Target CHO cells were transfected with pCDNA3 (empty vector), PILRα, or nectin-1, along with a reporter plasmid expressing luciferase under the control of the T7 promoter. Infection with a PRV recombinant that expresses both an mRFP-tagged capsid protein and a GFP-tagged envelope protein.We infected mice with PRV 341, a virulent PRV Becker recombinant in which the minor capsid protein VP26 was fused to mRFP and the envelope protein Us9 was fused to green fluorescent protein (GFP) (16). After maturation, only axons that could penetrate these barriers are found in the N compartment (Fig. 4). 3E to G). Lancaster KZ, Pfeiffer JK. Although it is assumed that PRV and HSV-1 do not produce the prodigious diversity generated by RNA viruses during replication, our study identified a distinct restriction of axon-epithelial cell spread of infection at or near the point of egress.
Data were normalized to the expression level of HSV-1 gH/gL, which was set at 100%. Mice as described in the legend for Fig. Zip codes near herpesvirus DNA ends The presence of zip codes near the ends of herpesvirus genomes is of interest as it suggests zip codes may be involved in two functions, uptake of virus DNA into the host cell nucleus and circularization of the DNA once it has entered. Reductions in the degree of homology of the HVP-2 sequence to that of SA8 were also observed in other locations in the genome. Based on previous electron microscopic and immunofluorescence analyses of axons (8, 13, 18, 24, 34, 37, 39), these puncta represent individual capsids and vesicles containing viral glycoproteins. Coexpression of HSV-1 US3 kinases with wild-type and mutant FLAG-HDAC2 produced results identical to that observed with VZV. SaHV-1 glycoprotein-mediated fusion with PILRα-expressing cells was greatly reduced compared to that for nectin-1 or CD155 fusion.
Intraaxonally, enveloped virions within vesicles associated with microtubules were observed (Fig. By 6 h, more and larger ORF29 punctae were visible and formed widely distributed clusters in the nuclei of infected cells. (1978). (B) Calculation of plaque diameters. Proteins that increased after IFN-β treatment were assigned to multiple functional categories, including cytoskeleton regulation, translation regulation, signal transduction, cell growth regulation, secretion and exocytosis, metabolism regulation, and cell adhesion. Also, deselect the checkbox for “Close Active Shutter during Stage movement”. (A) TCC were cultured for 6 h with autologous BLCL pulsed with the indicated peptides or medium as a negative control.
This will allow frequent image acquisition over long time periods without excessive photo-damage. A hallmark of WNV neuropathogenesis is the disruption of the BBB resulting in uncontrolled entry of immune cells into the brain. Considering that current knowledge is based on analyses of the functions of a few viral proteins, a wealth of data awaits to be discovered through analyses of the many multifunctional proteins expressed by virus. Second, we infected PK15 cells to make infected-cell lysates and Western blotted with antibodies against gD, and we verified that no gD could be detected in these lysates. These may represent previously described “light” or “L-particles” , , which are expected to have the same membrane topology as a virion. 1E). In neonatal kittens, the infection can generalize and is associated with neurological signs and a high mortality rate.
In addition, regions with an abundance of hydrophilic residues are generally more likely to be exposed to the solvent on the surface of proteins, and thus may be better insertion sites than buried hydrophobic regions. The HSV-1 miR-H1 miRNA, which has previously been detected only in cells undergoing productive HSV-1 replication (11, 39), was not recovered in this analysis of latently infected human TG. 3516; Corning, Corning, NY). FBKT1 and Huh-7 cells were maintained in RPMI 1640 medium supplemented with 10% FBS. PRV 758R (gE-null revertant) was constructed from PRV 758. Cells were fixed and stained as described previously (22). To ensure that axonal transport of other cargoes can still occur in these cultures, we simultaneously imaged capsids (labeled with GFP) and mitochondria (labeled with MitoTracker Red).
Lam gC− possesses a deletion in the gene encoding glycoprotein C (gC) (24), whereas the gene encoding gE is deleted in the Lam gE− mutant (59). The VZV DNA load, which varied among subjects (Fig. The activation of a local IFN response in axons represents a new paradigm for cytokine control of neuroinvasion. We found that axons respond differentially to type I and type II interferons. (B and D) After 20 days, the S compartments of chambers in panel A were superinfected with UV-inactivated PRV 959 (green capsids) at an MOI of 100 (B), and the S compartments of chambers in panel C were superinfected with non-UV-inactivated PRV 959 at an MOI of 0.1 (D). Closed arrowheads indicate doubly positive structures, whereas open arrowheads indicate singly positive structures. The magnified panel shows the approximate imaging window and the directions, defined as anterograde and retrograde.