scholarly journals Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

2019 ◽  
Author(s):  
Samantha R. Coy ◽  
Alyssa N. Alsante ◽  
James L. Van Etten ◽  
Steven W. Wilhelm
Keyword(s):  
Post Infection ◽  
Laboratory Culture ◽  
Paramecium Bursaria ◽  
Infection Cycle ◽  
Active Infection ◽  
Virion Assembly ◽  
Virus Particles ◽  
Chlorella Virus ◽  
Chlorella Variabilis ◽  
Reliable Mechanism

AbstractBest practices in laboratory culture management often include cryopreservation of microbiota, but this can be challenging with some virus particles. By preserving viral isolates researchers can mitigate genetic drift and laboratory-induced selection, thereby maintaining genetically consistent strains between experiments. To this end, we developed a method to cryopreserve the model, green-alga infecting virus,Paramecium bursaria Chlorella virus 1(PBCV-1). We explored cryotolerance of the infectivity of this virus particle, whereby freezing without cryoprotectants was found to maintain the highest infectivity (~2.5%). We then assessed the cryopreservation potential of PBCV-1 during an active infection cycle in itsChlorella variabilisNC64A host, and found that virus survivorship was highest (69.5 ± 16.5 %) when the infected host is cryopreserved during mid-late stages of infection (i.e., coinciding with virion assembly). The most optimal condition for cryopreservation was observed at 240 minutes post-infection. Overall, utilizing the cell as a vehicle for viral cryopreservation resulted in 24.9 – 30.1 fold increases in PBCV-1 survival based on 95% confidence intervals of frozen virus particles and virus cryopreserved at 240 minutes post-infection. Given that cryoprotectants are often naturally produced by psychrophilic organisms, we suspect that cryopreservation of infected hosts may be a reliable mechanism for virus persistence in non-growth permitting circumstances in the environment, such as ancient permafrosts.

scholarly journals Cryopreservation of Paramecium bursaria Chlorella Virus-1 during an active infection cycle of its host

PLoS ONE ◽  
2019 ◽  
Vol 14 (3) ◽  
pp. e0211755 ◽  
Author(s):  
Samantha R. Coy ◽  
Alyssa N. Alsante ◽  
James L. Van Etten ◽  
Steven W. Wilhelm
Keyword(s):  
Paramecium Bursaria ◽  
Infection Cycle ◽  
Active Infection ◽  
Chlorella Virus

scholarly journals Global Analysis of Chlorella variabilis NC64A mRNA Profiles during the Early Phase of Paramecium bursaria Chlorella Virus-1 Infection

PLoS ONE ◽  
2014 ◽  
Vol 9 (3) ◽  
pp. e90988 ◽  
Author(s):  
Janet M. Rowe ◽  
Adrien Jeanniard ◽  
James R. Gurnon ◽  
Yuannan Xia ◽  
David D. Dunigan ◽  
...  
Keyword(s):  
Early Phase ◽  
Global Analysis ◽  
Paramecium Bursaria ◽  
Chlorella Virus ◽  
Chlorella Variabilis

scholarly journals Paramecium bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural and Regulatory Features of a Giant Virus

2012 ◽  
Vol 86 (16) ◽  
pp. 8821-8834 ◽  
Author(s):  
David D. Dunigan ◽  
Ronald L. Cerny ◽  
Andrew T. Bauman ◽  
Jared C. Roach ◽  
Leslie C. Lane ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Chromatin Modification ◽  
Proteome Analysis ◽  
Virus Genome ◽  
Host Protein ◽  
Paramecium Bursaria ◽  
Content Type ◽  
Chlorella Virus ◽  
Protein Size ◽  
Chlorella Variabilis

The 331-kbp chlorovirusParamecium bursariachlorella virus 1 (PBCV-1) genome was resequenced and annotated to correct errors in the original 15-year-old sequence; 40 codons was considered the minimum protein size of an open reading frame. PBCV-1 has 416 predicted protein-encoding sequences and 11 tRNAs. A proteome analysis was also conducted on highly purified PBCV-1 virions using two mass spectrometry-based protocols. The mass spectrometry-derived data were compared to PBCV-1 and its hostChlorella variabilisNC64A predicted proteomes. Combined, these analyses revealed 148 unique virus-encoded proteins associated with the virion (about 35% of the coding capacity of the virus) and 1 host protein. Some of these proteins appear to be structural/architectural, whereas others have enzymatic, chromatin modification, and signal transduction functions. Most (106) of the proteins have no known function or homologs in the existing gene databases except as orthologs with proteins of other chloroviruses, phycodnaviruses, and nuclear-cytoplasmic large DNA viruses. The genes encoding these proteins are dispersed throughout the virus genome, and most are transcribed late or early-late in the infection cycle, which is consistent with virion morphogenesis.

