pUL135 and pUL138 impact phosphorylation of EGFR.

<p>Fibroblasts were infected with WT, <i>UL135</i>_STOP, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 1. At 48 hpi, infected cells were pulsed with 10nM EGF for 15min and then lysed. (A) EGFR was immunoprecipitated with ms α-EGFR and both IP and lysate samples were separated by SDS-PAGE. Blots were stained with rb α-EGFR, rb α-EGFR phosphotyrosine 1068, ms α-phosphotyrosine, and ms α-IE1/2. (B) The quantification of phosphorylation over three experiments is shown. To control for the variation in EGFR levels in different infections, we normalized signals associated with pY1068 or pY to total EGFR. Statistical significance relative to WT was calculated by student t-test (* p-value ≤ 0.05). (C) Serum-starved or fed fibroblasts expressing EGFR_3XFLAG were infected with WT CMV at 20 hours post transduction. Cells were stained with ms α-EGFR, rb α-EGFR pY1068 at 48 hpi. A merge of all three images in shown to the right. (D) Serum-starved, infected fibroblasts were pulsed with Alexa Fluor 647-conjugated EGF ligand on ice, fixed 20 min after a shift to 37C and stained with rb α-EGFR. Cells were imaged by deconvolution microscopy. For C and D, nuclei are stained with DAPI.</p

EGFR association with Rab 5 and Rab 11 vesicles increased in the context of infection.

<p>Fibroblasts over-expressing EGFR_3XFLAG were stained with ms α-EGFR and (A) rb α-Rab5 as a marker of sorting endosomes, or (B) rb α-Rab11 to stain the ERC at 48 hpi. Cells were imaged by deconvolution microscopy. For all panels, nuclei are stained with DAPI. A merge of all three images in shown to the right. The extent of colocalization between (C) Rab 5 or (D) Rab 11 vesicles was quantitated by the Sqaussh.workfkow in the Mosaic suite of Image J and Fiji. The y axes represent the average proportion of the indicated Rab that is coincident with EGFR. Statistical significance was determined by a one-way ANOVA with Tukey correction for percent overlap. Standard deviation is depicted by error bars. Asterisks represent statistically significant differences (p values <0.001).</p

EGFR signaling maintains latency.

<p>CD34⁺ HPCs were infected with WT, <i>UL135</i>_<i>STOP</i>, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 2. (A) At 1, 4, and 8 dpi cells were stained with Alexa Fluor 647-conjugated EGF ligand and BV421-conjugated ms α-CD34 for FACS analysis to measure EGFR surface levels. Bars represent the average of three independent experiments where EGFR surface levels are relative to mock infection. Standard error of the mean is shown. Asterisk indicates significance by Student’s t-test where p<0.05. (B) Pure populations of CD34⁺ HPCs infected with WT, <i>UL135</i>_STOP, or <i>UL138</i>_STOP at an MOI of 2 were isolated by FACS at 24 hpi and maintained in LTBMC with EGFR inhibitor AG1478 at 10 μM. At 10 dpi, viable CD34⁺ HPCs were seeded by limiting dilution onto monolayers of permissive fibroblasts (reactivation). An equivalent number of cells were mechanically disrupted and seeded in parallel to determine the infectious virus present in the culture prior to reactivation (pre-reactivation). Frequency of infectious centers formed pre and post reactivation was determined 14 days later from the number of GFP-positive wells at each dilution using ELDA software. Bars represent the average fold change in drug-treated cells relative to vehicle (DMSO) pre-reactivation control for three independent experiments. Standard error of the mean is shown. (C) WT-infected CD34+ cells treated with PI3K inhibitor LY294002 at 20 μM were analyzed as described for panel B. (B-C) Significance determined by two-way ANOVA with Bonferroni correction for differences between vehicle controls and drug treatments, asterisk indicates p-value<0.05.</p

Inhibition of EGFR signal promotes viral replication.

<p>Fibroblasts infected with 0.5 MOI of WT virus were treated with DMSO, (A) 5 μM AG1478 or (B) 20 μM LY294002 at 18hpi. Cells and media were collected over a time course and virus yields were determined by TCID₅₀. Values represent the average of three independent experiments with SEM shown. Statistical significance was determined by two-way ANOVA with Bonferroni correction for differences between vehicle controls and drug treatments are indicated by asterisks (* p-value<0.05; ** p-value<0.01).</p

Primer sequences.

