UL103 mutant virus genome structure and complementation. (A) Schematic depiction of the Towne-BAC genome, where boxes 1 to 7 identify herpesvirus-conserved gene clusters (31). The replacement of the US1-US12 region in Towne-BAC with a BAC and a GFP eukaryotic expression cassette has been previously described (30). The region encompassing UL103 in gene cluster 6 is shown expanded, with Towne-BAC, rescue, intermediate Kan^r-SacB constructs, and replacement mutant viruses schematically presented. (B) Ethidium bromide-stained electrophoretically separated AvrII-NheI restriction fragment digestion products of Towne-BAC, UL103-R, and UL103-Stop-F/S Bacmid DNA (left panel) and the same samples following DNA blot hybridization to detect the UL103 ORF (right panel). Insertion of AvrII restriction sites into the UL103-Stop-F/S virus resulted in a band at 18.9 kbp (black arrow) and one at 3 kbp (black arrowhead). The thick band at 3.3 kbp (asterisk) is plasmid pSim6. (C and D) Complementation assay using UL103myc-expressing and control HF. Towne-BAC, Δ_53-750UL103, UL103-R, and UL103-Stop-F/S virus infections were carried out at an MOI of 0.005 in the presence of 0.01% CMV antibody-positive pooled human IgG to prevent secondary plaque formation. Cells were fixed and stained for IE1/IE2 at 7 days postinfection (dpi), and results are graphed as the average numbers of cells per focus ± standard deviation (SD) (error bars).

Immunoblot analyses of viral proteins from HF infected with Towne-BAC, UL103-R, UL103-Stop-F/S, or Δ_53-750UL103 virus and mock-infected HF (MOI of 3). Detection of IE1/IE2, ppUL44, ppUL99 (pp28), and β-actin were carried out at 24, 48, and 72 hpi. Lane M, mock-infected HF.

Single-step growth curve (MOI of 3) of UL103-R (circles) and UL103-Stop-F/S (rectangles) viruses during block and reversal of BDCRB. Cells and medium were harvested individually every day for 7 days and frozen at −80°C. Virus titer was determined and graphed as log₁₀ mean value plus SD. The data point at 0 dpi indicates the input virus dose.

TEM of cells infected with UL103-R virus (A, C, and E) or with UL103-Stop-F/S virus (B, D, and F) at 5 dpi. (A to D) Virus-infected cells 1 day after the reversal (5 dpi) of BDCRB. (E and F) Virus-infected cells that were not treated with BDCRB. The small black arrows point to viral particles, and the black arrowheads point to DBs. The large black arrows in panels B and F point to aggregates. The nucleus (Nu), cytoplasm (Cyt), and extracellular space (ECS) are indicated. Bars, 5 μm (A and B) and 1 μm (C to F).

Multistep (A) (MOI of 0.3) or single-step (B) (MOI of 3) growth curves of Towne-BAC (squares), UL103-R (circles), and UL103-Stop-F/S (triangles) reconstituted viruses. The cells and medium were harvested at the indicated times postinfection and frozen at −80°C. The virus titer was determined in triplicate by plaque assay on HF and graphed as log₁₀ mean value ± SD. The data point at 0 dpi indicates the input virus dose.

Immunofluorescence localization of cellular and viral antigens in uninfected and UL103myc-infected cells. (A to L) Immunofluorescence localization of cellular antigens in UL103myc-expressing uninfected cells using mouse anti-golgin-97 (C), mouse-anti-GM130 (F), mouse anti-EEA-1 (I), and mouse anti-CD63 (L) and donkey anti-mouse conjugated to FITC (green). UL103-myc (B, E, H, and K) was visualized with chicken anti-myc and donkey anti-chicken secondary antibody conjugated to rhodamine (red). Nuclei were stained with Hoechst 33258 (blue). Panels A, D, G, and J display merged images of Hoechst 33258, rhodamine, and FITC. (M to O) Immunofluorescence localization of UL103myc (N) at 3 dpi of Δ_53-750UL103 virus-infected cells (MOI of 1) (O) using chicken anti-myc and anti-chicken secondary antibody conjugated to rhodamine (red). (M) Merged image of rhodamine, GFP fluorescence, and Hoechst 33258. (P to d) Immunofluorescence localization of cellular and viral antigens at 3 dpi of Townevar ATCC-infected cells (MOI of 1). pp28 (R), golgin-97 (U), GM130 (X), EEA-1 (a), and CD63 (d) were detected using donkey anti-mouse secondary antibody conjugated to FITC, while myc (Q, T, W, Z, and c) was detected with donkey anti-chicken secondary antibody conjugated to rhodamine. Panels P, S, V, Y, and b display merged images of Hoechst 33258, rhodamine, and FITC. UL103myc was functional in these cells, as shown in Fig. 1C and D. Original magnification obtained by confocal microscopy, ×1,000.

Characterization of viral components in cells treated with BDCRB and infected with UL103-R virus (A and C) or UL103-Stop-F/S virus (B and D) at 4 dpi by TEM. The nucleus (Nu), cytoplasm (Cyt), extracellular space (ECS), and assembly compartment (AC) are indicated. The arrows in panel B point to cytoplasmic aggregates. (C and D) TEM depicting size bar of 0.5 μm. In panels C and D, the arrows point to dense bodies (DBs) and the arrowhead points to a noninfectious enveloped particle (NIEP). Bars, 5 μm (A and B) and 0.5 μm (C and D).

Immunofluorescence localization of viral and cellular antigens in cells infected with UL103-R or UL103-Stop-F/S virus and treated with BDCRB. The cells were infected at an MOI of 0.3 and fixed 4 dpi. pp28, golgin-97, GM130, LAMP-1, and CD63 were visualized with donkey anti-mouse conjugated to rhodamine. Original magnification, ×1,000.