Cis-acting elements required for strong stop acceptor template selection during moloney murine leukemia virus reverse transcription

1998 ◽  
Vol 281 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Robert Topping ◽  
Marie-Ange Demoitie ◽  
Nam Hee Shin ◽  
Alice Telesnitsky
Keyword(s):  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Cis Acting ◽  
Template Selection ◽  
Murine Leukemia ◽  
Strong Stop

Replication-defective chimeric helper proviruses and factors affecting generation of competent virus: expression of Moloney murine leukemia virus structural genes via the metallothionein promoter

1987 ◽  
Vol 7 (5) ◽  
pp. 1797-1806
Author(s):  
R A Bosselman ◽  
R Y Hsu ◽  
J Bruszewski ◽  
S Hu ◽  
F Martin ◽  
...  
Keyword(s):  
Cell Line ◽  
Sequence Homology ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Helper Cell ◽  
Factors Affecting ◽  
Cis Acting ◽  
Virus Expression ◽  
Murine Leukemia

Two chimeric helper proviruses were derived from the provirus of the ecotropic Moloney murine leukemia virus by replacing the 5'long terminal repeat and adjacent proviral sequences with the mouse metallothionein I promoter. One of these chimeric proviruses was designed to express the gag-pol genes of the virus, whereas the other was designed to express only the env gene. When transfected into NIH 3T3 cells, these helper proviruses failed to generate competent virus but did express Zn2+-inducible trans-acting viral functions needed to assemble infectious vectors. One helper cell line (clone 32) supported vector assembly at levels comparable to those supported by the Psi-2 and PA317 cell lines transfected with the same vector. Defective proviruses which carry the neomycin phosphotransferase gene and which lack overlapping sequence homology with the 5' end of the chimeric helper proviruses could be transfected into the helper cell line without generation of replication-competent virus. Mass cultures of transfected helper cells produced titers of about 10(4) G418r CFU/ml, whereas individual clones produced titers between 0 and 2.6 X 10(4) CFU/ml. In contrast, defective proviruses which share homologous overlapping viral sequences with the 5' end of the chimeric helper proviruses readily generated infectious virus when transfected into the helper cell line. The deletion of multiple cis-acting functions from the helper provirus and elimination of sequence homology overlapping at the 5' ends of helper and vector proviruses both contribute to the increased genetic stability of this system.

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 Characterization of Intracellular Reverse Transcription Complexes of Moloney Murine Leukemia Virus

1999 ◽  
Vol 73 (11) ◽  
pp. 8919-8925 ◽  
Author(s):  
Ariberto Fassati ◽  
Stephen P. Goff
Keyword(s):  
Reverse Transcription ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Sedimentation Velocity ◽  
Micrococcal Nuclease ◽  
Moloney Murine Leukemia Virus ◽  
Velocity Sedimentation ◽  
Equilibrium Density ◽  
Transcription Complexes ◽  
Murine Leukemia

ABSTRACT To examine the early events in the life cycle of Moloney murine leukemia virus (MoMLV), we analyzed the intracellular complexes mediating reverse transcription. Partial purification of the reverse transcription complexes (RTCs) by equilibrium density fractionation and velocity sedimentation indicated that three distinct species of intracellular complexes are formed shortly after cell infection. Only one of these species is able to start and complete reverse transcription in the cell cytoplasm. This RTC is composed of at least the viral genome, capsid, integrase, and reverse transcriptase proteins. The RTC becomes permeable to micrococcal nuclease but not to antibodies. Shortly after initiation of reverse transcription, the viral strong stop DNA within the RTC is protected from nuclease digestion. The sedimentation velocity of the RTC decreases during reverse transcription. After entry into the nucleus, most capsid proteins are lost from the RTC and its sedimentation velocity decreases further.

scholarly journals Effects of Identity Minimization on Moloney Murine Leukemia Virus Template Recognition and Frequent Tertiary Template-Directed Insertions during Nonhomologous Recombination

2007 ◽  
Vol 81 (22) ◽  
pp. 12156-12168 ◽  
Author(s):  
Nisha K. Duggal ◽  
Leslie Goo ◽  
Steven R. King ◽  
Alice Telesnitsky
Keyword(s):  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Nonhomologous Recombination ◽  
Sequence Identity ◽  
Donor Acceptor ◽  
Template Selection ◽  
Donor Template ◽  
Murine Leukemia

ABSTRACT Homology requirements for Moloney murine leukemia virus recombination were addressed in this study by monitoring titer defects observed when acceptor/donor template identity lengths were systematically reduced. Recombination acceptors with at least 16 contiguous bases of donor template identity were recognized as efficiently as longer acceptors. In contrast, a sharp 1-log titer drop was observed for an acceptor of only 15 bases long, with an additional 1-log titer decline for an 8-base acceptor and further decreases for shorter acceptors. Eighty-three independent nonhomologous recombination products were sequenced to examine recombination template selection in the absence of significant sequence identity. These replication products contained a total of 152 nonhomologous crossover junctions. Forced copy choice models predict that forced nonhomologous recombination should result in DNA synthesis to the donor template's 5′ end, followed by microidentity-guided acceptor template selection. However, only a single product displayed this structure. The majority of examined nonhomologous recombination products contained junction-associated sequence insertions. Most insertions resulted from the use of one or more tertiary templates, recognizable as discontiguous portions of viral or host RNA or minus-strand DNA. The donor/acceptor template microidentity evident at most crossovers reconfirmed the remarkable capability of the reverse transcription machinery to recognize short regions of sequence identity. These results demonstrate that recruitment of discontiguous host or viral sequences is a common way for retroviruses to resolve nonhomologous recombination junctions and provide experimental support for the role of splinting templates in the generation of retroviral insertions.

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