin-resistant viruses. Expression of the selectable marker in these viruses can be regulated by the homologous transcriptional promoter of the gene, by the promoter contained within the Friend spleen focus-forming virus long terminal repeat, or by the simian virus 40 early region promoter. In all cases, the rescued viruses appeared to arise by recombination in vivo with Moloney murine leukemia virus sequences, resulting in the acquisition of the Moloney 3' long terminal repeat and variable amounts of the 3' adjacent Moloney genome. In two of the thymidine kinase constructs where tk was inserted 200 base pairs downstream from the long terminal repeat, the rescued viruses acquired a large part of the murine leukemia virus genome, including the region involved in packaging genomic RNA into virions. The generation of infectious neomycin-resistant virus is associated with deletions of simian virus 40 splicing and polyadenylation sequences. These results demonstrate that nonhomologous recombination and deletion events can take place in animal cells, resulting in the acquisition or removal of cis-acting sequences required for, or inhibitory to, retrovirus infectivity.

scholarly journals Moloney Murine Leukemia Virus Envelope Protein Subunits, gp70 and Pr15E, Form a Stable Disulfide-Linked Complex

1998 ◽  
Vol 72 (8) ◽  
pp. 6537-6545 ◽  
Author(s):  
Dirk-Jan E. Opstelten ◽  
Michael Wallin ◽  
Henrik Garoff
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Alkylating Agents ◽  
Moloney Murine Leukemia Virus ◽  
Virus Envelope ◽  
Disulfide Linkage ◽  
Virus Envelope Protein ◽  
Biochemical Analyses ◽  
Murine Leukemia

ABSTRACT The nature and stability of the interactions between the gp70 and Pr15E/p15E molecules of murine leukemia virus (MLV) have been disputed extensively. To resolve this controversy, we have performed quantitative biochemical analyses on gp70-Pr15E complexes formed after independent expression of the amphotropic and ecotropic Moloney MLVenv genes in BHK-21 cells. We found that all cell-associated gp70 molecules are disulfide linked to Pr15E whereas only a small amount of free gp70 is released by the cells. The complexes were resistant to treatment with reducing agents in vivo, indicating that the presence and stability of the disulfide interaction between gp70 and Pr15E are not dependent on the cellular redox state. However, disulfide-bonded Env complexes were disrupted in lysates of nonalkylated cells in a time-, temperature-, and pH-dependent fashion. Disruption seemed not to be caused by a cellular factor but is probably due to a thiol-disulfide exchange reaction occurring within the Env complex after solubilization. The possibility that alkylating agents induce the formation of the intersubunit disulfide linkage was excluded by showing that disulfide-linked gp70-Pr15E complexes exist in freshly made lysates of nonalkylated cells and that disruption of the complexes can be prevented by lowering the pH. Together, these data establish that gp70 and Pr15E form a stable disulfide-linked complex in vivo.

scholarly journals A retrovirus carrying the K-fgf oncogene induces diffuse meningeal tumors and soft-tissue fibrosarcomas.

1993 ◽  
Vol 13 (4) ◽  
pp. 1998-2010 ◽  
Author(s):  
D Talarico ◽  
M M Ittmann ◽  
R Bronson ◽  
C Basilico
Keyword(s):  
Growth Factor ◽  
Soft Tissue ◽  
Fusion Protein ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Oncogenic Potential ◽  
Meningeal Tumors ◽  
Murine Leukemia

The K-fgf/hst oncogene encodes a growth factor of the fibroblast growth factor (FGF) family and transforms cells through an autocrine mechanism which requires extracellular activation of its receptor(s). To identify the cell and tissue targets of K-fgf oncogenic potential in vivo, we constructed a recombinant retrovirus carrying the human K-fgf cDNA and injected it, together with helper Moloney murine leukemia virus, into immunocompetent as well as nude mice. The original construct was highly transforming in tissue culture but produced no detectable pathologies in vivo with the exception of a single fibrosarcoma which arose after a long latency. The virus produced by this tumor appears to have undergone a complex series of recombination events involving the helper Moloney murine leukemia virus. It encodes an Env/K-FGF fusion protein whose expression is under the control of a hybrid long terminal repeat. This virus (designated MFS, for meningeal fibrosarcoma) induces tumors in mice with high frequency and short latency. These neoplasms consist of aggressive fibrosarcomas of soft tissue as well as diffuse meningeal tumors originating from the dura mater that surround the whole central nervous system and cause severe hydrocephalus. The Env/K-FGF fusion protein expressed by the MFS virus has retained all of the biological properties of native K-FGF, including secretion, mitogenic activity, heparin binding, and neutralization by anti-K-FGF antibodies. These and other results indicate that the tumors induced by the MFS virus result from the oncogenic potential of K-FGF.

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