Structural Organization of DNA in Chlorella Viruses

Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes

Strukturelle Organisation der DNA in PBCV-1

Der Prozess der DNA-Kondensation stellt in allen lebenden Organismen einen komplizierten Prozess dar. Der Größenunterschied zwischen DNA-Molekül und Zelle bzw. Viruskapsid, sowie die negative Ladung des Phosphatrückgrates der DNA sind die größte Schwierigkeit einer erfolgreichen DNA-Aggregation. Im Laufe der Evolution haben sich unterschiedliche Prozesse entwickelt, um diese Aufgabe erfolgreich durchzuführen. So kodieren z.B. eukaryotische Zellen für Proteine, so genannte Histone, die wie in einer Spule von der DNA eng umschlungen werden. Dadurch ist es möglich die DNA zu kondensieren und eng zu packen. Dieser Prozess kann ebenfalls von positiv geladenen Molekülen, Polyaminen oder Kationen durchgeführt werden. Ebenso wurden Prozesse etabliert, die es ermöglichen Geninformation auf geringem Raum zu kodieren, die so genannten overlapping genes, um die Größe des Genoms zu reduzieren. Chlorellaviren sind ein positives Beispiel für eine erfolgreiche DNA-Kondensation wie am Prototyp dieser Familie, PBCV-1 zu erkennen ist. Die Herausforderung für diese Virengruppe besteht darin, ein Genom in ihrem Kapisd zu verpacken, welches 1000 mal länger ist. In topographischen Atomic force microscopy - und fluoreszenzmikroskopischen Messungen wurde festgestellt, dass die isolierte, virale DNA repetitive Strukturen aufweist. Des Weiteren zeigen Sequenzanalysen, dass eine periodische Verteilung GC-reicher Sequenzen vorliegt, die mögliche Bindungsstellen für basische Proteine sein können. Biochemische Experimente zeigen, dass keine signifikanten Konzentrationen an Polyaminen vorliegen und diese Menge lediglich 0.02 % der DNA kondensieren könnte. Ebenso liegt keine höhere Konzentration eines einzelnen Kations, z.B. Mg2+, vor, wie Energy dispersive X-ray spectroscopy und Inductively coupled plasma-mass spectrometry belegen. Es ist allerdings möglich, dass die Summe aller Kationen für die Kondensierung teilweise verantwortlich ist, da bereits 58 % der DNA neutralisiert werden könnte. Diese Erkenntnis steht im Gegensatz zu den Ergebnissen der Phage λ, die ausschließlich Kationen und Polyamine zur Aggregation nutzt. PBCV-1 kodiert für ein Protein, A278L, welches Kinaseaktivität aufweist. Des Weiteren ähnelt A278L in den physikalischen Eigenschaften Histonen und ist somit ein wichtiger Kandidat für die Aggregation der viralen DNA. Das rekombinante Protein zeigt in topographischen AFM-Messungen, im Vergleich zum neutralen Protein BSA, eine Verdopplung der Aggregation der viralen DNA. Kraftspektroskopische Messungen zeigen, dass das rekombinante Protein A278L (25 nN) eine ähnliche Bindungskraft zur DNA aufweist wie die an der DNA assoziierten Partikel (64 nN). Dieses Protein könnte, neben Kationen, für das Aggregieren der DNA verantwortlich sein, so dass eine Metastruktur und eine Organisation der viralen DNA gewährleistet werden kann

SDS-PAGE pattern of proteins associated with PBCV-1 DNA.

<p>DNA was released from capsids by osmotic shock and separated from soluble proteins by centrifugation. The DNA-containing pellet was treated with DNase to release DNA-bound proteins. The framed bands were excised and used for MALDI TOF analysis. Lane 1: weight marker, lane 2: proteins obtained after DNAse treatment.</p

Identification of DNA binding proteins and most abundant proteins in PBCV-1 virion from MALDI-TOF PMF analysis.

<p>The table lists (in bold) the PBCV-1 CDSs from which peptides were detected in MALDI-TOF spectra. The peptides correspond to DNA associated proteins described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030133#pone-0030133-g003" target="_blank">Fig. 3</a>. Only database entries with a protein score with a significance <0.05 are presented. The identity of the A278L protein was confirmed by the MS/MS sequencing of 3 peptides. Also included are the copy numbers per virion of the most abundant proteins in PBCV-1 virion. The copy numbers were estimated using the exponentially modified protein abundance index (emPAI) algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030133#pone.0030133-Ishihama1" target="_blank">[40]</a>. Based on the knowledge that the major capsid protein (A430L) is present in 1440 copies per virion, we calculated the abundances of the major proteins in the virion. For these proteins, parameters such as the molecular weight (MW), the isoelectric point (IP) and the functional annotation from the Greengene database (<a href="http://greengene.uml.edu/database/php/get_orf.php?genome_name=PBCV1" target="_blank">http://greengene.uml.edu/database/php/get_orf.php?genome_name=PBCV1</a>) are provided. Putative DNA binding sites were further identified by the BindN algorithm (<a href="http://bioinfo.ggc.org/bindn" target="_blank">http://bioinfo.ggc.org/bindn</a>). The data are presented as percentage of putative DNA binding sites relative to total protein (% DNA binding).</p

Histogram of the leading inverse Fourier-frequency (in bp) for all sequence motifs of length eight (round symbols).

<p>The most pronounced peaks occur at 9,935 bp, 2,138 bp, and 17,020 bp. For comparison we also illustrate the distribution (grey bars) of distances between individual fluorescence maxima from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030133#pone-0030133-g001" target="_blank">Fig. 1H</a>. We assume that 1 kb DNA is in the extended form 0.323 µm long.</p

AFM images of viral DNA and associated proteins, which were isolated by osmotic shock.

<p>Scan of single PBCV-1 particles after osmotic shock in a height image <b>A</b> and in amplitude image <b>B</b>. The images reveal emerging DNA and protein particles from the disrupted virus. Magnification of DNA from disrupted virus with protein particles <b>C</b>. Proteins are absent after the sample was treated with proteinase K <b>D</b>. 3 dimensional image of individual BSA protein <b>E</b> and of individual purified 70 kDa PBCV-1 protein A278L. The latter is a putative DNA-binding protein coded by virus PBCV-1. Scale bars 100 nm in A–D and 2 nm in E and F.</p

Ejection of viral DNA.

<p><b>A:</b> Fluorescence images of <i>C. variabilis</i> with ejected DNA molecules. The incubation medium contained <i>C. variabilis</i> cells and virus PBCV-1 at an m.o.i. of ∼100 plus the fluorescent DNA stain DAPI. The image shows a chlorella cell (cc) and the viral DNA molecule, which is propelled away from the alga cell. <b>B:</b> Magnification of the area indicated by the box in <b>A. Inset:</b> same area as in <b>B</b> with conventional light microscopy and phase contrast. <b>C:</b> same as in <b>A</b> but with two DNA bands projecting away from a chlorella cell (cc). <b>D:</b> Magnification of area indicated by box in <b>C</b> with loop like DNA structure. <b>E:</b> Electron micrograph of viral DNA projecting away from host cell wall. The cell wall of the alga exhibits the typical hole (*), which the viruses digest for infection. From this hole two linear structures project towards the left side. The part marked in <b>E</b> is magnified in <b>F</b> and presented in artificial colors in order to highlight the linear structures projecting away from the cell wall hole. <b>G:</b> fluorescence intensity profile along DNA molecule between arrows in <b>B</b>. <b>H:</b> Histogram of distances between individual fluorescence maxima as in <b>E</b> from 30 ejected DNA molecules.</p