For example, modeling of the structure of the H7N9 hemagglutinin (HA) helped predict the potential of the Chinese A/Hangzhou/1/2013 strain by predicting that the H7H9 strain could bind to human sialic acid receptors. COS cells transfected with the indicated gH plasmid plus a gL plasmid were metabolically labeled with a mixture of [35S]methionine and [35S]cysteine for 16 h. Confocal microscopic images of chimeric pUL34-containing proteins in the presence and absence of wild-type pUL31. Figure 3 shows the results obtained with the HSV-1 and HSV-2 substitution mutants, and Fig. Together, this work supplies a context for further investigation of the causes and effects of metabolic perturbations during infection. 2B, lanes 1, 2, and 4, respectively). First, biochemical assays using a recombinant US11 protein indicated that the protein can inhibit the phosphorylation of eIF-2α by activated PKR (5).
The open arrows in panels C1, D1, E1, and E2 mark extracellular virions. Vero cells were transfected with plasmids encoding the wild-type protein or no protein (empty vector) and with the different TN mutants. VP5 is the major capsid protein and serves as a loading control between samples. Processing of viral DNA. Similarly in lanes 8 and 12, 1.6 µg of VP19C was used and in lanes 9 and 13, 6.4 µg of VP19C was used. The sucrose gradients were fractionated and analyzed by SDS-PAGE followed by Coomassie staining (top panel, representative gel) and autoradiography (bottom panels). 5l to n and o to q show data for in69 and in104, respectively, as examples of this group.
The locations of the transposition inserts in the VP23 and VP19C polypeptides are shown in Fig. Although the precise sequence of the VP19C NLS has not been identified, it has been mapped to a region containing 33 amino acids, and database searching has revealed that it does not belong to any of the known classes of NLS. The UL41 polypeptides encoded by a number of mutant alleles are indicated, with only those amino acids at which the mutants differ from KOS indicated. coli. (C) Expression of the γ134.5 protein and its derivatives. The secondary structure of the IDE binding domain is likely important for its interaction with IDE.To determine whether amino acids 24 to 71 of gE are sufficient for binding to IDE, we fused the first 71 amino acids of VZV gE to a portion of another herpesvirus glycoprotein to determine if the VZV gE sequence conferred binding to IDE. The transferred proteins were probed with anti-pUL6 (B, D, and E) or anti-HA (A, C, and F) antibodies.
We suggest that tyrosine at 167 makes a hydrogen bond via its hydroxyl side chain to the purine ring and freezes GCV in a conformation not optimal for the phosphorylation. For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 293T cells were plated in 48-well plates and transfected similarly, with 0.5 μg of DNA and 2.5 μl of GENEPorter per well. Cells were then exposed to serial dilutions of β-gal-expressing virus diluted in PBS plus 0.1% glucose and 1% heat-inactivated serum. Confluent monolayers of Vero cells in 35-mm-diameter cell culture dishes were infected for 1 h with wt virus or mutant viruses (MOI, 10 PFU/ml). All proteins were expressed at similar levels. Briefly, supernatants and cells from 24 T-150 flasks of Vero cells infected with either pYEbac102 or YE102-VC1 were collected at 24 and 36 hpi, respectively, and treated with TNE (10 mM Tris; 500 mM NaCl; 1 mM EDTA; pH 7.5) buffer for 15 min at 4°C. Twelve hpi, cells were treated with Accutase (Innovative Cell Technologies, Inc., San Diego, CA) to obtain a single-cell suspension, and the numbers were adjusted to 106 cells/ml.
In both experiments, anti-gD Abs efficiently blocked cosedimentation of HVEMt with HSV-1 as evidenced by the absence of HVEMt from the virus-containing fraction of the sucrose gradient in one experiment (Fig. Transformants containing the appropriate mutant plasmids were selected for by resistance to kanamycin and carbenicillin and screened by restriction endonuclease and agarose gel analysis. Sequences encoding VP23 were fused to the yeast Gal4 DNA-binding domain (pGBT9-VP23), and the gene encoding VP19C was fused to the Gal4 transactivation domain (pGAD424-VP19C) (7). After 48 h, the spun cell pellet was re-seeded at a cell density of 5 × 105 vc ml−1 in basal medium containing 125 µg ml−1 G418. We have previously shown that VP16 and vhs form a stable complex in vitro and in vivo in infected cells (48), suggesting that physical association is necessary for VP16 to attenuate vhs activity during an infection. We have previously shown that VP16 plays another important regulatory role in viral growth, manifested through its ability to modulate the activity of the virion host shutoff protein (vhs) (28, 48). Viral glycoproteins gD and gK and the membrane protein UL20 were conserved between McKrae and F strains.
The selective transportation of proteins into and out of the nucleus is essential for proper cell function.