α-Amanitin-Resistant Viral RNA Synthesis in Nuclei Isolated from Nuclear Polyhedrosis Virus-Infected
            <i>Heliothis zea</i>
            Larvae and
            <i>Spodoptera frugiperda</i>
            Cells

Eighteen genes of Autographa californica nuclear polyhedrosis virus are necessary and sufficient to transactivate expression from the late vp39 promoter in transient-expression assays in SF-21 cells. These 18 genes, known as late expression factor genes (lefs), are also required to transactivate the very late promoter of the polyhedrin gene, polh, but expression from this promoter is relatively weak compared with expression from the vp39 promoter. To further define the factors required for late and very late promoter expression, we first determined that the eighteen lefs were also required for expression from two other major baculovirus promoters: the late basic 6.9-kDa protein gene, p6.9, and the very late 10-kDa protein gene, p10. We next examined the effect of the very late expression factor 1 gene (vlf-1), a gene previously identified by analysis of a temperaturesensitive mutant, in the transient-expression assay and found that vlf-1 specifically transactivated the two very late promoters but not the two late promoters. We then surveyed the Autographa californica nuclear polyhedrosis virus genome for additional genes which might specifically regulate very late gene expression; no additional vlf genes were detected, suggesting that VLF-1 is the primary regulator of very late gene expression. Finally, we found that the relative contribution of the antiapoptosis gene p35, which behaves as a lef in these transient-expression assays, depended on the nature of the other viral genes provided in the cotransfection mixtures, suggesting that other viral genes also contribute to the ability of the virus to block apoptosis.

mentioning

confidence: 99%

Factors regulating baculovirus late and very late gene expression in transient-expression assays

Todd

Passarelli

et al. 1996

“…Studies with inhibitors of DNA synthesis (56) and with a temperature-sensitive AcM-NPV mutant defective in DNA synthesis (13,16) demonstrate that expression of late and very late baculovirus genes is dependent on viral DNA replication. The switch from early to late gene expression is coincident with the appearance of an alpha-amanitin-resistant RNA polymerase activity (14,18,26,64).…”

mentioning

confidence: 99%

Differential requirements for baculovirus late expression factor genes in two cell lines

Miller

1995

A plasmid library of 18 late expression factor (LEF) genes (LEF library) from the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) supports transient expression from a late viral promoter in the SF-21 cell line, derived from Spodoptera frugiperda. We found, however, that this LEF library was unable to support expression from the same promoter in the TN-368 cell line, derived from Trichoplusia ni, which is also permissive for AcMNPV replication. To identify the additional factor(s) required for expression in TN-368 cells, we cotransfected the LEF library with clones representing portions of the AcMNPV genome not represented in the LEF library. A single additional gene was identified; this gene corresponded to ORF70 of the complete AcMNPV sequence and potentially encodes a 34-kDa cysteine-rich polypeptide. Because of its differential effect on late gene expression in the two cell lines, we renamed ORF70 hcf-1 (for host cell-specific factor 1). hcf-1 was involved in expression from reporter plasmids under late and very late but not early promoter control, indicating that it was also a LEF gene. Plasmid DNA replication assays indicated that HCF-1 was involved in virus origin-specific DNA replication in TN-368 cells. Three LEF genes, ie-2, lef-7, and p35, required for optimal virus origin-specific plasmid DNA replication or stability in SF-21 cells had little or no influence in TN-368 cells. Thus, as determined by transient-expression assays, cell line-specific and potentially host-specific factors are required for origin-specific DNA replication or stability.

“…Previous functional investigations of the polyhedrin gene promoter (25,26,29,30) and investigations of the plO gene promoter in transient expression systems (32,49,50) allowed the definition of sequences required for their maximum activity. Moreover, like most other late genes, the polyhedrin and plO genes are transcribed by an a-amanitinresistant RNA polymerase induced upon infection (8,13,18). The polyhedrin and plO gene promoters share a 12nucleotide consensus sequence (37) containing an ATAAG motif which is found at the transcription initiation sites of most characterized baculovirus late genes.…”

mentioning

confidence: 99%

Competition between baculovirus polyhedrin and p10 gene expression during infection of insect cells

Chaabihi

Ogliastro

Martin

et al. 1993

Polyhedrin and plO genes are expressed concurrently during the late stage of infection. To determine whether any competition occurs between these two genes at a transcriptional and/or translational level, a series of Autographa californica nuclear polyhedrosis recombinant viruses with deletions of promoter and coding sequences of the plO or polyhedrin gene was constructed. Two modified baculoviruses with only one of the very