Molecular analysis of soybean dwarf virus isolates in the eastern United States confirms the presence of both D and Y strains and provides evidence of mixed infections and recombination.

Soybean dwarf virus (SbDV), first identified as an agricultural problem in Japan, has emerged as a growing problem in the Midwestern United States. The majority of research on SbDV had been limited to four lab maintained strains from Japan. SbDV had been found in clover in the eastern United States, but these isolates rarely emerged into soybeans. These isolates were analyzed by multiplex PCR and sequencing, revealing that some were infections of both Y and D components, including a recombinant subisolate. Phylogenetic analyses for the US isolates revealed a broad diversity of SbDV, with selection pressure greater on the movement protein than the coat protein. The field isolates from the Eastern United States showed differences in symptoms, aphid transmission and host range, demonstrating that a study of field isolates is an important complement to laboratory maintained strains in understanding the biology and evolution of plant viruses.

Chlorella viruses encode most, if not all, of the machinery to glycosylate their glycoproteins independent of the endoplasmic reticulum and Golgi.

In contrast to all other viruses that use the host machinery located in the endoplasmic reticulum and Golgi to glycosylate their glycoproteins, the large dsDNA-containing chlorella viruses encode most, if not all, of the components to glycosylate their major capsid proteins. Furthermore, all experimental results indicate that glycosylation occurs independent of the endoplasmic reticulum and Golgi.

The A312L 5'-UTR of Chlorella virus PBCV-1 is a translational enhancer in Arabidopsis thaliana.

PBCV-1 (Paramecium bursaria Chlorella virus) is a large double stranded DNA virus that replicates in certain eukaryotic chlorella like green algae. The PBCV-1 A312L gene encodes a 33-kDa protein whose function currently is unknown. The 5'-UTR of the A312L mRNA is 153 nucleotides, longer than the 5'-UTR in any other PBCV-1 gene. The sequence 5'-AAAC was repeated 17 times within 156bp 5' to the A312L gene start codon and this sequence was repeated 13 times continuously in the 5'-UTR of the mRNA. Recombinant genes were constructed in vector pBI121 that contained the A312L 5'-UTR, in both the forward and inverse-complement orientations, fused to the GUS gene under the control of the CaMV 35S promoter. These constructs were introduced into Arabidopsis thaliana and the results indicated that the A312L 5'-UTR functions as a translational enhancer only in the forward orientation. Overall, the ratio of GUS enzyme activity to GUS mRNA was 15-fold higher in constructs derived from the A312L 5'-UTR in the forward orientation as compared to constructs containing the 5'-UTR in the inverse-complement orientation or those lacking the A312L 5'-UTR.

Putative gene promoter sequences in the chlorella viruses.

Three short (7 to 9 nucleotides) highly conserved nucleotide sequences were identified in the putative promoter regions (150 bp upstream and 50 bp downstream of the ATG translation start site) of three members of the genus Chlorovirus, family Phycodnaviridae. Most of these sequences occurred in similar locations within the defined promoter regions. The sequence and location of the motifs were often conserved among homologous ORFs within the Chlorovirus family. One of these conserved sequences (AATGACA) is predominately associated with genes expressed early in virus replication.

Towards predicting protein-protein interactions in novel organisms.

Machine learning methods are often used to predict Protein-Protein Interactions (PPI). It is common to develop methods using known PPI from well-characterised reference organisms, drawing from that organism data for inferring a predictive model and evaluating the model. We present evidence that this practice does not give a meaningful indication of the model's performance on genetically distinct organisms. We conclude that this practice cannot be applied to proteins inferred from the genetic sequence of a novel organism for which no PPI data is available, and that there is need for evaluating such methods on organisms distinct from their training organisms.

Sequence and annotation of the 288-kb ATCV-1 virus that infects an endosymbiotic chlorella strain of the heliozoon Acanthocystis turfacea.

Acanthocystis turfacea chlorella virus (ATCV-1), a prospective member of the family Phycodnaviridae, genus Chlorovirus, infects a unicellular, eukaryotic, chlorella-like green alga, Chlorella SAG 3.83, that is a symbiont in the heliozoon A. turfacea. The 288,047-bp ATCV-1 genome is the first virus to be sequenced that infects Chlorella SAG 3.83. ATCV-1 contains 329 putative protein-encoding and 11 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands and intergenic space is minimal. Thirty-four percent of the viral gene products resemble entries in the public databases, including some that are unexpected for a virus. For example, these unique gene products include ribonucleoside-triphosphate reductase, dTDP-d-glucose 4,6 dehydratase, potassium ion transporter, aquaglyceroporin, and mucin-desulfating sulfatase. Comparison of ATCV-1 protein-encoding genes with the prototype chlorella virus PBCV-1 indicates that about 80% of the ATCV-1 genes are present in PBCV-1.

