Ranjit Dasgupta

Structure of the black beetle virus genome and its functional implications

Abstract The black beetle virus (BBV) is an isometric insect virus whose genome consists of two messenger-active RNA molecules encapsidated in a single virion. The nucleotide sequence of BBV RNA1 (3105 bases) has been determined, and this, together with the sequence of BBV RNA2 (1399 bases) provides the complete primary structure of the BBV genome. The RNA1 sequence encompasses a 5′ non-coding region of 38 nucleotides, a coding region for a protein of predicted molecular weight 101,873 (protein A, implicated in viral RNA synthesis) and a 3′ proximal region encoding RNA3 (389 bases), a subgenomic messenger RNA made in infected cells but not encapsidated into virions. The RNA3 sequence starts 16 bases inside the coding region of protein A and contains two overlapping open reading frames for proteins of molecular weight 10,760 and 11,633, one of which is believed to be protein B, made in BBV-infected cells. A limited homology exists between the sequences of RNA1 and RNA2. Sequence regions have been identified that provide energetically favorable bonding between RNA2 and RNA1 possibly to facilitate their common encapsidation, and between RNA2 and negative strand RNA1 possibly to regulate the production of RNA3.

Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography

The genomic RNA2s of nodaviruses encode a single gene, that of protein alpha, the precursor of virion proteins beta and gamma. We compared the sequences of the RNA2s of the nodaviruses, black beetle virus (BBV), flock house virus, boolarra virus and nodamura virus, with the objective of identifying homologies in the primary and secondary structure of these RNAs and in the structure of their encoded protein. The sequences of the four RNAs were found to be similar, so that homologous regions relating to translation and RNA replication were readily identified. However, the overall, secondary structures in solution, deduced from calculations of optimal Watson-Crick base-pairing configurations, were very different for the four RNAs. We conclude that a particular, overall, secondary structure in solution within host cells is not required for virus viability. The partially refined X-ray structure of BBV (R = 26.4% for the current model) was used as a framework for comparing the structure of the encoded proteins of the four viruses. Mapping of the four protein sequences onto the BBV capsid showed many amino acid differences on the outer surface, indicating that the exteriors of the four virions are substantially different. Mapping in the beta-barrel region showed an intermediate level of differences, indicating that some freedom in choice of amino acid residues is possible there although the basic framework of the capsids is evidently conserved. Mapping onto the interior surface of the BBV capsid showed a high degree of conservation of amino acid residues, particularly near the protein cleavage site, implying that that region is nearly identical in all four virions and has an essential role in virion maturation, and also suggests that all four capsid interior surfaces have similar surfaces exposed to the viral RNA. Apart from a small portion of the C promoter, the amino terminus of the BBV protein (residues 1 to 60) is crystallographically disordered and the amino acid residues in that region are not well conserved. The disordered portion of the BBV protein clearly projects from the capsid inner surface into the interior of the virion, the region occupied by the viral RNA. In all four viruses, residues 1 to 60 had a high proportion of basic residues, suggesting a virus-specific interaction of the amino terminus with the virion RNA.

Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegypti

Glucosamine:fructose‐6‐phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6‐phosphate and is the first and rate‐limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP‐N‐acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat‐1) and its 5′ regulatory region from Aedes aegypti. There is no intron in AeGfat‐1 and there are two potential transcription start sites. AeGfat‐1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat‐1 message is constitutively expressed but is gradually up‐regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT‐1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids.

Replication of Flock House Virus in Three Genera of Medically Important Insects

Abstract Flock House Virus (family Nodaviridae, genus Alphanodavirus, FHV) was originally isolated from grass grubs Costelytra zealandica (White) (Coleoptera: Scarabaeidae) in New Zealand and belongs to a family of divided genome, plus-sense RNA insect viruses. FHV replicates in insects, a nematode, plants, and yeast. We previously reported replication of FHV in four genera of mosquitoes and expression of green fluorescent protein in Aedes aegypti (L.) produced by an FHV-based vector. We report here that FHV multiplies vigorously in vivo in the malaria vectors Anopheles gambiae Giles and An. stephensi Liston and in vitro in a cell line derived from An. gambiae. In addition, FHV multiplies extensively in two other medically important insects, the tsetse fly, Glossina morsitans morsitans Westwood, and the reduviid bug Rhodnius prolixus Stal, extending its host range to four orders of insects (Coleoptera, Lepidoptera, Diptera, and Hemiptera). The virus disseminates in all the major tissues of the insects studied. Anopheles and Glossina show mortality when FHV is injected at a dose above 104 plaque-forming units (pfu) or the virus accumulates to titer above 108 pfu. A lower dose (103 pfu) promotes more extensive virus multiplication and reduces mortality to <10%. No adverse effects are observed in Ae. aegypti, Culex pipiens pipiens L., and Armigeres subalbatus (Coquillett), when injected with a dose of up to 107 pfu. Mosquitoes orally fed with FHV exhibited slower virus growth rate with lower mortality. Our results indicate that FHV has uniquely broad insect host range and that the virus can be used to study virus host interactions in a variety of medically important insects.

