Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates.
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ABSTRACT:
SUBMITTER: Vreede FT
PROVIDER: S-EPMC506943 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:[This corrects the article DOI: 10.1371/journal.ppat.1003971.].
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Project description:The BM2 protein of influenza B virus functions as an ion channel, which is suggested to be important for virus uncoating in endosomes of virus-infected cells. Because direct support for this function is lacking, whether BM2 plays an essential role in the viral life cycle remains unknown. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutant viruses possessing M segments with the mutated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still expressed BM2. The introduction of multiple stop codons and a one-nucleotide deletion downstream of the stop-start pentanucleotide, in addition to disablement of the BM2 initiation codon, failed to generate viable mutant viruses, but the mutant M segments still expressed proteins that reacted with the BM2 peptide antiserum. To completely abolish BM2 expression, we generated a mutant M gene whose BM2 open reading frame was deleted. Although this mutant was not able to replicate in normal MDCK cells, it did replicate in a cell line that we established which constitutively expresses BM2. Furthermore, a virus possessing the mutant M gene lacking the BM2 open reading frame and a mutant NA gene containing the BM2 open reading frame instead of the NA open reading frame underwent multiple cycles of replication in MDCK cells, with exogenous sialidase used to supplement the deleted viral sialidase activity. These findings demonstrate that the BM2 protein is essential for influenza B virus replication.
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Project description:Ultraviolet (UV)-induced DNA damage causes an efficient block of elongating replication forks. The checkpoint kinase, CHK1 has been shown to stabilize replication forks following hydroxyurea treatment. Therefore, we wanted to test if the increased UV sensitivity caused by the unspecific kinase inhibitor caffeine--inhibiting ATM and ATR amongst other kinases--is explained by inability to activate the CHK1 kinase to stabilize replicative structures. For this, we used cells deficient in polymerase ? (Pol?), a translesion synthesis polymerase capable of properly bypassing the UV-induced cis-syn TT pyrimidine dimer, which blocks replication. These cells accumulate gaps behind progressing replication forks after UV exposure. We demonstrate that both caffeine and CHK1 inhibition, equally retards continuous replication fork elongation after UV treatment. Interestingly, we found more pronounced UV-sensitization by caffeine than with the CHK1 inhibitor in clonogenic survival experiments. Furthermore, we demonstrate an increased collapse of replicative structures after caffeine treatment, but not after CHK1 inhibition, in UV-irradiated cells. This demonstrates that CHK1 activity is not required for stabilization of gaps induced during replication of UV-damaged DNA. These data suggest that elongation and stabilization of replicative structures at UV-induced DNA damage are distinct mechanisms, and that CHK1 is only involved in replication elongation.
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