scholarly journals A virus-encoded potassium ion channel is a structural protein in the chlorovirus Paramecium bursaria chlorella virus 1 virion

2013 ◽  
Vol 94 (11) ◽  
pp. 2549-2556 ◽  
Author(s):  
Giulia Romani ◽  
Adrianna Piotrowski ◽  
Stefan Hillmer ◽  
James Gurnon ◽  
James L. Van Etten ◽  
...  
Keyword(s):  
Lipid Membrane ◽  
Experimental Finding ◽  
Cross Reactivity ◽  
Paramecium Bursaria ◽  
Structural Protein ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Chlorella Virus ◽  
Viral Particles ◽  
Extracellular Side

Most chloroviruses encode small K+ channels, which are functional in electrophysiological assays. The experimental finding that initial steps in viral infection exhibit the same sensitivity to channel inhibitors as the viral K+ channels has led to the hypothesis that the channels are structural proteins located in the internal membrane of the virus particles. This hypothesis was questioned recently because proteomic studies failed to detect the channel protein in virions of the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1). Here, we used a mAb raised against the functional K+ channel from chlorovirus MA-1D to search for the viral K+ channel in the virus particle. The results showed that the antibody was specific and bound to the tetrameric channel on the extracellular side. The antibody reacted in a virus-specific manner with protein extracts from chloroviruses that encoded channels similar to that from MA-1D. There was no cross-reactivity with chloroviruses that encoded more diverse channels or with a chlorovirus that lacked a K+ channel gene. Together with electron microscopic imaging, which revealed labelling of individual virus particles with the channel antibody, these results establish that the viral particles contain an active K+ channel, presumably located in the lipid membrane that surrounds the DNA in the mature virions.

The Fine Structure of Replicating Simian Adenoviruses in LLC-MK2

1968 ◽  
Vol 26 ◽  
pp. 220-221
Author(s):  
Matias Pardo ◽  
Malcolm Slifkin ◽  
Leonard Merkow ◽  
Marie Sanchez
Keyword(s):  
Tissue Culture ◽  
Post Infection ◽  
Cesium Chloride ◽  
Longitudinal Section ◽  
Tubular Structures ◽  
Virus Particles ◽  
Ultrastructural Studies ◽  
Culture Cells ◽  
Simian Adenovirus ◽  
Infectivity Titer

The simian adenoviruses SV20, SV30 and SA7 have been found to be oncogenic in the Syrian hamster. The growth characteristics and replicative cycle of these viruses in tissue culture therefore appeared appropriate to investigate. Cesium chloride purified simian adenovirus with an infectivity titer of 100 TCID50, was inoculated into monolayers of LLC-MK2 cells. Cells were fixed in osmium tetroxide and embedded for ultrastructural studies at 1, 3, 6, 9, 18, 24, 48, 72, 120 and 192 hours post-infection.At the first hour post-infection, virus particles were adsorbed to the plasmalemma and found within the peripheral cytoplasm of many LLC-MK2 cells (Fig. 1). Although the first detection of infectious virus occurred at 14 hours and infectivity titers did not reach a maximum until 30 hours, intranuclear virus particles were observed by 3 hours in typical adenovirus crystalline array (Fig. 2) by means of electron microscopy. These typical honeycomb arrayed virus particles at 3 hours provided evidence of significant replication in approximately 5 percent of tissue culture cells examined. Simultaneously, a classical nuclear inclusion manifested by peripheral condensation of nuclear chromatin was evident by light microscopy. As early at 6 to 9 hours, unusual intranuclear concentric membranes formed “tubes” which contained linear arranged virus particles (Fig. 3). In transverse or tangential sections, these “tubes” appeared cochlear-like in shape. In longitudinal section, these intranuclear tubular structures contained individual virus particles at various stages of maturation in a linear arranged order. This arrangement resembled “peas in a pod”.

scholarly journals ER-Shaping Atlastin Proteins Act as Central Hubs to Promote Flavivirus Replication and Virion Assembly

