Temperature strongly influenced percent mortality and time to death of salamanders exposed to the Ambystoma tigrinum virus (iridovirus) (ATV). Most salamanders survived when exposed at 26°C, whereas all died at 18°C and nearly all died at 10°C. Some asymptomatic salamanders that survived 60 d at 10 or 26°C were found to be carrying virus. Polymerase chain reaction (PCR) confirmed the presence of virus in ATV-exposed salamanders but was found to be less sensitive than cell culture in detecting ATV at low concentrations. PCR products were 100% identical to ATV in the major capsid protein sequence. Virus titer was higher in salamanders held at 10°C than at 18°C but little virus, if any, was present in the small number of salamanders that died at 26°C. These results may help explain periodic viral epizootics in field populations of A. tigrinum where water temperatures fluctuate widely. KEY WORDS: Epizootic · Cell culture · Iridovirus · Mortality Resale or republication not permitted without written consent of the publisherDis Aquat Org 63: [95][96][97][98][99][100] 2005 MATERIALS AND METHODSGeneral methods. ATV was cultured using epithelioma papilloma cyprini (EPC) cells (Fijan et al. 1983). Plaque and TCID 50 (tissue culture infectious dose 50 ; Reed & Muench 1938) assays employing EPC cells were used to estimate the concentration of virus used in each experiment. Three to 4 mo old Ambystoma tigrinum nebulosum larvae originated from stock held in the laboratory for at least 2 generations. No mortality due to possible virus infection was observed in the rearing facility during this period. During the experiments, each salamander was held individually in 300 ml of water in a Zip Lock ® (S. C. Johnson) container and water was changed weekly. Salamanders were fed brine shrimp Artemia sp. 3 times a week and observed daily. Salamanders that died were stored at -70°C. At the end of each experiment, body wall samples were taken from all dead larvae and tail clips from surviving salamanders for virus detection. Each sample was placed in a Stomacher ® (Seward) bag containing 2 ml of Eagle's Minimum Essential Medium (MEM) + 2% Fetal Bovine Serum (FBS) + penicillin-streptomycin-neomycin (PSN; Sigma) + diatomaceous earth powder, and homogenized in a Stomacher device. The homogenate was centrifuged at 9000 × g for 10 min and the supernatant was stored at -80°C. Supernatant (100 µl) was inoculated onto EPC cells in each well of 12-or 24-well plates, the preparation was rocked for 1 h and then 1 ml of MEM + 10% FBS + PSN was added. Cells were incubated at 22°C under 5% CO 2 and observed for 2 wk for cytopathic effect (CPE). Samples showing no CPE were passed through 2 further sets of EPC cells to confirm the presence or absence of virus.To determine the multiplication of virus at the 3 experimental temperatures, replicate EPC cultures in 24-well plates were inoculated with dilutions of ATV and held at 10, 18 or 26°C for 10 to 16 d. Development of CPE was observed and TCID 50 was calculated during incubation.In order ...
Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is phosphorylated. Phosphorylation can affect protein localization, protein interactions, and protein function. The major sites of ICP27 that are phosphorylated are serine residues 16 and 18, within a CK2 site adjacent to a leucine-rich region required for ICP27 export, and serine 114, within a PKA site in the nuclear localization signal. Viral mutants bearing serine-to-alanine or glutamic acid substitutions at these sites are defective in viral replication and gene expression. To determine which interactions of ICP27 are impaired, we analyzed the subcellular localization of ICP27 and its colocalization with cellular RNA export factors Aly/REF and TAP/ NXF1. In cells infected with phosphorylation site mutants, ICP27 was confined to the nucleus even at very late times after infection. ICP27 did not colocalize with Aly/REF or TAP/NXF1, and overexpression of TAP/NXF1 did not promote the export of ICP27 to the cytoplasm. However, in vitro binding experiments showed that mutant ICP27 was able to bind to the same RNA substrates as the wild type. Nuclear magnetic resonance (NMR) analysis of the N terminus of ICP27 from amino acids 1 to 160, compared to mutants with triple substitutions to alanine or glutamic acid, showed that the mutations affected the overall conformation of the N terminus, such that mutant ICP27 was more flexible and unfolded. These results indicate that these changes in the structure of ICP27 altered in vivo protein interactions that occur in the N terminus but did not prevent RNA binding.ICP27 is a multifunctional protein that acts at both the transcriptional and posttranscriptional levels (47). At the transcriptional level, ICP27 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAP II) and recruits RNAP II to sites of herpes simplex virus 1 (HSV-1) transcription/ replication (13, 59). The interaction of ICP27 with RNAP II requires the N-terminal leucine-rich region (LRR) of ICP27, and the viral mutant dLeu, in which this region is deleted, cannot interact with and recruit RNAP II (13). As a result, viral early and late transcript levels are reduced to 10 to 20% of the levels seen in wild-type HSV-1 infection (32). At the posttranscriptional level, ICP27 interacts with SR proteins, which are essential splicing factors, and with SRPK1, an SR protein-specific kinase, to mediate the aberrant phosphorylation of SR proteins (50). Inappropriately phosphorylated SR proteins are unable to participate in spliceosome assembly, and host cell pre-mRNA splicing is inhibited (19, 50), which contributes to the shutoff of host protein synthesis (18). Beginning about 5 h after infection, ICP27 leaves splicing speckles and recruits the cellular mRNA export factor Aly/REF to viral transcription/replication sites (8, 9). ICP27 then binds viral RNAs (46) and begins shuttling between the nucleus and cytoplasm in its role as an RNA export factor (8,9,28,39,46,52). ICP27 is exported to the cytoplasm through the TAP/ ...
Herpes simplex virus 1 (HSV-1) protein ICP27 is a multifunctional regulatory protein that is posttranslationally modified by phosphorylation during viral infection. ICP27 has been shown to be phosphorylated on three serine residues, specifically serine residues 16 and 18, which are within casein kinase 2 (CK2) sites, and serine residue 114, which is within a protein kinase A (PKA) site. Phosphorylation is an important regulatory mechanism that is reversible and controls many signaling pathways, protein-protein interactions, and protein subcellular localization. To determine the role of phosphorylation in modulating the activities of ICP27, we constructed phosphorylation site mutations at each of the three serine residues. Single, double, and triple viral mutants were created in which alanine or glutamic acid was substituted for serines 16, 18, and 114. ICP27 phosphorylation site mutants were defective in viral replication and viral gene expression. Notably, ICP4-containing replication compartment formation was severely compromised, with the appearance of small ring-like structures that persisted even at late times after infection. Neither the colocalization of ICP27 with RNA polymerase II nor the formation of Hsc70 nuclear foci was observed during infection with the phosphorylation site mutants, both of which occur during wild-type HSV-1 infection. These data indicate that several key events in which ICP27 plays a role are curtailed during infection with ICP27 phosphorylation site mutants.Herpes simplex virus 1 (HSV-1) ICP27 is a multifunctional regulatory protein that interacts with a number of proteins and that binds RNA (32). The different activities of ICP27 are regulated in a temporal manner during infection. At early times, ICP27 is localized to the nucleus, whereas beginning about 5 to 6 h after infection, ICP27 continuously shuttles between the nucleus and cytoplasm. ICP27 mediates its nuclear activities through a series of protein-protein interactions. At very early times during infection, ICP27 recruits the predominantly cytoplasmic splicing factor kinase SRPK1 into the nucleus (34). This results in the aberrant phosphorylation of a family of essential splicing factors termed SR proteins, which are specifically phosphorylated by SRPK1. The inappropriate phosphorylation of SR proteins prevents their participation in spliceosome assembly, which causes splicing complex formation to stall, and host cell pre-mRNA splicing is impaired (15,34). This contributes to the shutoff of host protein synthesis (14). Also at early times after infection, ICP27 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNAP II) and causes RNAP II relocalization to sites of HSV-1 transcription/replication. This recruitment of RNAP II is necessary for highly active and efficient viral transcription (5). Beginning about 5 h after infection, ICP27 disassociates from splicing speckles, taking the cellular mRNA export factor Aly/REF with it to viral transcription/replication compartments (3, 4). ICP27 then binds viral tr...
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