The imperfect hairpins transcribed from pmidsEGFP (dsEGFP) were designed such that a 28-nt antisense portion would possess perfect complementarity with a region within the 5′ coding portion of the EGFP mRNA. Tissues were immediately processed or stored at −80°C until use. Cultured hMSCs that were exposed to HSV-1(KOS) gL86 (panel (B)) were 100% stained blue, indicating 100% viral infection, whereas uninfected cells did not show any staining. (A) Diagram of the PLK2 gene, with the position of chromatin immunoprecipitation (ChIP) primer pairs indicated. These studies argue that ICP0 does not function efficiently in neuronal cells and thus productive infection is inhibited. Therefore, instead we expressed luciferase from an HCMV IE1 promoter in the Us5 locus of a relatively virulent strain, SC16 (18) (Fig. After centrifugation (13000×g for 30 min) the supernatants were collected and assayed to determine their protein concentration (Bradford method, Bio-Rad).
Inflammasome complexes assemble in response to pathogen infection, cation flux, or ROS generation. Assays (50-μl volume) were performed in duplicate, and assay mixtures were incubated at 37°C for 40 min. Subconfluent HEL cells on coverslips were infected with KOS at an MOI of 10 PFU/cell. Although these results suggest that HSV-1 may be latent in CG, there are several caveats about this study that should be considered in interpretation of these results. (A) HSV-1 infection induces the cleavage of caspase 1. Therefore, we note that the previously proposed functions of UL12 based on its nuclease activities may be important for HSV-1 pathogenesis in vivo. Primers for IFN-γ-inducible protein-10 (IP-10) were 5′-CAGCACCATGAACCCAAGTGC-3′ (sense) and 5′-GCTGGTCACCTTTCAGAAGACC-3′ (antisense).
The cells were incubated in 250 μl lysis buffer (0.1% Triton X-100, 0.1% SDS, 20 mg/ml proteinase K, 10 mM Tris-HCl-1 mM EDTA) at 56°C for 1 h and boiled for 10 min. IL-6 levels were measured as above. (B) DCs were mock-infected or infected with HSV-1 at a MOI of 1 and harvested at the indicated time points after infection (pi). An MOI-response study showed that autophagy is affected only minimally at the different MOIs tested (Fig 3D). Samples were harvested at 16 to 18 h postinfection. Pediatric Clinics of North America, 51(4), 889-908. Protein concentrations were determined by Bradford assay (Bio-Rad).
To construct the SPA BAC, the 3′ portion of UL12 was recoded and fused to a 3′ SPA tag-coding sequence. P1 contains the HSV-1 LAT promoter from NotI to the PvuI site just downstream of the TATA box (the 5′ end of the region replaced in HSV2/LAT1); S1 contains the HSV-1 LAT sequence from the PvuI site through ∼1.5 kb downstream of the 5′ splice site of the intron (the 3′ end of the region replaced in HSV2/LAT1) but excludes ICP0 coding sequences; E1 contains HSV-1 LAT exon 1 from the TATA box to the 5′ splice site of the intron. In the present study, we provide experimental evidence for the absence of apoptosis in sensory neurons latently infected with HSV-1. At 5 hours post infection cells were treated with UV and allowed to recover for one hour. These cells were chosen because they naturally express very low levels of tetherin and are highly permissive for HSV-1 (28). aureus pellet was resuspended in serum-free medium to a concentration of 20×106 CFU/ml for use in subsequent experiments involving infection of nasal turbinate tissue ex-vivo. Genomic DNA from HSV-1 particles was extracted either by a phenol-chloroform method, as previously described (4), or in agarose plugs with a slightly modified procedure.
This figure describes activated … Again, HSV-2 failed to infect double-KO mice in this model. HSV-1 (McKrae) stock was prepared as previously described (23). Retrograde transport of the closely related alphaherpesvirus pseudorabies virus (PRV) has been observed in live cells to be saltatory, with brief, rapid transport events (10). Such advances in understanding the involvement of receptors in tissue are essential preconditions for unraveling HSV invasion of skin, which in turn will allow the development of antiviral reagents. Peripheral blood lymphocytes (PBLs) were isolated by density gradient centrifugation on Ficoll-Hypaque Plus (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Conclusion: The Us3 protein is a crucial factor to suppress T cell activation in HSV-1 infection.
Moreover, we used genetically engineered keratinocytes lacking O-glycan elongation capacity to demonstrate that O-linked glycans are indeed important for HSV-1 biology as HSV-1 particles produced in these cells had significantly lower titers compared to wild-type keratinocytes. Full Text The Full Text of this article is available as a PDF (363K). The restriction was partially overcome by treatment of the cells with polyethylene glycol after adsorption of the low-pH inactivated HSZP virions. These findings suggest that at low temperatures the virus-coded enzyme is dispensable for virus growth in actively dividing tissue culture cells but at high temperatures the enzyme is essential for virus replication. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection.