Lonal antibody specific for the ICP4 epitope was used to detect
Lonal antibody specific for the ICP4 epitope was used to detect the tagged pUL114 molecules. A protein with a predicted molecular weight of 32 kDa was specifically detected from the recombinant virus in which pUL114 was tagged at the carboxyl terminus (Fig. 6A). A very light band with the same migration rate wasFigure 2 Repair of RC2620 Repair of RC2620. (A) HEL cells were infected at an MOI of 5 PFU/cell and total DNA was harvested at the indicated times. The quantity of viral DNA for AD169 (black squares), RC2620 (black circles), and RQ2620 (open circles) were determined by dot blot hybridization as described in materials and methods. (B) Titers of AD169 (black squares), RC2620 (black circles), and RQ2620 (open circles) are shown. The time point at 0 hpi represents the titer of the input virus.Page 5 of(page number not for citation purposes)Virology Journal 2005, 2:http://www.virologyj.com/content/2/1/detect the coprecipitated protein. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26240184 This experiment confirmed the earlier result and demonstrated that it was also possible to specifically coprecipitate ppUL44 with pUL114 fusion proteins (Fig. 6B). Consistent with the previous result, the coprecipitation appeared to be less efficient for pUL114 labeled at the amino terminus. To confirm these results, plasmids expressing ppUL44 (pMP62) and pUL114 with a carboxyl terminal EGFP tag were transfected into monolayers of primary foreskin fibroblast cells. In cells transfected with pMP62 alone, ppUL44 localized exclusively to the nucleus and is shown merged with DAPI image (Fig 7A), which was similar to the localization observed in infected cells early in infection. Cells expressing either the full length pUL114-EGFP fusion protein (pMP39), PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28250575 or the fusion protein in which aa 3?4 were deleted from pUL114 (pMP41) exhibited punctate cytoplasmic fluorescence (Fig 7B, C). This localization pattern was Isoarnebin 4 site distinct from the nuclear staining observed with the UL114 CTAG recombinant virus. However, when ppUL44 and full length pUL114 fusion proteins were coexpressed in the same cell, pUL114 was recruited to the nucleus with ppUL44 (Fig 7D ), consistent with its nuclear localization in the context of infected cells. A small quantity of ppUL44 also appeared to localize to a subset of the cytoplasmic punctae containing pUL114. Deletion of aa 3?4 from the pUL114 fusion protein eliminated its recruitment to the nucleus by ppUL44, suggesting that this domain is required for the interaction ppUL44 (Fig 7G ). This interpretation of the data is consistent with the impaired nuclear localization observed with UL114 NTAG-infected cells, in which the amino terminal domain of pUL114 was altered through the addition of the ICP4 epitope tag (data not shown). Also consistent with this result, is the inefficient coprecipitation of ppUL44 with pUL114 fusion proteins when the tags were fused to the amino terminus (Fig. 6). These data suggest that these proteins associate in a manner that is dependent on aa 3?4 of pUL114, and independent of other viral proteins or viral DNA. These experiments do not, however, eliminate the possibility that they might associate in an indirect manner through cellular proteins.Characterizing the defect in DNA synthesis The localization of pUL114 to replication compartments, and its apparent association with ppUL44, which is known to interact with the DNA polymerase [9] imply that this molecule is part of the viral DNA replication complex. This interpretation of the data is consistent with the obser.