Functional characterization of a portion of the Moloney murine leukemia virus
            <i>gag</i>
            gene by genetic footprinting

Auerbach, Marcy R.; Shu, Chang; Kaplan, Artem; Singh, Ila R.

doi:10.1073/pnas.2034020100

Cited by 35 publications

(42 citation statements)

References 24 publications

(19 reference statements)

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“…We show that modified MLV particles tolerate foreign sequences in the N-terminus of p12 and the C-termini of MA and NC, partly confirming earlier studies (36,37). Although cleavage between MA and p12 is not critical for the maturation of MLV, cleavage of p12/CA and CA/NC is essential for normal core structure and RNA dimer stabilization, respectively (7).…”

Section: Discussionsupporting

confidence: 87%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

125

118

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Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

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Section: Discussionsupporting

confidence: 87%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

125

118

View full text Add to dashboard Cite

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“…The PIC of the Moloney murine leukemia virus (Mo-MuLV) notably retains CA for many hours (5,15). The presence of CA in the PIC is consistent with the effects of many gag mutations in the CA domain on early stages of infection (1,2,30). CA is even more strongly implicated in early events of infection by its role as a target of the Fv1 gene, a dominantacting restriction system present in many mouse strains (41,55).…”

mentioning

confidence: 60%

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

Yueh

Leung

Bhattacharyya

et al. 2006

J Virol

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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.

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“…Gag proteins from other retroviruses contain nuclear trafficking signals as well; putative NLS and NES sequences were reported for the human immunodeficiency virus type 1 (HIV-1) Gag protein (5,14). The murine leukemia virus (MLV) Gag polyprotein is present in the nucleus, the MLV NC protein localizes to the nucleoli (39), and the p12 domain was implicated to have a role in the nuclear import of the viral genome, presumably owing to intrinsic karyophilic properties (1,53). Thus, the subcellular targeting signals within Gag proteins, although poorly characterized, are likely to play important roles during infection.…”

mentioning

confidence: 99%

Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

Scheifele¹,

Kenney

Cairns

et al. 2007

J Virol

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Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.Retroviral assembly is directed by the viral Gag polyprotein precursor. Despite containing very little sequence homology, all Gag proteins share three canonical domains, the MA (matrix), CA (capsid), and NC (nucleocapsid) domains, as well as additional regions that vary for each retrovirus. Immature virus particles contain unprocessed Gag and Gag-Pol fusion proteins arranged in a concentric array just inside the viral envelope, with the NC domain of Gag toward the center, bound to the dimeric, positive-sense viral RNA (vRNA) genome (4, 18, 51). The cleavage of Gag during the maturation process occurs during or immediately after budding, leading to a dramatic change in morphology, with the appearance of an electrondense core near the particle center. This dense core is comprised of vRNA coated by the NC protein, together forming the ribonucleoprotein (RNP) complex. The RNP is enclosed by a shell composed of CA multimers forming the virion core, which is in turn surrounded by the MA protein in association with the lipid envelope (reviewed in references 45 and 46). The molecular events that govern RNP and core formation during virus assembly and maturation are still not well understood.Functional mapping of the Gag polyprotein identified three discrete motifs, known as assembly domains, that coordinate virion formation and...

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Functional characterization of a portion of the Moloney murine leukemia virus gag gene by genetic footprinting

Cited by 35 publications

References 24 publications

Protein transduction from retroviral Gag precursors

Protein transduction from retroviral Gag precursors

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

Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

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