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Initial Events Associated With Virus Pbcv-1 Infection Of Chlorella Nc64a, Gerhard Thiel, Anna Moroni, David Dunigan, James L. Van Etten Oct 2013

Initial Events Associated With Virus Pbcv-1 Infection Of Chlorella Nc64a, Gerhard Thiel, Anna Moroni, David Dunigan, James L. Van Etten

David D Dunigan Ph. D.

Chlorella viruses (or chloroviruses) are very large, plaque-forming viruses. The viruses are multilayered structures containing a large double-stranded DNA genome, a lipid bilayered membrane, and an outer icosahedral capsid shell. The viruses replicate in certain isolates of the coccal green alga, Chlorella. Sequence analysis of the 330-kbp genome of Paramecium bursaria Chlorella virus 1 (PBCV-1), the prototype of the virus family Phycodnaviridae, reveals <365 protein-encoding genes and 11 tRNA genes. Products of about 40% of these genes resemble proteins of known function, including many that are unexpected for a virus. Among these is a virus-encoded protein, called Kcv, which forms …


Paramecium Bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural And Regulatory Features Of A Giant Virus, David Dunigan, Ronald Cerny, Andrew T. Bauman, Jared C. Roach, Leslie C. Lane, Irina V. Agarkova, Kurt William Wulser, Giane M. Yanai-Balser, James R. Gurnon, Jason C. Vitek, Bernard J. Kronschnabel, Adrien Jeannard, Guillaume Blanc, Chris Upton, Gary Duncan, O. William Mcclung, Fangrui Ma, James L. Van Etten Oct 2013

Paramecium Bursaria Chlorella Virus 1 Proteome Reveals Novel Architectural And Regulatory Features Of A Giant Virus, David Dunigan, Ronald Cerny, Andrew T. Bauman, Jared C. Roach, Leslie C. Lane, Irina V. Agarkova, Kurt William Wulser, Giane M. Yanai-Balser, James R. Gurnon, Jason C. Vitek, Bernard J. Kronschnabel, Adrien Jeannard, Guillaume Blanc, Chris Upton, Gary Duncan, O. William Mcclung, Fangrui Ma, James L. Van Etten

David D Dunigan Ph. D.

The 331 kilobase pairs chlorovirus PBCV-1 genome was re-sequenced and annotated to correct errors in the original 15 year old sequence; forty codons was considered the minimum protein size of an open reading frame. PBCV-1 encodes 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 host Chlorella variabilis NC64A 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 one host …


Virion-Associated Restriction Endonucleases Of Chloroviruses, Irina V. Agarkova, David Dunigan, James L. Van Etten Oct 2013

Virion-Associated Restriction Endonucleases Of Chloroviruses, Irina V. Agarkova, David Dunigan, James L. Van Etten

David D Dunigan Ph. D.

Chloroviruses are large, double-stranded-DNA, plaque-forming viruses that infect certain eukaryotic chlorella- like green algae. The prototype of the genus is Paramecium bursaria chlorella virus 1 (PBCV-1). Chlorovirus genomes contain various amounts of methylated nucleotides due to virus-encoded DNA methyltransferases (MTases); about 25% of the MTases are associated with companion DNA site-specific (restriction) endonucleases (REases). These enzymes constitute virally encoded restriction-modification (R/M) systems. Although several of the chlorovirus R/M systems are characterized, their biological functions are unknown. The PBCV-1 proteome reveals that two virus-encoded REases, but not their companion MTases, are virion associated, suggesting that viral REases might help degrade the …


Chlorovirus: A Genus Of Phycodnaviridae That Infects Certain Chlorella-Like Green Algae, Ming Kang, David Dunigan, James L. Van Etten Oct 2013

Chlorovirus: A Genus Of Phycodnaviridae That Infects Certain Chlorella-Like Green Algae, Ming Kang, David Dunigan, James L. Van Etten

David D Dunigan Ph. D.

Taxonomy: Chlorella viruses are assigned to the family Phycodnaviridae, genus Chlorovirus, and are divided into three species: Chlorella NC64A viruses, Chlorella Pbi viruses and Hydra viridis Chlorella viruses. Chlorella viruses are large, icosahedral, plaque-forming, dsDNA viruses that infect certain unicellular, chlorella-like green algae. The type member is Paramecium bursaria chlorella virus 1 (PBCV-1).

