scholarly journals An ac34 Deletion Mutant of Autographa californica Nucleopolyhedrovirus Exhibits Delayed Late Gene Expression and a Lack of Virulence In Vivo

2012 ◽  
Vol 86 (19) ◽  
pp. 10432-10443 ◽  
Author(s):  
Y. Cai ◽  
Z. Long ◽  
J. Qiu ◽  
M. Yuan ◽  
G. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Deletion Mutant ◽  
Autographa Californica ◽  
Late Gene ◽  
Late Gene Expression ◽  
Autographa Californica Nucleopolyhedrovirus

scholarly journals Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication

2010 ◽  
Vol 84 (12) ◽  
pp. 6153-6162 ◽  
Author(s):  
Mei Yu ◽  
Eric B. Carstens
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Viral Genome ◽  
Virus Production ◽  
Autographa Californica ◽  
Late Gene ◽  
Late Gene Expression ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Budded Virus

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) lef-3 is one of nine genes required for viral DNA replication in transient assays. LEF-3 is predicted to contain several domains related to its functions, including nuclear localization, single-strand DNA binding, oligomerization, interaction with P143 helicase, and interaction with a viral alkaline nuclease. To investigate the essential nature of LEF-3 and the roles it may play during baculovirus DNA replication, a lef-3 null bacmid (bKO-lef3) was constructed in Escherichia coli and characterized in Sf21 cells. The results showed that AcMNPV lef-3 is essential for DNA replication, budded virus production, and late gene expression in vivo. Cells transfected with the lef-3 knockout bacmid produced low levels of early proteins (P143, DNA polymerase, and early GP64) and no late proteins (P47, VP39, or late GP64). To investigate the functional role of domains within the LEF-3 open reading frame in the presence of the whole viral genome, plasmids expressing various LEF-3 truncations were transfected into Sf21 cells together with bKO-lef3 DNA. The results showed that expression of AcMNPV LEF-3 amino acids 1 to 125 was sufficient to stimulate viral DNA replication and to support late gene expression. Expression of Choristoneura fumiferana MNPV lef-3 did not rescue any LEF-3 functions. The construction of a LEF-3 amino acid 1 to 125 rescue bacmid revealed that this region of LEF-3, when expressed in the presence of the rest of the viral genome, stimulated viral DNA replication and late and very late protein expression, as well as budded virus production.

scholarly journals Characterization of late gene expression factors lef-9 and lef-8 from Bombyx mori nucleopolyhedrovirus

2002 ◽  
Vol 83 (8) ◽  
pp. 2015-2023 ◽  
Author(s):  
Asha Acharya ◽  
Karumathil P. Gopinathan
Keyword(s):  
Gene Expression ◽  
Bombyx Mori ◽  
Late Gene ◽  
Late Gene Expression ◽  
Reading Frame ◽  
Early Transcription ◽  
A Site ◽  
Translational Termination

Late gene expression factors, LEF-4, LEF-8, LEF-9 and P47 constitute the primary components of the Autographa californica multinucleocapsid polyhedrovirus (AcMNPV)-encoded RNA polymerase, which initiates transcription from late and very late promoters. Here, characterization of lef-9 and lef-8, which encode their corresponding counterparts, from Bombyx mori NPV is reported. Transcription of lef-9 initiated at two independent sites: from a GCACT sequence located at −38 nt and a CTCTT sequence located at −50 nt, with respect to the +1 ATG of the open reading frame. The 3′ end of the transcript was mapped to a site 17 nt downstream of a canonical polyadenylation signal located 7 nt downstream of the first of the two tandem translational termination codons. Maximum synthesis of LEF-9 was seen from 36 h post-infection (p.i.). The transcription of lef-8 initiated early in infection from a GTGCAAT sequence that differed in the corresponding region from its AcMNPV counterpart (GCGCAGT), with consequent elimination of the consensus early transcription start site motif (underlined). Peak levels of lef-8 transcripts were attained by 24 h p.i. Immunocopurification analyses suggested that there was an association between LEF-8 and LEF-9 in vivo.

trans Activation of the simian virus 40 late transcription unit by T-antigen

1985 ◽  
Vol 5 (6) ◽  
pp. 1391-1399
Author(s):  
J Brady ◽  
G Khoury
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
T Antigen ◽  
Late Gene ◽  
Antigen Binding ◽  
Late Gene Expression ◽  
Antigen Binding Site

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

scholarly journals Early c-Fos Induction after Cerebral Ischemia: A Possible Neuroprotective Role

2001 ◽  
Vol 21 (5) ◽  
pp. 550-556 ◽  
Author(s):  
Sunghee Cho ◽  
Eun-Mi Park ◽  
Yoonseong Kim ◽  
Nian Liu ◽  
Judit Gal ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cerebral Ischemia ◽  
Fusion Gene ◽  
Glucose Deprivation ◽  
Late Gene ◽  
Late Gene Expression ◽  
Ca1 Neurons ◽  
Fos Expression

The role of c-Fos in neurodegeneration or neuroprotection after cerebral ischemia is controversial. To investigate whether early c-Fos induction after ischemia is associated with neuroprotection, rats were subjected to 10 minutes of transient forebrain ischemia and c-Fos expression was examined. Resistant dentate granule cells and neurons in CA2–4 displayed more robust immunoreactivity than vulnerable neurons in the CA1 region of hippocampus during early hours of reperfusion. By 6 hours after reperfusion, c-Fos immunoreactivity was greatly diminished in all areas of the hippocampus. Administration of N-acetyl-O-methyldopamine (NAMDA), a compound previously shown to protect CA1 neurons against ischemia, increased c-Fos immunoreactivity in the CA1 vulnerable region at 6 hours after ischemia and protected SK-N-BE(2)C neurons from oxygen glucose deprivation. Further in vitro study showed that NAMDA potentiated phorbol-12 myristate-13 acetate (PMA)-induced c-Fos expression, AP1 binding activity, and late gene expression determined by chloramphenicol acetyltransferase (CAT) activity from AP1 containing tyrosine hydroxylase promoter-CAT fusion gene in SK-N-BE(2)C neurons. In vivo and in vitro results showed that a neuroprotectant, NAMDA, in concert with another stimulus (for example, ischemia or PMA) up-regulates c-Fos expression and suggested that the early rise of NAMDA-induced c-Fos expression in vulnerable CA1 neurons may account for neuroprotection by means of up-regulating late gene expression for survival.

scholarly journals trans Activation of the simian virus 40 late transcription unit by T-antigen.

1985 ◽  
Vol 5 (6) ◽  
pp. 1391-1399 ◽  
Author(s):  
J Brady ◽  
G Khoury
Keyword(s):  
Gene Expression ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
T Antigen ◽  
Late Gene ◽  
Antigen Binding ◽  
Late Gene Expression ◽  
Antigen Binding Site

We have investigated the role of simian virus 40 (SV40) T-antigen in the induction of late gene expression independent of its function in amplifying templates through DNA replication. Northern blot and S1 nuclease analyses showed that stimulation occurred at the transcriptional level. At least two template elements, the T-antigen-binding sites and the 72-base-pair repeats, appeared to be important for this induction. Using template mutants, we demonstrated that deletions within T-antigen-binding site II decreased T-antigen-mediated late gene expression approximately 10- to 20-fold. In addition, multiple point mutations within a single retained copy of the SV40 72-base-pair repeat decreased T-antigen-mediated late gene expression. Using in vivo competition studies, we demonstrated that competitor DNA fragments containing the SV40 control region (nucleotides 5171 through 272) quantitatively decreased SV40 late gene expression in COS-1 cells. In contrast, competition with a plasmid containing SV40 nucleotides 1 through 294 (which removes all of T-antigen-binding site I and half of site II) was much less efficient. Finally, we demonstrated that in vivo competition experiments employing competitor fragments distal to the T-antigen-binding sites within the late template region (SV40 nucleotides 180 through 2533) resulted in superinduction of late gene expression in COS-1 cells. This finding suggests that negative factors such as repressors or attenuators may modulate late SV40 gene expression before induction. Our results are consistent with a model in which induction of late gene expression involves an interaction of the SV40 origin region with DNA-binding proteins, one of which may be T-antigen. Activation of the SV40 late transcription unit may involve induction of the SV40 enhancer or removal of a repressor-like protein or both.

ORF98 ofAutographa californicanucleopolyhedrosisvirusis an auxiliary factor in late gene expression

2003 ◽  
Vol 49 (3) ◽  
pp. 157-163 ◽  
Author(s):  
Kathleen L Hefferon
Keyword(s):  
Gene Expression ◽  
Autographa Californica ◽  
Late Gene ◽  
Late Promoter ◽  
Late Gene Expression ◽  
Baculovirus Infection ◽  
The Family ◽  
Auxiliary Factor ◽  
Late Expression ◽  
Early Late

Autographa californica nucleopolyhedrosisvirus (AcMNPV) is the type member of the family Baculoviridae. Gene expression of AcMNPV during virus infection is temporally regulated. A series of late expression factors (LEFs) are required for late gene expression to take place. A number of additional factors have also been shown to more modestly influence late gene expression. Using the LEF transient assay, we scanned the AcMNPV genome for such factors by replacing plasmids using the LEF genes with larger clones and then looked for increases in late gene expression using a reporter plasmid under the control of a late promoter. Using this approach, ORF98 was identified as having a stimulatory effect on late gene expression. The ability of ORF98 to influence early, late, and very late gene expression was established. Furthermore, tagged versions of ORF98 were localized to the nuclei of transfected cells and were shown to interact with each other as homo-oligomers. Potential roles of ORF98 in baculovirus infection are discussed.Key words: AcMNPV, late expression factors, transactivator, gene expression.

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