Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice

Yeung, Douglas; Brown, Grant W.; Tam, Patrick; Russnak, Roland; Wilson, Gary; Clark‐Lewis, Ian; Astell, Caroline R.

doi:10.1016/0042-6822(91)90467-p

Cited by 51 publications

(32 citation statements)

References 41 publications

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“…The fourth intracellular loop or the carboxyl-terminal tail of the receptor might interact with other proteins necessary for efficient CCR5 targeting to the plasma membrane, and the proper folding of these domains might require cysteine palmitoylation. For several GPCRs, such as olfactory receptors in Caenorhabditis elegans, opsins, and adrenomedullin receptor, specific chaperones or cargo receptors, necessary for their efficient transport to the cell surface, have been identified (43)(44)(45)(46)(47)(48). Alternatively, unpalmitoylated receptors might leave accessible patches of hydrophobic residues, favoring interaction with chaperones or aggregation of the receptor, leading to its degradation (49).…”

Section: Discussionmentioning

confidence: 99%

Palmitoylation of CCR5 Is Critical for Receptor Trafficking and Efficient Activation of Intracellular Signaling Pathways

Blanpain

Wittamer

Vanderwinden

et al. 2001

Journal of Biological Chemistry

133

132

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CCR5 is a CC chemokine receptor expressed on memory lymphocytes, macrophages, and dendritic cells and also constitutes the main coreceptor for macrophagetropic (or R5) strains of human immunodeficiency viruses. In the present study, we investigated whether CCR5 was palmitoylated in its carboxyl-terminal domain by generating alanine substitution mutants for the three cysteine residues present in this region, individually or in combination. We found that wild-type CCR5 was palmitoylated, but a mutant lacking all three Cys residues was not. Through the use of green fluorescent fusion proteins and immunofluorescence studies, we found that the absence of receptor palmitoylation resulted in sequestration of CCR5 in intracellular biosynthetic compartments. By using the fluorescence recovery after photobleaching technique, we showed that the non-palmitoylated mutant had impaired diffusion properties within the endoplasmic reticulum. We next studied the ability of the mutants to bind and signal in response to chemokines. Chemokines binding and activation of G i -mediated signaling pathways, such as calcium mobilization and inhibition of adenylate cyclase, were not affected. However, the duration of the functional response, as measured by a microphysiometer, and the ability to increase [ 35 S]guanosine 5-3-O-(thio)triphosphate binding to membranes were severely affected for the non-palmitoylated mutant. The ability of RANTES (regulated on activation normal T cell expressed and secreted) and aminooxypentane-RANTES to promote CCR5 endocytosis was not altered by cysteine replacements. Finally, we found that the absence of receptor palmitoylation reduced the human immunodeficiency viruses coreceptor function of CCR5, but this effect was secondary to the reduction in surface expression. In conclusion, we found that palmitoylated cysteines play an important role in the intracellular trafficking of CCR5 and are likely necessary for efficient coupling of the receptor to part of its repertoire of signaling cascades.

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Section: Discussionmentioning

confidence: 99%

Palmitoylation of CCR5 Is Critical for Receptor Trafficking and Efficient Activation of Intracellular Signaling Pathways

Blanpain

Wittamer

Vanderwinden

et al. 2001

Journal of Biological Chemistry

133

132

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“…with 4% paraformaldehyde (PFA; 15 min at room temperature). Viral proteins were detected with an NS1-specific monoclonal antibody (MAb) (generous gift from Caroline Astell) (29), an intact capsid MAb, and a polyclonal capsid-protein VP2 antibody (Ab; generous gifts from Colin R. Parrish) (27), followed by goat anti-mouse or anti-rabbit Alexa-555-or Alexa-633-conjugated secondary Abs (Molecular Probes, Life technologies, Grand Island, NY). Modified histones were labeled with rabbit Abs against H3K9me3 and H3K27ac (Abcam, Cambridge, MA) followed by goat anti-rabbit Alexa 633-conjugated secondary Abs.…”