Physical properties: Chlorella virus particles are large (molecular weight ~1 × 109 Da) and complex. The virion of PBCV-1 contains more than 100 different proteins; the major capsid protein, Vp54, comprises ~40% of the virus protein. Cryoelectron microscopy and three-dimensional image reconstruction of PBCV-1 virions …


Evaluation Of Higher Plant Virus Resistance Genes In The Green Alga, Chlorella Variabilis Nc64a, During The Early Phase Of Infection With Paramecium Bursaria Chlorella Virus-1, Janet M. Rowe, David D. Dunigan, Guillaume Blanc, James R. Gurnon, Yuannan Xia, James L. Van Etten Oct 2013

Evaluation Of Higher Plant Virus Resistance Genes In The Green Alga, Chlorella Variabilis Nc64a, During The Early Phase Of Infection With Paramecium Bursaria Chlorella Virus-1, Janet M. Rowe, David D. Dunigan, Guillaume Blanc, James R. Gurnon, Yuannan Xia, James L. Van Etten

David D Dunigan Ph. D.

With growing industrial interest in algae plus their critical roles in aquatic systems, the need to understand the effects of algal pathogens is increasing. We examined a model algal host–virus system, Chlorella variabilis NC64A and virus, PBCV-1. C. variabilis encodes 375 homologs to genes involved in RNA silencing and in response to virus infection in higher plants. Illumina RNA-Seq data showed that 325 of these homologs were expressed in healthy and early PBCV-1 infected (≤60 min) cells. For each of the RNA silencing genes to which homologs were found, mRNA transcripts were detected in healthy and infected cells. C. variabilis, …


Phycodnaviruses: A Peek At Genetic Diversity, David Dunigan, Lisa Fitzgerald, James L. Van Etten Oct 2013

Phycodnaviruses: A Peek At Genetic Diversity, David Dunigan, Lisa Fitzgerald, James L. Van Etten

David D Dunigan Ph. D.

The family Phycodnaviridae encompasses a diverse collection of large icosahedral, dsDNA viruses infecting algae. These viruses have genomes ranging from 160 to 560 kb. The family consists of six genera based initially on host range and supported by sequence comparisons. The family is monophyletic with branches for each genus, but the phycodnaviruses have evolutionary roots that connect with several other families of large DNA viruses, referred to as the nucleocytoplasmic large DNA viruses (NCLDV). The genomes of members in three genera in the Phycodnaviridae have recently been sequenced and the purpose of this manuscript is to summarize these data. The …


Microarray Analysis Of Paramecium Bursaria Chlorella Virus 1 Transcription, Giane M. Yanai-Balser, Garry A. Duncan, James D. Eudy, Dong Wang, Xiao Li, Irina V. Agarkova, David Dunigan, James L. Van Etten Oct 2013

Microarray Analysis Of Paramecium Bursaria Chlorella Virus 1 Transcription, Giane M. Yanai-Balser, Garry A. Duncan, James D. Eudy, Dong Wang, Xiao Li, Irina V. Agarkova, David Dunigan, James L. Van Etten

David D Dunigan Ph. D.

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 …


Fungal Symbiosis From Mutualism To Parasitism: Who Controls The Outcome, Host Or Invader?, Regina S. Redman, David Dunigan, Rusty J. Rodriguez Oct 2013

Fungal Symbiosis From Mutualism To Parasitism: Who Controls The Outcome, Host Or Invader?, Regina S. Redman, David Dunigan, Rusty J. Rodriguez

David D Dunigan Ph. D.

• Plant symbiotic fungi are generally thought to express a single lifestyle that might increase (mutualism), decrease (parasitism), or have no influence (commensalism) on host fitness. However, data are presented here demonstrating that plant pathogenic Colletotrichum species are able to asymptomatically colonize plants and express nonpathogenic lifestyles. • Experiments were conducted in growth chambers and plant colonization was assessed by emergence of fungi from surface sterilized plant tissues. Expression of symbiotic lifestyles was assessed by monitoring the ability of fungi to confer disease resistance, drought tolerance and growth enhancement. • Several pathogenic Colletotrichum species expressed either mutualistic or commensal lifestyles …


Three-Dimensional Structure And Function Of The Paramecium Bursaria Chlorella Virus Capsid, Xinzheng Zhang, Ye Xiang, David Dunigan, Thomas Klose, Paul R. Chipman, James L. Van Etten, Michael G. Rossmann Oct 2013

Three-Dimensional Structure And Function Of The Paramecium Bursaria Chlorella Virus Capsid, Xinzheng Zhang, Ye Xiang, David Dunigan, Thomas Klose, Paul R. Chipman, James L. Van Etten, Michael G. Rossmann

David D Dunigan Ph. D.

