Kinetics of virus entry into Vero cells. Deletion of the predicted membrane α-helix contained in HSV gH ectodomain or mutational disruption of the α-helix inhibits HSV infectivity and cell-cell fusion.To attain a preliminary characterization of the membrane α-helix contained in HSV gH ectodomain, a loss-of-function approach was applied. In all cases, glass coverslips were examined with an Olympus confocal laser scanning microscope using 10× ocular and 40× stage objectives and excitation wavelengths of 488 and 568 nm. This cell fusion assay provided a convenient way to test the functional activity of multiple gD mutants and yielded results likely to be predictive of the effects of the mutations on viral entry. To confirm expression of HSV-2 tegument epitopes, COS-7 cells were co-transfected with plasmids expressing HSV-2 genes and cDNA encoding human HLA class I heavy chain. This metabolic rerouting model was also supported by the increased pools of metabolites of nucleotide synthesis in knockdown cells with replenished aspartate ( and ). This preferential drug-resistance mechanism might explain the high number of amino acid changes in the UL54 exonuclease domain, which is in contrast to drug-resistant HSV-1 strains that harbor mutations in the UL30 polymerase domain.
Permeabilized human TK143 fibroblasts infected with a recombinant vaccinia virus expressing human TAP1+2 were incubated with 125I-labeled 1–35 Tpa. 6C. To identify the amino acid domain in US11 phosphorylated by activated PKR, a recombinant US11 protein was incubated with purified activated PKR as well as nonactivated PKR in the presence of [γ-32P]ATP and isolated by SDS-polyacrylamide gel electrophoresis (PAGE). The yield of the ΔgKhpd-3 virus was approximately fivefold higher than those of the ΔgK, ΔgKhpd-1, and ΔgKhpd-2 viruses, while the yield of the ΔgKhpd-4 virus was approximately 10-fold higher than that of ΔgKhpd-3 and identical to that of KOS virus. The UL38 fragments that carried the 15-bp transprimer were cloned into the yeast two-hybrid plasmid, pGAD424, which encodes the Gal4 transcriptional activation domain. 2). We concluded from these Southern blots that the desired UL28 mutants had been generated.
This region also encodes a nuclear localization sequence  . Similarly, mutant virus McK(gKΔ31-68) did not show any apparent defect in envelopment, and fully enveloped virion particles were excreted out of infected cells. The UL25 transposon mutants were examined for their ability to complement the growth of the UL25-null virus. The results are shown in Fig. The kanamycin gene was removed from the plasmid by digestion with a unique Pme1 site, leaving a 15-bp insertion (10 bp of restriction site sequence and 5 bp of duplicated sequence of the target). The most N-terminal severely disabled mutant, in113, occurs almost immediately after the first evidence of conservation between the sequences of the alphaherpesvirus α-subunits (Fig. The chimera K/3/K(208;243) encodes a polypeptide with amino acids 208 to 243 from strain 333 sandwiched between amino acids 1 to 135 and 244 to 489 from KOS.
Co-expression of the HSV-1 triplex proteins in the same cell. Thus, while it is not essential, the amino-terminal domain of the γ134.5 protein may facilitate eIF-2α dephosphorylation. Since the amino terminus of HSV gD is important for receptor binding, we prepared truncation mutations and synthetic peptides that target the amino portion of VZV gE to investigate the structural requirements for its interaction with IDE. Bound immunoglobulins were revealed by enhanced chemiluminescence. In contrast, deletion of 709 aa from amino acid positions 2087 to 2795 of the 3,084-aa PrV (p)UL36 proved to retain function of the protein. The aromatic amino acids resulted in an even better GCV-phosphorylating activity than the corresponding Ala-167-mutated TKs, except for A168W. In this study, we examined the contribution of each HveA contact residue to gD binding, virus entry, and HveA oligomerization.
The coding sequence was identical to that described before (GenBank M12197) (32). The desired mutations were introduced into the plasmid containing the 903-bp MscI fragment by using the Gene Editor In Vitro Site-Directed Mutagenesis system (Promega) and the 5′-phosphorylated mutagenic oligonucleotides PC-1 (GCGCGCCTGCGCATGGCCCGCCGCGGCGCCCGCGGTGTCG), PC-2 (CGTGCAGTGCGCAGTGGCCTGGCCGGCGGC), PC-3 (GCCATGGCGCCCGCGGCGCCGGCCTTCTGCGAGG), PC-4 (GACTTCTGCGAGGCGGAGGCCGCCTCGGCCGCCTGCGCGCG), PC-5 (GCCGCCTGCGCGGCCTGGGGCCTGGCGGCGCCGCTGCGG), PC-6 (CATGTCCCCGGCCGAGTACCGCCG), and PC-7 (CTTCGGCCCGGACGCACCGGTGCCCATG). Protein integrity, purity, and relative concentration were determined by Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). (B) Schematic representation of recombinant viruses constructed for TEV-specific proteolytic activity. Plaque morphology and one-step viral growth kinetics.Visual analysis of plaque morphology of mutant viruses was performed essentially as we have described previously (27, 44, 46). Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140. The predicted translation products of HSV-1 strain KOS, HSV-2 strain HG42, and herpes B virus strain E2490 ICP4 molecules were aligned using Vector NTI.
The interaction between the two triplex proteins VP23 and VP19C is important for assembly of the herpesvirus icosahedron. HSV-1 and HSV-2 are the most common human viral infections worldwide; they can be transmitted by casual or sexual contact, but HSV-2 is mostly transmitted sexually.