Section: Methodsmentioning

confidence: 99%

Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

Mäntylä

Salokas

Oittinen

et al. 2016

J Virol

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The association of host histones with parvoviral DNA is poorly understood. We analyzed the chromatinization and histone acetylation of canine parvovirus DNA during infection by confocal imaging and in situ proximity ligation assay combined with chromatin immunoprecipitation and high-throughput sequencing. We found that during late infection, parvovirus replication bodies were rich in histones bearing modifications characteristic of transcriptionally active chromatin, i.e., histone H3 lysine 27 acetylation (H3K27ac). H3K27ac, in particular, was located in close proximity to the viral DNA-binding protein NS1. Importantly, our results show for the first time that in the chromatinized parvoviral genome, the two viral promoters in particular were rich in H3K27ac. Histone acetyltransferase (HAT) inhibitors efficiently interfered with the expression of viral proteins and infection progress. Altogether, our data suggest that the acetylation of histones on parvoviral DNA is essential for viral gene expression and the completion of the viral life cycle. IMPORTANCEViral DNA introduced into cell nuclei is exposed to cellular responses to foreign DNA, including chromatinization and epigenetic silencing, both of which determine the outcome of infection. How the incoming parvovirus resists cellular epigenetic downregulation of its genes is not understood. Here, the critical role of epigenetic modifications in the regulation of parvovirus infection was demonstrated. We showed for the first time that a successful parvovirus infection is characterized by the deposition of nucleosomes with active histone acetylation on the viral promoter areas. The results provide new insights into the regulation of parvoviral gene expression, which is an important aspect of the development of parvovirus-based virotherapy. N uclear chromatin is composed of DNA and histone proteins (1). The histone proteins assemble DNA into nucleosomes, the composition and spacing of which contribute to higher-order chromatin packing. The chromatin is organized into regions of less-condensed actively transcribed chromatin (euchromatin) and highly condensed transcriptionally repressed chromatin (heterochromatin). Epigenetic modifications of histone proteins have been shown to correlate with the spatial distribution of active and repressed chromatin (2, 3). The acetylation of lysine 9 or 27 of histone H3 (H3K9ac and H3K27ac, respectively) and trimethylation of lysine 4 (H3K4me3) correlate with transcriptional activity, while repressed chromatin is characterized by, e.g., trimethylation or dimethylation of the same H3 lysine residues (H3K9me3 and H3K27me3 as well as H3K9me2 and H3K27me2) (4-8).Foreign DNA introduced into mammalian cells can be recognized as a threat by the host cell. The cellular responses to the foreign DNA, such as viral DNA, include the chromatinization of the entering DNA, leading to its transcriptional silencing (9-11). How viruses resist cellular chromatinization and silencing is known for only a few viruses (9, 12, 13). These include h...

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“…The viruses' ability to productively infect A9 cells was determined using a transfection-based virus expansion assay in which transfected A9 cells, seeded on Teflon-coated spot slides at 20% confluence, were cultured in the presence or absence of 1% neutralizing polyclonal anti-MVM capsid antibody for 2 days at 37°C or 3 days at 32°C. Cells were fixed, permeabilized, and stained for NS1 by indirect immunofluorescence, using the murine monoclonal antibody CE10B10 (32). Nuclei were counterstained with 4=,6-diamidino-2-phenylindole (DAPI), and the percentages of cells with NS1-positive nuclei were scored using a Zeiss Axio Imager M2 fluorescence microscope.…”

Section: Methodsmentioning

confidence: 99%

Mutations at the Base of the Icosahedral Five-Fold Cylinders of Minute Virus of Mice Induce 3′-to-5′ Genome Uncoating and Critically Impair Entry Functions

Cotmore

Tattersall

2012

J Virol

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The linear single-stranded DNA genome of minute virus of mice can be ejected, in a 3=-to-5= direction, via a cation-linked uncoating reaction that leaves the 5= end of the DNA firmly complexed with its otherwise intact protein capsid. Here we compare the phenotypes of four mutants, L172T, V40A, N149A, and N170A, which perturb the base of cylinders surrounding the icosahedral 5-fold axes of the virus, and show that these structures are strongly implicated in 3=-to-5= release. Although noninfectious at 37°C, all mutants were viable at 32°C, showed a temperature-sensitive cell entry defect, and, after proteolysis of externalized VP2 N termini, were unable to protect the VP1 domain, which is essential for bilayer penetration. Mutant virus yields from multipleround infections were low and were characterized by the accumulation of virions containing subgenomic DNAs of specific sizes. In V40A, these derived exclusively from the 5= end of the genome, indicative of 3=-to-5= uncoating, while L172T, the most impaired mutant, had long subgenomic DNAs originating from both termini, suggesting additional packaging portal defects. Compared to the wild type, genome release in vitro following cation depletion was enhanced for all mutants, while only L172T released DNA, in both directions, without cation depletion following proteolysis at 37°C. Analysis of progeny from single-round infections showed that uncoating did not occur during virion assembly, release, or extraction. However, unlike the wild type, the V40A mutant extensively uncoated during cell entry, indicating that the V40-L172 interaction restrains an uncoating trigger mechanism within the endosomal compartment.

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Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice

Cited by 51 publications

References 41 publications

Palmitoylation of CCR5 Is Critical for Receptor Trafficking and Efficient Activation of Intracellular Signaling Pathways

Palmitoylation of CCR5 Is Critical for Receptor Trafficking and Efficient Activation of Intracellular Signaling Pathways

Promoter-Targeted Histone Acetylation of Chromatinized Parvoviral Genome Is Essential for the Progress of Infection

Mutations at the Base of the Icosahedral Five-Fold Cylinders of Minute Virus of Mice Induce 3′-to-5′ Genome Uncoating and Critically Impair Entry Functions

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