Similarity in structure and function of the 3′-terminal region of the four brome mosaic viral RNAs☆

Abstract A 3′-terminal fragment, about 160 nucleotides long, was cleaved by limited nuclease digestion from each of the four RNA components of brome mosaic virus, and purified by two cycles of gel electrophoresis. These fragments accepted tyrosine in reactions catalyzed by wheat germ aminoacyl-tRNA synthetase. Analyses of nuclease digests suggested that the sequences of the fragments from brome mosaic virus RNA 3 and 4 were identical and that the fragments from RNA 1 and 2 differed from that of RNA 4 only in the positions of two and one nucleotides, respectively. A fragment isolated in a similar way from cowpea chlorotic mottle virus was similar in size to the brome mosaic virus RNA fragments, accepted tyrosine in the presence of wheat germ aminoacyl-tRNA synthetase, but had a substantially different nucleotide sequence.

Seed Transmission of Soybean vein necrosis virus: The First Tospovirus Implicated in Seed Transmission

Soybean vein necrosis virus (SVNV; genus Tospovirus; Family Bunyaviridae) is a negative-sense single-stranded RNA virus that has been detected across the United States and in Ontario, Canada. In 2013, a seed lot of a commercial soybean variety (Glycine max) with a high percentage of discolored, deformed and undersized seed was obtained. A random sample of this seed was planted in a growth room under standard conditions. Germination was greater than 90% and the resulting seedlings looked normal. Four composite samples of six plants each were tested by reverse transcription polymerase chain reaction (RT-PCR) using published primers complimentary to the S genomic segment of SVNV. Two composite leaflet samples retrieved from seedlings yielded amplicons with a size and sequence predictive of SVNV. Additional testing of twelve arbitrarily selected individual plants resulted in the identification of two SVNV positive plants. Experiments were repeated by growing seedlings from the same seed lot in an isolated room inside a thrips-proof cage to further eliminate any external source of infection. Also, increased care was taken to reduce any possible PCR contamination. Three positive plants out of forty-eight were found using these measures. Published and newly designed primers for the L and M RNAs of SVNV were also used to test the extracted RNA and strengthen the diagnosis of viral infection. In experiments, by three scientists, in two different labs all three genomic RNAs of SVNV were amplified in these plant materials. RNA-seq analysis was also conducted using RNA extracted from a composite seedling sample found to be SVNV-positive and a symptomatic sample collected from the field. This analysis revealed both sense and anti-sense reads from all three gene segments in both samples. We have shown that SVNV can be transmitted in seed to seedlings from an infected seed lot at a rate of 6%. To our knowledge this is the first report of seed-transmission of a Tospovirus.

Three regulatory regions of the Aedes aegypti glutamine synthetase gene differentially regulate expression: identification of a crucial regulator in the first exon

Aedes aegypti glutamine synthetase (GS) is expressed constitutively at various developmental stages and its relative mRNA abundance increases in the midgut following blood feeding in support of the biosynthesis of chitin, a component of the peritrophic matrix. To understand the regulation of GS expression better, GS‐luciferase reporter fusion genes were constructed and analysed in transiently transfected C6/36 cells. These studies have identified three GS regions: GS‐A, ‐B and ‐C (C1, C2) that are required for efficient transcription. The crucial regulatory DNA sequence is located within 140 nucleotides of the GS‐C region in the first exon. GS‐B region between −209 and +4 contains a negative modulator that represses transcription of the GS‐C promoter, but the 5′‐GS‐A region, between −476 and −282, can negate the transcription inhibition of GS‐B and promote GS transcription of the GS‐C promoter. Electrophoretic mobility shift assays showed that nuclear proteins for GS‐A, GS‐B and GS‐C1 are present in the C6/36 cells, and therefore that GS‐A, GS‐B and GS‐C1 indeed possess regulatory function. By contrast, nuclear proteins isolated from both cultured cells and midgut tissues bound to GS‐C2, suggesting that GS‐C2 plays an important role in GS transcription and that GS‐C2 is regulated by several different and redundant transcription factors to achieve constitutive expression in a wide variety of tissues.

Re-emergence of Tobacco streak virus Infecting Soybean in the United States and Canada

Melissa D. Irizarry, Department of Plant Pathology, Iowa State University, Ames 50011; Carol L. Groves, Department of Plant Pathology, University of Wisconsin-Madison 53706; Manjula G. Elmore, Department of Plant Pathology, Iowa State University, Ames 50011; Carl A. Bradley, Department of Crop Sciences, University of Illinois, Urbana 61801 (current address: Department of Plant Pathology, University of Kentucky, Princeton 42445); Ranjit Dasgupta and Thomas L. German, Department of Entomology, University of WisconsinMadison 53706; Douglas J. Jardine, Department of Plant Pathology, Kansas State University, Manhattan 66506; Erika Saalau Rojas, Department of Plant Pathology, Iowa State University, Ames 50011 (current address: UMass Cranberry Station, University of MassachusettsAmherst, East Wareham 02538); Damon L. Smith, Department of Plant Pathology, University of Wisconsin-Madison 53706; Albert U. Tenuta, Ontario Ministry of Agriculture, Food, and Rural Affairs; University of Guelph, Ridgetown N0P 2C0, Canada; Steven A. Whitham and Daren S. Mueller, Department of Plant Pathology, Iowa State University, Ames 50011