We have shown previously that the gKΔ31–68 virus enters Vero cells with slower entry kinetics than the wild-type virus (26, 32). In the luciferase-based assay, the target cells express the T7-promoter-driven luciferase gene and the effector cells express the quartet of gB, gD, gL, and gH plus a T7 RNA polymerase (1, 25, 33, 38). Some of the results are shown in Fig. Nectin-2 exhibits the same cellular localization (30, 31). Similarly, sera from HSV-2 seropositive individuals bound recombinant tegument proteins (Fig. Conceivably, the increased production of virion constituents generates virions with enhanced infectivity () and facilitates their release from the cell (). Most GCVR-mutations conferring cross-resistance to CDV are in the exonuclease domain.
We explored a similar strategy for 125I-Tyr21 labeled 1–35 polypeptide. The anti-FLAG (anti-UL20p) antibody did not react with antigen obtained from mock- or V3032 virus-infected cells (the V3032 virus has only gK tagged with the ProtC epitope tag) but strongly reacted with UL20p obtained from V3024-infected cells, migrating with the expected molecular mass of 25 to 27 kDa (Fig. In addition, there were peaks of radioactivity associated with threonine 71, serine 76, and threonine 83. Examination of Vero cells infected with the ΔgKhpd-1 mutant virus revealed the presence of large double-membrane vesicles containing tens to hundreds of nucleocapsids in the cytoplasm located proximal to the nuclear membrane (Fig. Using double enzyme digests, we were able to crudely map the location of the Pme1 site in the sequence. The vFH499 virus contains a nonsense mutation of amino acid 741. Characterization of the UL28 mutants.Virus stocks of the UL28 mutants grown on C1 cells were examined for their abilities to form plaques on UL28-complementing C1 cells and noncomplementing Vero cells (Table 1).
Previous attempts to purify full-length VP19C from E.coli were not successful. Nucleus (n), cytoplasm (c), and extracellular space (e) are marked. The N terminus of UL25 is required for capsid attachment in vitro.Previously we showed that [35S]methionine-labeled UL25 synthesized in a coupled in vitro transcription-translation system will attach to the surface of UL25-deficient capsids during in vitro incubation (24). Ten of the 15 insertion mutants (in14, in34, in37, in44, in79A, in79B, in84, in100, in111, and in116) similarly facilitated coprecipitation of UL28-Myc by R148, whereas the five remaining mutants (in51, in55, in69, in74, and in104) failed to do so. The mutations as judged by restriction enzyme analysis using Pme1 appeared to have randomly integrated throughout the UL38 and UL18 genes (Fig. Only one insertion (in143) mapped within the insert 1 locus, and it was also severely disabling, indicating that this inserted sequence is functionally important. (A) The UL41 polypeptide encoded by HSV-1 (strain KOS) is represented by the open rectangle, and that encoded by HSV-2 (strain 333) is represented by the hatched rectangle.
Rixon and colleagues had previously demonstrated in an elegant biophysical study using E. This indicates that deletions of both the amino terminus and the triplet repeats abolished the activity of the γ134.5 protein. 1B). Expression plasmids bearing these constructs were contransfected with plasmids expressing full-length pUL15-HA into CV1 cells, and lysates of the cells were immunoprecipitated with the HA- and pUL6-specific antibodies. Besides yielding a truncated protein which is significantly better to handle and analyze than the full-length (p)UL36, this also indicates that in the UL36 proteins of herpesviruses large nonessential regions are retained for as-yet-unknown reasons. 3). Although eight mutants showed reduced gD binding and one mutant, HveA-R75A, exhibited enhanced binding, mutation of the majority of contact residues had little effect on the ability of HveA to act as an entry receptor.
The nectin-2 protein expressed from pWM31 is indistinguishable from that expressed from Mph-pcDNA3 in viral entry assays (data not shown). All transfection and marker rescue experiments were carried out in 35-mm-diameter culture dishes by using LipofectAMINE PLUS reagent (Gibco BRL) according to the manufacturer’s instructions. Cells (5 × 107) were washed and lysed in 1 ml of a buffer containing 10 mM Tris (pH 7.6), 10 mM NaH2PO4 (pH 7.6), 500 mM NaCl, 10 mM NaF, 10 mM Na pyrophosphate, 5 mM 2-mercaptoethanol, 1% Triton X-100, 10% glycerol, 500 μg of Pefablock per ml, and 10 mM imidazole for 1 h on ice with periodic vortexing. The virus inoculum was subsequently removed, warm medium (34°C) was added, and the cultures were shifted to 34°C to allow virus penetration. Distributions were examined for normality using the univariate procedure with a Shapiro-Wilks test of normality. The latter suggests that soluble HVEM binds to the virus and competes with the cellular form of HVEM. The N- and C-terminal transactivation domains are necessary to various degrees for the transactivation and repression functions of ICP4.
It is important for stabilizing the capsid shell by interactions with the adjoining capsomeres made up of the major capsid protein, VP5. Therefore, the development of vaccines and novel therapeutic strategies against HSV has become an important research objective.