Vaccinia virus produces late mRNAs by discontinuous synthesis

Bertholet, Christine; Meir, Erwin G. Van; Heggeler-Bordier, Béatrice ten; Wittek, Riccardo

doi:10.1016/0092-8674(87)90211-x

Cited by 124 publications

(77 citation statements)

References 58 publications

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“…First, the vaccinia virus p l l K gene product contributes about 10% of the total protein mass present in the purified virion (Bertholet et al, 1987). Consistent with this observation, our data clearly establish the p l l K promoter as an alternative for high expression of secreted proteins.…”

Section: Discussionsupporting

confidence: 87%

Cellular Processing Limits the Heterologous Expression of Secretory Component in Mammalian Cells

Cottet¹,

Corthésy²

1997

European Journal of Biochemistry

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Recombinant vaccinia-virus-based expression systems are very popular for the overproduction of proteins in mammalian cell lines. Both the double virus T7/vaccinia hybrid system and the single recombinant strategy based on the p l l K late promoter were evaluated for their ability to govern expression and secretion of recombinant human secretory component (SC), a glycoprotein associated with IgA in mucosal secretions. We report here that, while the T7 promoter is transcriptionally 3.4-fold more active than the p l l K promoter, no difference in levels of secreted recombinant human SC is observed using either vaccinia system to infect CV-1 cells. High transcription, and thus translation levels, lead to saturation of early processing steps involved in protein export. Both systems exhibit transient accumulation of comparable amount of recombinant human SC in the endoplasmic reticulum andor the cis Golgi network, as demonstrated by immunofluorescence and endoglycosidase H (EndoH) sensitivities. Exposure of infected cells to tunicamycin results in similar inhibition of recombinant human SC export, further arguing that N-linked glycosylation is necessary for proper folding and subsequent secretion. Moreover, pulse-chase experiments indicate that newly synthesized recombinant human SC is not completely processed in a mature glycoprotein and that a portion of overexpressed SC might be degraded before it can be secreted. Recombinant human SC behaves identically to native SC in terms of kinetics of secretion and IgAbinding capacity.Our results indicate that optimization of expression systems should not only rely on the design of effective vectors, but also on the identification and clearance of the cellular bottlenecks associated with maturation of the secreted proteins.

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Section: Discussionsupporting

confidence: 87%

Cellular Processing Limits the Heterologous Expression of Secretory Component in Mammalian Cells

Cottet¹,

Corthésy²

1997

European Journal of Biochemistry

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“…5). The range of primer extension product lengths for both mRNAs was about 60 to 85 nucleotides, consistent with 50 nucleotides of mRNA plus the length of the non-template-encoded oligo(A) tract previously described for late mRNAs (3,20). Primer extension products also included longer products that we call readthrough products, although we do not know whether they reflect transcripts originating upstream of each promoter or are the result of primer hybridization to other transcripts.…”

supporting

confidence: 81%

“…Intermediate and late transcripts are moderately heterogeneous at their 5Ј ends because of apparent slippage of the RNA polymerase on the TTT in the initiator motif on the template DNA, producing mRNAs with 15 to 35 non-template-encoded A residues (3,20). They are also extremely heterogeneous at their 3Ј ends because the RNA polymerase terminates randomly (for example, see reference 24).…”

mentioning

confidence: 99%

Antiviral Activity of Distamycin A against Vaccinia Virus Is the Result of Inhibition of Postreplicative mRNA Synthesis

Broyles

Kremer

Knutson

2004

J Virol

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Distamycin A has been described as an inhibitor of the cellular pathogenesis of vaccinia virus in culture. Distamycin is an antibiotic that specifically targets the minor groove of DNA. We show here that distamycin is a potent inhibitor of vaccinia virus replication. Pulse-labeling experiments showed that most major late proteins failed to accumulate in the presence of the antibiotic. We characterized the effect of distamycin on vaccinia virus nucleic acid biosynthesis with the goal of determining the inhibitor's target. Early gene transcription was unaffected. DNA synthesis proceeded at normal rates, but DNA accumulated in large masses in the cytoplasm with no evidence of virion assembly. Transcription from the intermediate class promoter for the I1L gene was partially reduced by distamycin; however, transcription from the intermediate promoters for the three late transcription factor genes was severely inhibited. The accumulation of the late transcripts for the viral F17R and A10L genes also was severely impaired and was shown to be a direct inhibition of late promoter activity. These results indicate that inhibition of postreplicative intermediate and late transcription is the basis for inhibition of vaccinia virus by distamycin and indicate that DNA minor-groove ligands hold promise for effective anti-poxvirus drugs.Distamycin A is an antibiotic that reduces the pathogenic effects of vaccinia virus. Distamycin blocks virus-induced cell syncytium formation (22) and inhibits the visually observed cytopathic effects of the virus in cultured cells (16). Distamycin targets the minor groove of DNA (10), preferentially binding DNA sequences that are five consecutive A-T pairs, with affinity that varies with the particular sequence (1). The base composition of the DNA genome of vaccinia virus is 66% A-T, with transcriptional promoter regions being close to 90% A-T, making them ideal targets for the antibiotic.Vaccinia virus replicates its genome and assembles progeny virions exclusively in the cytoplasm of the infected cell. The processes of DNA synthesis and transcription of the three different classes of genes are coordinated in a sequential manner (reviewed in reference 4). Upon entry of the cell, viral early gene transcription begins immediately. The early gene products include factors participating in DNA synthesis that begins about an hour after infection. As DNA synthesis initiates, early gene transcription ceases and intermediate gene transcription ensues. Shortly thereafter, late gene transcription begins and intermediate gene transcription wanes. The virus-encoded proteins required for the transcription of each gene class are products of the preceding class in a gene expression cascade. The promoters for vaccinia virus early, intermediate, and late genes have A-T-rich motifs whose interaction with transcription factors potentially could be affected by a DNA minorgroove ligand. The early promoter element is targeted by the early transcription factor (6). We have shown that distamycin impairs DNA binding by ...

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“…Intermediate promoters are more prevalent in the vaccinia virus genome than previously appreciated, because many have the TAAATG motif at the start of their open reading frames, which was previously attributed to late gene promoters (X. Liu and S. S. Broyles, unpublished results). The RNA polymerase initiates on this motif within the A triplet (actually on the T triplet on the template strand) and slips repeatedly while attempting to initiate transcription (Bertholet et al, 1987;Schwer et al, 1987). The result is mRNA with a 59 end bearing a heterogeneous oligo(A) tail, averaging about 30 nt in length that is not template encoded.…”

Section: Intermediate Gene Transcriptionmentioning

confidence: 99%