<p>Primer sequences.</p

Activated EGFR localizes to the viral assembly compartment irrespective of stimulation.

<p>Mock- or WT-infected fibroblasts were stained with rb α-EGFR pY1068 and (A) ms α-GM130 to stain the Golgi or (B) ms α-pp28 as a marker for the viral assembly compartment. Cells were imaged by deconvolution microscopy. For all panels, nuclei are stained with DAPI. A merge of all three images in shown to the right.</p

A model for pUL135/pUL138-mediated regulation of EGFR trafficking.

<p>We hypothesize that pUL135 and pUL138 oppose one another in the regulation of EGFR to affect replicative or latent states of infection, respectively. pUL135 impedes recycling of EGFR back to the cell surface or promotes its turnover, whereas pUL138 sustains EGFR surface levels and activity. Within productively infected cells, EGFR is associated with endocytic vesicles localized to the VAC. As mTOR and transferrin have also been shown to localize in the VAC, we further propose that the VAC may function as a “viral signaling compartment” in addition to its role in virus maturation. This compartment may allow HCMV to insulate host signaling from extracellular up or downregulation or be a means by which the virus sense and responds to host cues.</p

Antibody description and sources.

<p>Antibody description and sources.</p

pUL135 and pUL138 regulate EGFR trafficking.

<p>Fibroblasts were infected with WT, <i>UL135</i>_STOP, and <i>UL138</i>_<i>STOP</i> virus at an MOI of 1. At 48hpi, cell were stimulated with EGF, collected over a time course of 0–180 minutes post pulse, and stained with BV421 conjugated ms α-EGFR. EGFR surface levels were measured in infected (GFP+) cells by FACS. (A and B) EGFR surface levels over the time course in mock- and WT-infected cells. The WT curve is replotted in panel B on a scale to better discern trafficking dynamics. (C-E) EGFR trafficking during <i>UL135</i>_<i>STOP</i> and <i>UL138</i>_<i>STOP</i> infection in fibroblasts relative to WT. Panel D expands the 0–60 minute time points and panel E expands 0–20 min. (F) Data from selected timepoints were chosen and compared to initial EGFR levels summarize the differences within each infection.</p

pUL138 and pUL135 interact with EGFR.

<p>(A) pUL138 fused with a C-terminal 3XFlag epitope tag was immunoprecipitated with a Flag-specific antibody from lysates derived from fibroblasts infected with TB40/E-<i>UL138</i>_{<i>3XFLAG</i>} at 48 hpi. Following tryptic digest, peptides were identified by LC-MS/MS. Candidates were subtracted from a parallel Flag antibody pull-down from infected cell lysates without a Flag-tagged protein. 128 total candidates remained after subtraction. High priority candidates belonging to a network of interactions were identified by STRING and NCBI analysis. UL138 interacted with EGFR with a number of proteins associated with EGFR signaling. (B) Interaction between EGFR and either pUL135 or pUL138 was confirmed by the reciprocal co-immunoprecipitation. Fibroblasts were transduced with lentiviruses expressing <i>UL135</i>_<i>MYC</i>, <i>UL138</i>_<i>MYC</i> or <i>UL37</i>_<i>MYC</i> (control). EGFR was precipitated using ms α-EGFR and interactions were detected by blotting with chk α-myc or rb α-EGFR. (C) EGFR was immunoprecipitated from lysates derived from fibroblasts mock-infected or infected with 1 MOI of WT, <i>UL138</i>_STOP, <i>UL135/8</i>_STOP, or <i>UL133</i>_MYC (control). Interactions were detected by blotting for rb α-pUL135, rb α-pUL138, and chk α-myc. D. pUL135_V5 was immunoprecipitated using ms α-V5 from lystates derived from HEK cells overexpressing pUL135_V5, pUL138_MYC, or both. Interactions were detected by immunoblotting with chk α-myc and chk α-V5. For B-D, total lysates are shown.</p