JVI Accepted Manuscript Posted Online 17 February 2016 J. Virol. doi:10.1128/JVI.00179-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Expression of oncogenic alleles induces multiple blocks to HCMV infection

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Shihao Xu, Xenia Schafer, and Joshua Munger*

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Department of Biochemistry and Biophysics

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University of Rochester Medical Center

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Rochester, New York 14642, USA

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*Corresponding author:

Phone: (585) 273-4800

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Fax: (585) 275-6007

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E-mail:[email protected]

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Abstract

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In contrast to most viruses, Human cytomegalovirus (HCMV) is unable to productively infect

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most cancer-derived cell lines. The mechanisms of this restriction are unclear. To explore this

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issue, we tested whether defined oncogenic alleles including the Simian virus 40 (SV40) T

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antigen (TAg) and oncogenic H-Ras inhibit HCMV infection. We find that expression of the

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SV40 TAg blocks HCMV infection in human fibroblasts, whereas the replication of a related

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herpes virus, herpes simplex virus 1 (HSV-1), is not impacted. The earliest restriction of HCMV

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infection involves a block of viral entry, as TAg expression prevented the nuclear delivery of viral

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DNA and pp65. Subsequently, we found that TAg expression reduces the abundance of

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platelet-derived growth factor receptor α (PDGFRα), a host protein important for HCMV entry.

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Viral entry into TAg-immortalized fibroblasts could largely be rescued by PDGFRα over-

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expression. Similarly, PDGFRα over-expression in HeLa cells markedly increased HCMV gene

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expression and DNA replication. However, robust production of viral progeny was not restored

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by PDGFRα over-expression in either HeLa or in TAg-immortalized fibroblasts, suggesting

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additional restrictions associated with transformation and TAg expression. In TAg-expressing

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fibroblasts, the immediate early 2 (IE2) protein was not rescued to the same extent as the

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immediate early 1 (IE1) protein, suggesting that TAg expression impacts the accumulation of

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major immediate early (MIE) transcripts. Transduction of IE2 largely rescued HCMV gene

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expression in TAg-expressing fibroblasts, but did not rescue the production of infectious virions.

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Collectively, our data indicate that oncogenic alleles induce multiple restrictions to HCMV

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replication.

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Importance

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HCMV cannot replicate in most cancerous cells, yet the causes of this restriction are not clear.

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The mechanisms that restrict viral replication in cancerous cells represent viral vulnerabilities

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that can be potentially therapeutically exploited in other contexts. Here we find that SV40 T

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antigen-mediated transformation inhibits HCMV infection at multiple points in the viral life cycle,

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including proper viral entry, normal expression of immediate early genes, and viral DNA

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replication. Our results suggest that the SV40 T antigen could be a valuable tool to dissect

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cellular activities that are important for successful infection, thereby potentially informing novel

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anti-viral development strategies. This is an important consideration given that HCMV is a

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leading cause of birth defects and causes severe infection in immunocompromised individuals.

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Introduction

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HCMV is a ubiquitous opportunistic β-hepesvirus that infects ~50-70% of the global population.

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While infection of healthy individuals is frequently resolved without severe complications, HCMV

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poses a major threat to immune-compromised individuals such as AIDS patients and organ-

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transplant recipients (1, 2). Further, HCMV is a leading cause of birth defects, affecting

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approximately five thousand newborns in the United States every year (3). In the

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immunocompetent host, an efficient antiviral immune response limits viral infection, and HCMV

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enters a latent phase in hematopoietic progenitor cells, which is characterized by silencing of

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the major immediate early (MIE) promoter and subsequent limitation of viral gene expression

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(4).