Sequence and annotation of the 314-kb MT325 and the 321-kb FR483 viruses that infect Chlorella Pbi.

Viruses MT325 and FR483, members of the family Phycodnaviridae, genus Chlorovirus, infect the fresh water, unicellular, eukaryotic, chlorella-like green alga, Chlorella Pbi. The 314,335-bp genome of MT325 and the 321,240-bp genome of FR483 are the first viruses that infect Chlorella Pbi to have their genomes sequenced and annotated. Furthermore, these genomes are the two smallest chlorella virus genomes sequenced to date, MT325 has 331 putative protein-encoding and 10 tRNA-encoding genes and FR483 has 335 putative protein-encoding and 9 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands, and intergenic space is minimal. Approximately 40% of the viral gene products resemble entries in public databases, including some that are the first of their kind to be detected in a virus. For example, these unique gene products include an aquaglyceroporin in MT325, a potassium ion transporter protein and an alkyl sulfatase in FR483, and a dTDP-glucose pyrophosphorylase in both viruses. Comparison of MT325 and FR483 protein-encoding genes with the prototype chlorella virus PBCV-1 indicates that approximately 82% of the genes are present in all three viruses.

Sequence and annotation of the 369-kb NY-2A and the 345-kb AR158 viruses that infect Chlorella NC64A.

Viruses NY-2A and AR158, members of the family Phycodnaviridae, genus Chlorovirus, infect the fresh water, unicellular, eukaryotic, chlorella-like green alga, Chlorella NC64A. The 368,683-bp genome of NY-2A and the 344,690-bp genome of AR158 are the two largest chlorella virus genomes sequenced to date; NY-2A contains 404 putative protein-encoding and 7 tRNA-encoding genes and AR158 contains 360 putative protein-encoding and 6 tRNA-encoding genes. The protein-encoding genes are almost evenly distributed on both strands, and intergenic space is minimal. Two of the NY-2A genes encode inteins, the large subunit of ribonucleotide reductase and a superfamily II helicase. These are the first inteins to be detected in the chlorella viruses. Approximately 40% of the viral gene products resemble entries in the public databases, including some that are unexpected for a virus. These include GDP-d-mannose dehydratase, fucose synthase, aspartate transcarbamylase, Ca(++) transporting ATPase and ubiquitin. Comparison of NY-2A and AR158 protein-encoding genes with the prototype chlorella virus PBCV-1 indicates that 85% of the genes are present in all three viruses.

The structure and evolution of the major capsid protein of a large, lipid-containing DNA virus.

Paramecium bursaria Chlorella virus type 1 (PBCV-1) is a very large, icosahedral virus containing an internal membrane enclosed within a glycoprotein coat consisting of pseudohexagonal arrays of trimeric capsomers. Each capsomer is composed of three molecules of the major capsid protein, Vp54, the 2.0-A resolution structure of which is reported here. Four N-linked and two O-linked glycosylation sites were identified. The N-linked sites are associated with nonstandard amino acid motifs as a result of glycosylation by virus-encoded enzymes. Each monomer of the trimeric structure consists of two eight-stranded, antiparallel beta-barrel, "jelly-roll" domains related by a pseudo-sixfold rotation. The fold of the monomer and the pseudo-sixfold symmetry of the capsomer resembles that of the major coat proteins in the double-stranded DNA bacteriophage PRD1 and the double-stranded DNA human adenoviruses, as well as the viral proteins VP2-VP3 of picornaviruses. The structural similarities among these diverse groups of viruses, whose hosts include bacteria, unicellular eukaryotes, plants, and mammals, make it probable that their capsid proteins have evolved from a common ancestor that had already acquired a pseudo-sixfold organization. The trimeric capsid protein structure was used to produce a quasi-atomic model of the 1,900-A diameter PBCV-1 outer shell, based on fitting of the Vp54 crystal structure into a three-dimensional cryoelectron microscopy image reconstruction of the virus.