A cryoelectron microscopy 8.5 Å resolution map of the 1,900 Å diameter, icosahedral, internally enveloped Paramecium bursaria chlorella virus was used to interpret structures of the virus at initial stages of cell infection. A fivefold averaged map demonstrated that two minor capsid proteins involved in stabilizing the capsid are missing in the vicinity of the unique vertex. Reconstruction of the virus in the presence of host chlorella cell walls established that the spike at the unique vertex initiates binding to the cell wall, which results in the enveloped nucleocapsid moving closer to the cell. This process is concurrent with the …


‘Strawberry Festival’ Strawberry, C. K. Chandler, D. E. Legard, David Dunigan, T. E. Crocker, C. A. Sims Oct 2013

‘Strawberry Festival’ Strawberry, C. K. Chandler, D. E. Legard, David Dunigan, T. E. Crocker, C. A. Sims

David D Dunigan Ph. D.

Most of the strawberries (Fragaria xananassa Duchesne) produced in Florida are shipped fresh to locations throughout the eastern United States and Canada (Florida Agricultural Statistics [www.nass.usda.gov/ fl]). Therefore, Florida growers need cultivars that produce fruit that are attractive and flavorful, and maintain these qualities during and after long-distance shipment. 'Strawberry Festival' strawberry has produced commercially acceptable yields of firm, attractive, and flavorful fruit in trials at the Univ. of Florida's Gulf Coast Research and Education Center in Dover (GCREC-Dover) and in two commercial fields in west central Florida. It is recommended for trial in areas where strawberries are grown in …


Chlorovirus-Mediated Membrane Depolarization Of Chlorella Alters Secondary Active Transport Of Solutes, Irina V. Agarkova, David Dunigan, James R. Gurnon, Timo Greiner, Julia Barres, Gerhard Thiel, James L. Van Etten Oct 2013

Chlorovirus-Mediated Membrane Depolarization Of Chlorella Alters Secondary Active Transport Of Solutes, Irina V. Agarkova, David Dunigan, James R. Gurnon, Timo Greiner, Julia Barres, Gerhard Thiel, James L. Van Etten

David D Dunigan Ph. D.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding …


Chloroviruses Encode A Bifunctional Dcmp-Dctp Deaminase That Produces Two Key Intermediates In Dttp Formation, Yuanzheng Zhang, Frank Maley, Gladys F. Maley, Garry Duncan, David Dunigan, James L. Van Etten Oct 2013

Chloroviruses Encode A Bifunctional Dcmp-Dctp Deaminase That Produces Two Key Intermediates In Dttp Formation, Yuanzheng Zhang, Frank Maley, Gladys F. Maley, Garry Duncan, David Dunigan, James L. Van Etten

David D Dunigan Ph. D.

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments …


Capping Of Tobacco Mosaic Virus Rna: Analysis Of Viral-Coded Guanylyltransferase-Like Activity, David Dunigan, Milton Zaitlin Oct 2013

Capping Of Tobacco Mosaic Virus Rna: Analysis Of Viral-Coded Guanylyltransferase-Like Activity, David Dunigan, Milton Zaitlin

David D Dunigan Ph. D.

The 5’ end of tobacco mosaic virus (TMV) genomic RNA is capped with 7- methylguanosine. A virus-coded polypeptide with guanylyltransferase activity has been investigated. This enzyme is responsible for forming the 5’→5’ linkage of guanosine 5’-monophosphate to the 5’- diphosphate of an acceptor RNA, thereby forming the cap. A critical step in the mechanism for cap formation in the eukaryotic nucleus is for guanylyltransferase to bind covalently to guanosine 5’- monophosphate with the hydrolysis of pyrophosphate when guanosine 5’- triphosphate is the substrate. The TMV 126-kilodalton protein, which is most probably a component of the TMV replicase, was found to …


Cloning Of Nt.Cviqii Nicking Endonuclease And Its Cognate Methyltransferase: M.Cviqii Methylates Ag Sequence, Siu-Hong Chan, Zhenyu Zhu, David Dunigan, James L. Van Etten, Shuang-Yong Xu Oct 2013

Cloning Of Nt.Cviqii Nicking Endonuclease And Its Cognate Methyltransferase: M.Cviqii Methylates Ag Sequence, Siu-Hong Chan, Zhenyu Zhu, David Dunigan, James L. Van Etten, Shuang-Yong Xu

David D Dunigan Ph. D.

Chlorella virus NY-2A has a large, highly methylated dsDNA genome (45% of the cytosines are 5-methylcytosine and 37% of the adenines are N6-methyladenine). Here, we report the cloning, expression, and characterization of the NY-2A-encoded CviQII nicking-modification (N-M) system. The nicking endonuclease, Nt.CviQII, recognizes R ↓ AG (R = A or G, ↓ indicating cleavage site) sequences and cleaves the phosphodiester bond 5′ to the adenosine. Because of the difficulty in cloning and expressing the wild-type Nt.CviQII, C-terminal truncation mutants were generated and full-length Nt.CviQII was reconstructed by intein-mediated peptide ligation. The truncation mutants and the reconstructed full-length Nt.CviQII have the …