(A and B) HEK 293T cells were either mock transfected or transfected twice with PAPII or PAPγ duplex siRNA oligos (si #1, si #2, or a mixture of #1+#2) or one of two nonspecific control siRNA oligos (scr si #1 or scr si #2). Twenty-four hours after the final siRNA transfection, the cells were transfected with a DNA plasmid expressing SOX; each sample was split in half and, 24 h later, either harvested for protein and immunoblotted with PAPII and PAPγ antibodies to gauge the efficiency of siRNA-mediated knockdown (A), or processed for oligo(dT) in situ hybridization and α-SOX immunofluorescence to monitor the efficiency of SOX-induced poly(A) RNA accumulation (B). Arrows denote the location of PAPII and PAPγ protein on the western blots in (A). Nonspecific cross-reactive bands serve as loading controls. (C and D) HEK 293T cells were transfected with the indicated siRNA as described above, followed by subsequent transfection with either a plasmid expressing GFP alone or together with a SOX expression plasmid. Cells were treated with 5 ng/ml LMB for 6 h prior to harvesting either protein for western blotting with PAPII, PAPγ, and SOX antibodies (C), or RNA for northern blotting with GFP and 18S probes (D).

TIME cells were either mock infected, latently infected with KSHV, or infected with KSHV and lytically reactivated using an adenoviral expression vector containing the viral lytic transactivator RTA (Ad-RTA) for 8, 12, or 24 h. Mock-infected cells were similarly treated with Ad-RTA. PABPC and SOX proteins were detected by immunofluorescence analysis with polyclonal PABPC antibodies and affinity purified SOX polyclonal antibodies.

(A) Diagram of reporter constructs either containing the WT polyadenylation signal sequence (GFP) or lacking the polyadenylation signal and terminating in either a hammerhead ribozyme element (GFP-HR) with or without a preceding templated 60-nt poly(A) or poly(U) tail (GFP-A₆₀-HR and GFP-U₆₀-HR, respectively), or the histone 3′ SL (GFP-hisSL). (B) A total of 200 ng of each GFP plasmid was transfected into HEK 293T cells, which were harvested 24 h later and subjected to western blotting with GFP antibodies to show relative protein expression from each construct. (C and D) HEK 293T cells were transfected with the indicated GFP construct alone or together with a SOX expression construct at a 1∶2 ratio (50 ng of GFP, 100 ng of SOX). Total RNA was harvested from each sample 24 h post-transfection and northern blotted with GFP and 18S probes. (E and F) HEK 293T cells were transfected twice sequentially with the indicated duplex siRNA oligos. Twenty-four hours after the final siRNA transfection, the cells were transfected with the GFP-A₆₀-HR plasmid alone or together with a SOX expression plasmid at a 1∶2 ratio, and incubated in media containing 5 ng/ml LMB for 6 h prior to harvesting. Twenty-four hours later, the cells were harvested either for RNA and northern blotted with GFP and 18S probes (E), or for protein and western blotted with PABPC, PABPN, and actin (loading control) antibodies to monitor the efficiency of siRNA-induced knockdown (F). Quantification (normalized to 18S levels) is shown below each northern blot. The level of each GFP mRNA in the absence of SOX was set to 1.0, and the corresponding level of that particular mRNA in the presence of SOX was then calculated.

(A and B) HEK 293T cells were transfected with a plasmid expressing GFP alone or together with a plasmid expressing SOX and either left untreated (A) or treated with 5 ng/ml LMB for 6 h (A and B). Twenty-four hours post-transfection, total RNA was harvested, resolved on an agarose-formaldehyde gel, and northern blotted with a ³²P-labeled GFP probe. The line through the gel indicates where an intervening lane was cropped out of the image (B). (C) HEK 293T cells were transfected with the indicated plasmids and treated with LMB as described above. Total RNA was prepared from the cells and digested with RNaseH in the presence or absence of oligo(dT), then resolved by agarose-formaldehyde electrophoresis, and then northern blotted with a ³²P-labeled GFP probe. In (A–C) 18S rRNA serves as a loading control. (D) HEK 293T cells were transfected with the indicated plasmid and, 24 h later, subjected to oligo(dT) in situ hybridization (upper panels) followed by staining with SOX antibodies (for vector, SOX, P176S samples) or HA antibodies (for HA-HSV AE sample) (center). The lower panels show overlap of the oligo(dT) and antibody staining. The right column shows a magnified version of two cells (one expressing SOX and one lacking SOX) from an inset derived from the SOX-transfected sample.

(A) HEK 293T cells were transfected with empty vector or with a plasmid expressing SOX and, 24 h later, subjected to double-label immunofluorescence analysis with monoclonal 10E10 PABPC antibodies (left) and SOX polyclonal antisera (center). The overlap of PABPC and SOX staining can be viewed in the right column. (B) HEK 293T cells were transfected with the indicated single-function SOX mutant and subjected to immunofluorescence as described above using 10E10 PABPC antibodies and SOX antisera. (C) HEK 293T cells were transfected with HA-tagged WT SOX or the HSV AE SOX homolog and subjected to immunofluorescence as described above using polyclonal PABPC antisera and monoclonal HA antibodies. (D) HEK 293T cells were transfected with a plasmid expressing GFP alone or together with a SOX expression plasmid and, 24 h later, treated with 5 ng/ml LMB for 12 h. The cells were then incubated in media lacking LMB but containing 1 µg/ml actinomycin D to block transcription, and the cytoplasmic fraction was isolated at the indicated times. RNA was then northern blotted with GFP and 18S probes, and the half-life (t_1/2) of the cytoplasmic GFP mRNA with and without SOX was calculated. Error bars show the standard error between samples. The graph represents a compilation of three independent experiments.

HEK 293T cells were transfected twice with PABPC or PABPN duplex siRNA oligos or a nonspecific control siRNA oligo (scr si). Twenty-four hours after the final siRNA transfection, the cells were transfected with a DNA plasmid expressing the GFP reporter alone or together with SOX; each sample was split in half and, 24 h later, either harvested for protein and immunoblotted with PABPC, PABPN, and SOX antibodies to gauge the efficiency and specificity of siRNA-mediated knockdown (A), or harvested for RNA and either northern blotted with GFP and 18S probes (B) or subjected to qPCR analysis (C) to monitor SOX activity. Each qPCR reaction was run in triplicate, and GFP mRNA levels were normalized to 18S RNA, because cellular housekeeping genes are subject to host shutoff by SOX. Error bars show standard error between sample replicates. Nonspecific cross-reactive bands serve as loading controls for the western blots in (A). Lines through gels indicate where intervening lanes were cropped out of the image.

SOX stimulates mRNA hyperadenylation in a manner dependent on the activity of both PAPII and PABPN. This aberrant 3′ processing event may trigger destruction of these mRNAs by quality control ribonucleases (shown as a pacman), possibly recruited to the mRNAs by PABPN and the nuclear relocalized PABPC. Within the cytoplasm, removal of PABPC could also be envisioned to negatively affect the stability of mRNAs by rendering their 3′ termini unprotected from ribonuclease digestion and decreasing translation efficiency.