RTA inhibits vFLIP-induced expression of TNFα and ICAM1 in TREx BCBL1 RTA cells.

<p>A–C. TREx BCBL1 cells were induced with doxycycline (1 ug/ml) and harvested at the indicated time points. Cells were split for western to assess RTA induction and for RNA isolation for qPCR as described above. Results are expressed as fold regulation 2∧(-ΔΔCt) A. Cells harvested for protein were analyzed by SDS-PAGE followed by western blot against RTA and B-actin. B. qPCR analysis of TNFα and ICAM1 expression as explained above. C. qPCR analysis of vFLIP expression at the indicated time points post doxycycline induction. Error bars represent standard error.</p

vFLIP is not visible in cells expressing RTA.

<p>vFLIP expression is decreased in 293T cells cotransfected with RTA. 293T cells were transfected with myc tagged vFLIP and RTA where indicated. vFLIP was detected with anti-myc antibody (Millipore) followed by a rhodamine conjugated secondary antibody (Jackson ImmunoResearch). RTA was detected with our own antibody against RTA followed by a FITC conjugated secondary antibody. Nuclei were visualized using a DAPI containing mounting media.</p

Amino acids 11–149 in RTA are required for degradation of vFLIP.

<p>A. Illustration depicting the RTA truncation mutants used in this study. Note that the rabbit RTA antibody recognizes amino acids 527–539, so not all mutants were detected by western blot. B. 293T cells were transfected with vFLIP and wild type or mutant RTA where indicated. 72 h post-transfection cells were harvested and analyzed by SDS-PAGE followed by western blot against myc(vFLIP), RTA and B-actin. C. 293T cells were transfected with V5 tagged RTA 11–149 and vFLIP where indicated. 72 h post-transfection cells were harvested and analyzed by SDS-PAGE followed by western blot against myc(vFLIP), RTA and B-actin.</p

RTA expression alters vFLIP expression.

<p>A. vFLIP protein is decreased in the presence of RTA. 293T cells were transfected with myc-vFLIP, RTA, or empty vector where indicated. 48 hrs post-transfection, cells were harvested and analyzed by SDS-PAGE followed by western blot with antibodies against myc(vFLIP), RTA, and tubulin. B. RTA induced degradation of vFLIP is dose dependent. 293T cells were transfected with myc-vFLIP and increasing levels of RTA or empty vector control where indicated. 48 hrs post transfection, cells were harvested and analyzed by SDS-PAGE followed by western blot with antibodies against myc(vFLIP), RTA, and tubulin. C. RTA induces proteasomal degradation of vFLIP. 293T cells were transfected with myc-vFLIP and RTA or empty vector control where indicated. The proteasome inhibitor MG132 was added 12 hrs post transfection at the indicated concentrations. DMSO was added in the absence of MG132 in lanes 1 and 2. 48 hrs post transfection, cells were harvested and analyzed by SDS-PAGE followed by western blot with antibodies against myc(vFLIP), RTA, and tubulin.</p

RTA inhibits vFLIP-induced expression of TNFα and ICAM1 in 293T cells.

<p>A-D. 293T cells were transfected with myc-vFLIP, RTA or empty vector control where indicated. At 72 hrs post-transfection, cells were harvested and split for RNA isolation and western blot (see D). For qPCR total RNA was isolated, reverse transcribed and quantified on an ABI7000 with using primers for TNFα and ICAM1 and Sybr green. The housekeeping genes used in the analysis were B-actin and GAPDH. Data was analyzed using the ΔΔCt method. A. vFLIP induced expression of TNFα and ICAM shown as fold regulation 2∧(-ΔΔCt). B and C. vFLIP induced expression of (B) TNFα, p = 0.0001 and (C) ICAM1, p = 0.0001 in the presence or absence of RTA calculated relative to vFLIP alone. Error bars represent standard error. D. Representative western blot from the same experiment, showing vFLIP and RTA protein expression, and RTA induced degradation of vFLIP.</p

The ubiquitin ligase activities of RTA and RAUL are not required for RTA induced degradation of vFLIP.

<p>A. 293T cells were transfected with myc-vFLIP and wild type or mutant (M1-C141S and M3-H145L) RTA or control vector where indicated. 72 hrs post transfection, cells were harvested and analyzed by SDS-PAGE followed by western blot with antibody against RTA, myc(vFLIP) and B-actin. B. 293T cells were transfected with myc-vFLIP, RTA, and myc-RAUL mutant C1051A where indicated. 72 hrs post transfection, cells were harvested and analyzed by SDS-PAGE followed by western blot with antibody against myc(vFLIP), RAUL, RTA, myc(RAUL) mutant and B-actin.</p

vFLIP induces expression of NFκB associated genes.

<p>vFLIP induces expression of NFκB associated genes.</p

Inhibition of the PIAS1-mediated IE1 SUMOylation by IE2 <i>in vitro</i>.

<p>(A) <i>In vitro</i> SUMOylation reactions for IE1 were conducted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103308#pone-0103308-g005" target="_blank">Fig. 5C</a> using His-IE1, GST-SAE2/1, His-Ubc9, GST-SUMO-1_GG, and increasing amounts (0.1, 0.5, and 1 µg) of GST or GST-IE2(346–542) with (left panel) or without (right panel) immunoprecipitated flag-PIAS1. The reaction products were analyzed by SDS-PAGE (8%) and immunoblot assays with anti-IE1 antibody. (B) The GST and GST-IE2(346–542) proteins added to <i>in vitro</i> SUMOylation reactions were detected by immunoblotting with an anti-GST antibody.</p

Evaluation of SUMOylation in cotransfection and <i>in vitro</i> assays.

<p>(A) 293T cells in six-well plates were cotransfected with 0.5 µg of plasmid expressing myc-US34A, myc-UL150, flag-SUMO-1, or flag-SUMO-2 as indicated. At 48 h, total cell lysates were prepared and immunoblotted with an anti-myc antibody. The bands corresponding to unmodified and SUMO-modified forms of myc-US34A and unmodified myc-UL150 are indicated. NS, non-specific bands. (B) <i>In vitro</i> SUMOylation reactions. Myc-UL123(IE1) and myc-US34A produced by <i>in vitro</i> transcription/translation were incubated with GST-SAE2/1, His-Ubc9, and His-SUMO-1_GG or GST-SUMO-1_GG as indicated. The reaction products were analyzed by SDS-PAGE (8%) and immunoblot assays with a myc-IE1 antibody. Unmodified and SUMO-modified forms of IE1 and US34A are indicated. (C) The disorder in UL122 (IE2), UL123 (IE1), and US34A was predicted with the IUPred program (<a href="http://iupred.enzim.hu" target="_blank">http://iupred.enzim.hu</a>). The lysine residues modified by SUMO (for IE1 and IE2) or predicted to be SUMOylation sites (for US34A) are indicated.</p

Alignment of gB subtypes.

<p>Amino acid alignment encompassing the proteolytic cleavage site of gB subtypes detected in the 53 CMV-infected women. Variants with amino acid changes within a subtype are depicted: gB1 (1 and 1**), and gB3 (3 and 3*). The gB1 prototype is Towne, gB2 prototype is AD169, and gB3 prototype is Toledo. The gB1* and the two variants of gB2 are associated with nucleotide changes only. Considering the five major gB subtypes, the two women with multiple strains had gB5/gB1, and gB4/gB1. The subtype distribution among the 51 women who shed one strain was as follows: 15 women shed gB1 (including variants), 9 women - gB2, 10 women - gB3, 4 women - gB4 and 13 women -gB5.</p