Deletion viruses despite the comparable single-step replication of these viruses. This
Deletion viruses regardless of the related single-step replication of those viruses. This suggests that pUL51 plays a crucial function in CCS in Vero cells and that this function is usually partly uncoupled from its previously described function in virus replication and from the virus release function observed right here. The defect in plaque formation was due particularly to the deletion in pUL51, because it was identical in the two independently constructed deletion recombinants and because it was totally corrected inside the complementing cell line that expresses wild-type pUL51 (Fig. 2D). In HEp-2 cells, there was no important virus replication defect for any of your viruses in comparison with the wild form (Fig. 2E). The UL51-FLAG virus along with the two deletion viruses showed a small but significant (P 0.05) release defect compared to the wild type but weren’t drastically unique from every other (Fig. 2F). The two deletion viruses did, however, show a CCS defect compared to both the wild-type and UL51-FLAG viruses (Fig. 2G). This defect was not as dramatic as that seen on Vero cells. Mutant virus plaques were about 6-fold smaller than the plaques formed by the wild-type and UL51-FLAG viruses. Since the deletion viruses plus the UL51-FLAG virus did not differ from each and every other in single-step development or virus release, this suggests that the difference in plaque size is as a result of the loss of a distinct CCS function of pUL51 in the deletion viruses. UL51 consists of a highly conserved YXX motif close to the N terminus. The UL51 protein is thought to localize towards the cytoplasmic face of Golgi membranes, and this localization suggests a possible function in DYRK Purity & Documentation trafficking of viral proteins or virions in transport vesicles that bud from this compartment. We hypothesized that pUL51 contains sequence motifs for this function. A search of the UL51 protein sequence making use of the Eukaryotic Linear Motif online Procollagen C Proteinase drug resource (24) revealed many membrane-trafficking motifs that might be expected to play a function in virion or virus glycoprotein sorting for CCS. A lot of of those motifs, however, have really low sequence complexity and therefore may be anticipated to appear by chance, no matter protein function. To determine likely func-April 2014 Volume 88 Numberjvi.asm.orgRoller et al.FIG 2 Development and spread of UL51 deletions on Vero and HEp-2 cells. (A) Single-step development of BAC-derived HSV-1(F), UL51-FLAG, and two independently isolated UL51 deletion viruses was measured on Vero cells. Stocks have been prepared from the total infected culture (cells and medium). (B) Virus released into the medium throughout the single-step growth experiment shown in panel A. (C) Sizes of plaques formed by wild-type and mutant viruses on Vero cells. Plaque areas were measured 2 days following low-multiplicity infection as described in Supplies and Methods. Each oval represents the region of a single plaque. Twenty plaques were measured for every virus. Note that the y axis has a logarithmic scale. (D) Very same as panel C except that plaques were measured on Vero and UL51complementing cells, as indicated under the graph. (G to H) Very same as panels A to C except that measurements had been performed by using HEp-2 cells. Note that the y axis in panel F features a linear scale. For replication and release measurements (A, B, E, and F), every point represents the mean of three independent experiments, along with the error bars represent the ranges of values obtained. Statistical significance for replication and release experiments, where noted in the text, was determi.