Expression of Baculovirus Late and Very Late Genes Depends on LEF-4, a Component of the Viral RNA Polymerase Whose Guanyltransferase Function Is Essential

Knebel-Mörsdorf, Dagmar; Quadt, Ilja; Li, Yang; Montier, Laura; Guarino, Linda A.

doi:10.1128/jvi.80.8.4168-4173.2006

J Virol

2006

DOI: 10.1128/jvi.80.8.4168-4173.2006

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Expression of Baculovirus Late and Very Late Genes Depends on LEF-4, a Component of the Viral RNA Polymerase Whose Guanyltransferase Function Is Essential

Dagmar Knebel-Mörsdorf

Ilja Quadt

Yang Li

et al.

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Cited by 19 publications

(15 citation statements)

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“…As expected for an RNA polymerase subunit, LEF-4 is essential for viral replication, as revealed by studies of a temperature-sensitive virus with a mutation in lef-4 and by RNA silencing experiments (30,47). LEF-4 is also necessary for transient expression of late and very late genes (48).…”

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“…These enzymes are defined by two essential glutamate-containing motifs that bind divalent cation. The substitution of any one of these glutamates abolishes enzymatic activity.As expected for an RNA polymerase subunit, LEF-4 is essential for viral replication, as revealed by studies of a temperature-sensitive virus with a mutation in lef-4 and by RNA silencing experiments (30,47). LEF-4 is also necessary for transient expression of late and very late genes (48).…”

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Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs

Guarino

2008

J Virol

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The baculovirus Autographa californica nucleopolyhedrovirus encodes two proteins with RNA triphosphatase activity. Late expression factor LEF-4, which is an essential gene, is a component of the RNA polymerase and also encodes the RNA capping enzyme guanylyltransferase. PTP/BVP is also an RNA triphosphatase, but is not essential for viral replication, possibly because its activity is redundant to that of LEF-4. To elucidate the role of these proteins in mRNA cap formation, a mutant virus that lacked both RNA triphosphatase activities was constructed. Infection studies revealed that the double-mutant virus was viable and normal with respect to the production of budded virus. Pulse-labeling studies and immunoblot analyses showed that late gene expression in the double mutant was equivalent to that in the wild type, while polyhedrin expression was slightly reduced. Direct analysis of the mRNA cap structure indicated no alteration of cap processing in the double mutant. Together, these results reveal that baculoviruses replicate and express their late genes at normal levels in the absence of its two different types of RNA triphosphatases.Baculoviruses are unique among eukaryotic DNA viruses in their use of both cellular and viral transcription machinery (19,25). Viral infection is initiated by the host RNA polymerase II that recognizes and transcribes early promoters which are structurally similar to cellular promoters. Late and very late genes are transcribed by a virus-encoded RNA polymerase composed of four late expression factors (LEFs) called LEF-4, -8, and -9 and P47. These genes were first described for the type species Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), and homologs have been identified in all the baculovirus genomes that have been sequenced thus far (45). LEF-8 and LEF-9 are believed to form the catalytic pocket, because they contain motifs common to ␤ and ␤Ј RNA polymerases (38,49,59). LEF-4 is an RNA capping enzyme with both guanylyltransferase and RNA triphosphatase activities (14,17,28), and the function of P47 is unknown.Late and very late baculovirus mRNAs have a typical methyl-7-guanosine cap structure at their 5Ј ends (53). In eukaryotic cells, this cap structure is essential and is required for efficient pre-mRNA splicing, export, stability, and translation initiation (11). Three enzymatic activities are required for cap formation: hydrolysis of the 5Ј triphosphate end of the nascent transcript to a diphosphate by RNA triphosphatase, capping of the diphosphate end with GMP by RNA cap guanylyltransferase, and N-7 methylation of guanine cap by RNA cap methyltransferase (11, 57).Baculoviruses have evolved a novel strategy for capping their late mRNAs. Even though they replicate in the nuclei of infected cells, baculoviruses are unable to use the cellular capping machinery, because these enzymes find their substrates through interactions with the highly repetitive carboxyl-terminal domain of the cellular RNA polymerase II (6,23,43). Since the baculovirus RNA polymerase la...

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confidence: 98%

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confidence: 99%

Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs

Guarino

2008

J Virol

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“…The two other factors influenced by the loss of DBP are not directly involved in viral DNA replication. LEF-4, a component of the viral RNA polymerase, is essential for late gene expression as well as for production of viral progeny (6,11). P35 acts as an apoptotic suppressor and is required to prevent apoptosis in AcMNPV-infected S. frugiperda cells (1,2,8).…”

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“…The cloned cDNA of the AcMNPV dbp gene served as a DNA template to generate dsRNA corresponding to 609 bp at the 5Ј terminus of dbp (15) (primer 1a, 5Ј-GGGTAATACGACTCACTATAGGATGGCAACTAAA CGCAAG-3Ј; primer 1b, 5Ј-GGGCACACGTTTGGTTCCA T-3Ј; primer 2a, 5Ј-GGGTAATACGACTCACTATAGGGC ACACGTTTGGTTCCAT-3Ј; and primer 2b, 5Ј-GGATGGC AACTAAACGCAAG-3Ј). As a control, we used dsRNA corresponding to a 600-bp fragment of the green fluorescent protein (GFP) gene as described recently (11). S. frugiperda cells were transfected with 20 g of DBP dsRNA or 5 g of GFP dsRNA as calcium phosphate precipitates (BD BaculoGold) followed by infection with AcMNPV (10 PFU/cell).…”

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Studies of the Silencing of Baculovirus DNA Binding Protein

Quadt

Lent

Knebel-Mörsdorf

2007

J Virol

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Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P10 and GP64 was unaffected. In the absence of DBP, viral DNA replication sites were formed, indicating replication of viral DNA. Electron microscopy studies, however, revealed a loss of formation of polyhedra and virus envelopment, suggesting that the primary role of DBP is viral formation rather than viral DNA replication.

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Genomic analysis of Oryctes rhinoceros virus reveals genetic relatedness to Heliothis zea virus 1

et al. 2006

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Oryctes rhinoceros virus (OrV) is an unassigned invertebrate dsDNA virus with enveloped and rod-shaped virions. Two cloned PstI fragments, C and D, of OrV DNA have been sequenced, consisting of 19,805 and 17,146 bp, respectively, and comprising about 30% of the OrV genome. For each of the two fragments, 20 open reading frames (ORFs) of 150 nucleotides or greater with no or minimal overlap were predicted. Ten of the predicted 40 ORFs revealed significant similarities to Heliothis zea virus 1 (HzV-1) ORFs, of which five, lef-4, lef-5, pif-2, dnapol and ac81, are homologues of conserved core genes in the family Baculoviridae, and one is homologous to baculovirus rr1. A baculovirus odv-e66 homologue is also present in OrV. Five ORFs encode proteins homologous to cellular thymidylate synthase (TS), patatin-like phospholipase, mitochondrial carrier protein, Ser/Thr protein phosphatase, and serine protease, respectively. TS is phylogenetically related to those of eukarya and nucleo-cytoplasmic large dsDNA viruses. However, the remaining 25 ORFs have poor or no sequence matches with the current databases. Both the gene content of the sequenced fragments and the phylogenetic analyses of the viral DNA polymerase suggest that OrV is most closely related to HzV-1. These findings and the re-evaluation of the relationship of HzV-1 to baculoviruses suggest that a new virus genus, Nudivirus, should be established, containing OrV and HzV-1, which are genetically related to members of the family Baculoviridae.

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Expression of Baculovirus Late and Very Late Genes Depends on LEF-4, a Component of the Viral RNA Polymerase Whose Guanyltransferase Function Is Essential

Cited by 19 publications

References 30 publications

Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs

Roles of LEF-4 and PTP/BVP RNA Triphosphatases in Processing of Baculovirus Late mRNAs

Studies of the Silencing of Baculovirus DNA Binding Protein

Genomic analysis of Oryctes rhinoceros virus reveals genetic relatedness to Heliothis zea virus 1

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