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DAPI was utilized to stain total DNA

DAPI was utilized to stain total DNA. complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation Sipeimine of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes Rabbit Polyclonal to E2F6 into an MVA 68k-ank deletion mutant, we discovered that M2, Sipeimine a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-B, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in Sipeimine addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess. synthesis of viral DNA in cytoplasmic factories. As shown in Fig. 2, nuclear EdU fluorescence was bright in mock-infected HeLa cells but dim or absent by 3 h after infection with either MVA or MVA-186. Distinct cytoplasmic DNA factories were evident by bright EdU labeling in the cells infected with MVA at each of the 3-, 4-, and 5-h time points; I3 was present throughout the cytoplasm but was highly concentrated in the replication sites (Fig. 2). In the cells infected with MVA-186, small EdU-labeled viral factories were seen only occasionally, and there was always diffuse cytoplasmic I3 staining (Fig. 2). These data confirmed the deficiency in viral DNA replication in human cells infected with MVA-186, which would account for the defect in postreplicative protein Sipeimine synthesis. Interestingly, the data also show that viral DNA replication was not necessary for the decrease in nuclear DNA synthesis. Open in a separate window FIG 2 Deletion of MVA ORF 186 encoding the 68k-ank protein prevents viral genome replication. HeLa cells were mock infected or infected with 5 PFU/cell of MVA or MVA-186 (186) and incubated with EdU (10 M) for 1-h periods at 3, 4, and 5 h after infection. Following each incubation time, the cells were fixed, permeabilized, and reacted with Alexa Fluor 647 azide. The I3 single-stranded DNA binding Sipeimine protein was visualized by staining with a specific MAb, followed by an anti-mouse secondary antibody conjugated to Alexa Fluor 568 and DAPI to stain total DNA. Images were captured with a confocal microscope. Each panel is divided into quadrants: upper left, I3 (red); upper right, EdU (green); lower left, DAPI (blue); and lower right, merge. The scale bar represents 10 m. hpi, hours postinfection. 68k-ank contributes to MVA genome uncoating in human cells. Since uncoating of the viral genome precedes DNA replication, it was important to determine whether the defect in DNA synthesis might be a secondary effect. We addressed this question by quantification of punctate viral cores, which are destabilized after uncoating and can be detected by immunostaining with antibody to the A4 core protein (12, 14) and by detection of viral genomes which are accessible to the early I3 single-stranded DNA binding protein only after uncoating (14, 28, 29). In the experiment depicted in Fig. 3, HeLa cells were infected with MVA or MVA-186 in the absence of drugs or in the presence of the protein synthesis inhibitor cycloheximide (CHX) or the DNA synthesis inhibitor cytosine arabinoside (AraC). At 1 and 4 h after infection, the cells were fixed and stained with DAPI and antibodies to the A4 and I3 proteins. During the first hour of a normal VACV infection, the numbers of cores represent a balance due to entry and degradation. Without inhibitors, low numbers of cores that stained with A4 antibody were present in cells infected with MVA or MVA-186 at 1 h (Fig. 3A and ?andB).B). The numbers.