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 31
Author(s):  
Christopher J Neufeldt ◽  
Mirko Cortese ◽  
Pietro Scaturro ◽  
Berati Cerikan ◽  
Jeremy Wideman ◽  
...  
Keyword(s):  
Viral Genome ◽  
Viral Assembly ◽  
Host Factors ◽  
Genome Replication ◽  
Membrane Structures ◽  
Virion Assembly ◽  
Virus Particles ◽  
Membrane Invagination ◽  
Viral Genome Replication ◽  
Flavivirus Infection

Members of the Flavivirus genus rely extensively on the host cell endomembrane network to generate complex membranous replication organelles (ROs) that facilitate viral genome replication and the production of virus particles. For dengue virus and Zika virus, these ROs included vesicles which are formed through membrane invagination into the endoplasmic reticulum (ER) lumen, termed invaginated vesicles or vesicle packets (VPs), as well as large areas of bundled smooth ER, termed convoluted membranes. Though the morphology of these virus-induced membrane structures has been well characterized, the viral and host constituents that make up flaviviral ROs are still poorly understood. Here, we identified a subset of ER resident proteins (atlastins), normally required for maintaining ER tubule networks, as critical host factors for flavivirus infection. Specific changes in atlastin (ATL) levels had dichotomous effects on flaviviruses with ATL2 depletion, leading to replication organelle defects and ATL3 depletion to changes in viral assembly/release pathways. These different depletion phenotypes allowed us to exploit virus infection to characterize non-conserved functional domains between the three atlastin paralogues. Additionally, we established the ATL interactome and show how it is reprogrammed upon viral infection. Screening of specific ATL interactors confirmed non-redundant ATL functions and identified a role for ATL3 in vesicle trafficking. Our data demonstrate that ATLs are central host factors that coordinate the ER network and shape the ER during flavivirus infection.

scholarly journals Microarray Analysis of Paramecium bursaria Chlorella Virus 1 Transcription

2009 ◽  
Vol 84 (1) ◽  
pp. 532-542 ◽  
Author(s):  
Giane M. Yanai-Balser ◽  
Garry A. Duncan ◽  
James D. Eudy ◽  
Dong Wang ◽  
Xiao Li ◽  
...  
Keyword(s):  
Life Cycle ◽  
Virus Infection ◽  
Dna Synthesis ◽  
Comprehensive Evaluation ◽  
Paramecium Bursaria ◽  
Chlorella Virus ◽  
Double Stranded Dna ◽  
Unicellular Green Alga ◽  
Protein Encoding ◽  
Encoding Genes

ABSTRACT Paramecium bursaria chlorella virus 1 (PBCV-1), a member of the family Phycodnaviridae, is a large double-stranded DNA, plaque-forming virus that infects the unicellular green alga Chlorella sp. strain NC64A. The 330-kb PBCV-1 genome is predicted to encode 365 proteins and 11 tRNAs. To monitor global transcription during PBCV-1 replication, a microarray containing 50-mer probes to the PBCV-1 365 protein-encoding genes (CDSs) was constructed. Competitive hybridization experiments were conducted by using cDNAs from poly(A)-containing RNAs obtained from cells at seven time points after virus infection. The results led to the following conclusions: (i) the PBCV-1 replication cycle is temporally programmed and regulated; (ii) 360 (99%) of the arrayed PBCV-1 CDSs were expressed at some time in the virus life cycle in the laboratory; (iii) 227 (62%) of the CDSs were expressed before virus DNA synthesis begins; (iv) these 227 CDSs were grouped into two classes: 127 transcripts disappeared prior to initiation of virus DNA synthesis (considered early), and 100 transcripts were still detected after virus DNA synthesis begins (considered early/late); (v) 133 (36%) of the CDSs were expressed after virus DNA synthesis begins (considered late); and (vi) expression of most late CDSs is inhibited by adding the DNA replication inhibitor, aphidicolin, prior to virus infection. This study provides the first comprehensive evaluation of virus gene expression during the PBCV-1 life cycle.

scholarly journals Duplication of the Primary Encapsidation and Dimer Linkage Region of Human Immunodeficiency Virus Type 1 RNA Results in the Appearance of Monomeric RNA in Virions

2001 ◽  
Vol 75 (6) ◽  
pp. 2557-2565 ◽  
Author(s):  
Jun-ichi Sakuragi ◽  
Tatsuo Shioda ◽  
Antonito T. Panganiban
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Intramolecular Interaction ◽  
Genomic Rna ◽  
Virion Assembly ◽  
Virus Particles ◽  
Rna Dimerization ◽  
Immunodeficiency Virus ◽  
Dimer Linkage

ABSTRACT The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed whenpol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.

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