VEPLA was utilized to detect wild-type McKrae and gKΔ31–68 entry into Vero cells. In gHΔ378-397 the α-helix was collapsed, after insertion of two SphI sites at residues 377 and 397. Lane 1, protein standard; lane 2, full-length pUL34-GST. 2 for the human receptors (data not shown). Under both conditions, there is an accumulation of multiple intermediates of upper glycolysis, a decrease in TCA cycle compounds, and increased nucleotides ( and ). The mutations might modify the catalytic aspartates or interactions with the single-strand DNA, allowing new dNTP incorporation and elongation. Residue 21 and adjoining residues (17–24) are particularly intolerant of alanine substitution (see Fig.
Reverse immunoprecipitation experiments were also performed to test whether gB could immunoprecipitate UL20p. The boxed residues represent the required 30-amino-acid PKR binding domain (amino acids 91 to 121). 3C and D). The Y2H assay (9) was used to rapidly screen mutants for loss or decrease of interaction (Tables and ). We previously reported that this truncation precludes the interaction of both the UL15 and UL33 proteins with UL28 (13). Figure 3 shows that the 14 UL28 BglII linker insertion viruses expressed a UL28 protein that was of a size similar to that of the UL28 protein expressed from wild-type KOS virus. 2B).
These entry defects were particularly pronounced in PILRα-expressing CHO cells, in which both gK mutant viruses entered up to 8-fold less efficiently than the McKbac virus (Fig. Capsid binding was determined by adding an aliquot of the [35S]methionine-labeled UL25 to pooled A and B capsids derived from the nuclei of cells infected with the UL25-null mutant, vΔUL25 (Table 2). Interestingly, two of the mutants which did not support growth and DNA packaging (in111 and in116) retained the ability to interact with UL28, and these both had insertions near the C terminus of the protein. The arrows show the sites of the transposition insertions. When considered alongside the behavior of in463, which completely fails to support virus growth, this seems to indicate that sequences very near the C terminus of VP19C play essential roles in capsid assembly. Error bars represent standard errors of the means. Spencer et al.
In order to analyze the effect of this deletion in the context of the HSV genome, a recombinant virus KY0112 was constructed by using the bacterial artificial chromosome (BAC) system (24). Deletion of gE amino acids 32 to 71 abolishes binding to IDE. As shown in Fig. Surprisingly, also two putative late domain sequences (PSAP and PPKY), which hitherto have been described only for RNA viruses, in particular in retroviral Gag proteins, and have been shown to be necessary for efficient budding (7, 23), are also present in PrV (p)UL36. To reveal whether the mutant TK enzymes that show the highest ratio of GCV versus dThd phosphorylating potential (i.e. Weller (21). pWM36 (Ch 4) contains the two C2 domains of human nectin-2 (SacII-BlpI fragment of pWM28, a plasmid similar to pWM33) ligated between the V domain and TM sequences of mouse nectin-2 (BlpI-SacII fragment of pWM31).
These transfections were performed in the same way as those for the complementation experiments. Protein-DNA binding was performed in 50-μl reaction mixtures containing 2 × 104 cpm of DNA probe, 10 mM Tris-HCl (pH 7.9), 50 mM KCl, 2 mM dithiothreitol, 1 mM EDTA, 0.1% NP-40, 2% glycerol, 2% Ficoll, 3 mg of fetal bovine serum per ml, 50 ng of fish sperm DNA, 8.0 μg of unsonicated poly(dI-dC) (Pharmacia), and 2% polyvinyl alcohol. The percent positive cells for each time point (10, 20, 30, and 60 min) was determined for a total number of 5,000 Vero cells infected at an MOI of 1. Any remaining extracellular virus was inactivated by low-pH treatment (pH 3.0), and cells were incubated at 37°C and 5% CO2. , lane 3). In order to more accurately define regions of importance, their functions, and whether the C terminus can contribute to these functions, several deletion mutants within the N terminus of ICP4 were constructed both in the presence and in the absence of the C-terminal activation domain. All the mutations were analyzed for interaction with VP19C using the yeast two-hybrid assay and for genetic complementation of the VP23 null mutant.
The conformational changes of gD upon receptor binding initiate binding of the heterodimer gH/gL to gB, thereby activating the fusogenic activity (Atanasiu et al., 2010 ▶). The most prominent of these is the viral transactivator VP16, an abundant 490-amino-acid phosphoprotein contained in the viral tegument, an amorphous protein layer present between the viral capsid and envelope (5). In conclusion, the combination of methods described here represents a useful tool to evaluate the significance of aa substitutions for resistance of clinical HSV-1 strains. Five 17 syn+ x 17 hep syn syncytial recombinant viruses, R1-R5, generated in marker transfer experiments with cloned 17 hep syn fragments containing gB sequences, produced 17 hep syn-like disease in mice.