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HCMV is somewhat unique compared to many viruses that can be propagated in vitro in that it

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is incapable of productively infecting most commonly used transformed cell lines (1, 5). In

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contrast to other herpes viruses such as the Herpes Simplex Viruses, HCMV must be

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propagated in non-transformed cells, most commonly in primary fibroblasts. One known

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contributing factor is the inability of laboratory adapted strains of HCMV to infect a broad range

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of cell types including epithelial cells whereas clinical isolates of HCMV exhibit a wide tropic

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range, and are typically capable of infecting fibroblasts, macrophages, epithelial and endothelial

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cells (1). Continued propagation of laboratory strains in fibroblasts results in the inability to

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productively infect epithelial and endothelial cells. This tropic defect results from the

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accumulation of mutations in the UL128-131 region (6, 7). This region encodes for proteins that

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form the gH/gL/UL128-131 complex, which is essential for viral entry into epithelial and

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endothelial cells (6-10). While repair of this region enables entry into transformed epithelial cell

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lines such HeLa cells, production of viral progeny is still minimal (6), suggesting additional host

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cell restriction mechanisms. Further, fibroblastic cells are also unable to support productive

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HCMV infection (5). The mechanisms of this viral cellular restriction are unclear.

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To explore how oncogenic signaling might limit HCMV replication, we employed a genetically

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defined model of tumorigenesis, which consists of primary human foreskin fibroblasts, life-

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extended telomerase-expressing fibroblasts, and life-extended fibroblasts that express the

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Simian virus 40 (SV40) T antigens (TAg), either alone or in conjunction with expression of an

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oncogenic H-Ras G12V allele (11). This genetically defined model enables the deconvolution of

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the genetic complexity associated with transformation, and therefore permits study of the

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mechanisms of oncogenic viral restriction. We found that the expression of the SV40 TAg

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severely attenuated HCMV but not herpes simplex virus replication. This restriction involved

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inhibition of the nuclear delivery of viral DNA and the pp65 tegument protein, suggesting that

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TAg expression interferes with viral entry. TAg expression was found to substantially down-

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regulate PDGFRα, a cell surface protein that plays a major role during viral entry (12). Viral

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entry could largely be rescued by PDGFRα over-expression, whereas viral gene expression and

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DNA replication were only partially rescued, and the production of infectious viral progeny was

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not rescued at all. In the presence of increased PDGFRα, expression of the SV40 TAg inhibited

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the accumulation of IE2, the major viral transcriptional activator. Transduction of IE2 further

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rescued viral gene expression, but not production of infectious HCMV. Combined, these results

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indicate that oncogenic signaling induces several blocks to the HCMV life cycle, including

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reduced expression of an important host entry factor, and down-regulation of viral gene

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expression.

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Materials and methods

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Cell culture and viral infection. Ras-transformed BJ fibroblasts, telomerase-SV40 TAg

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immortalized BJ fibroblasts, and primary BJ fibroblasts (ATCC CRL-2522) were courteously

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provided by Robert Weinberg (Whitehead Institute for Biomedical research). Telomerase-

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expressing BJ fibroblasts were created via lentiviral transduction (see below). Primary

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fibroblasts, their telomerase-expressing, immortalized and transformed derivatives were

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cultured in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) supplemented with 10%

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fetal bovine serum, 4.5 g/L glucose and 1% penicillin–streptomycin (Pen-Strep; Life

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Technologies). Primary fibroblasts, telomerase-expressing, immortalized, and transformed

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derivatives were seeded at a density of 5000 cells per well of standard 12-well plate 24 h before

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viral infection. Unless indicated otherwise, the strain utilized in all studies was BADwt derived

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from a bacterial artificial chromosome (BAC) clone of HCMV AD169 laboratory strain (13).

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BADsubUL21.5, which expresses EGFP driven by the SV40 early promoter, replicates with

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similar kinetics as wild-type virus (14). HCMV TB40/e strain that expresses EGFP driven by the

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SV40 early promoter was provided by Dr. Eain Murphy (15). Viral genomes were BrdU labeled

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as previously described (16). Briefly, MRC-5 fibroblasts were infected with AD169 at multiplicity

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of infection (MOI) of 0.05. When the cells exhibited ~90% cytopatheic effect (CPE) (~5 dpi),

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fresh medium containing 10 µM BrdU was added. Forty-eight h later, the medium was spiked

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with an additional 10 µM BrdU for an additional 24 h before harvesting for a viral stock. This

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stock was designated as BrdU-AD169, and previously found to replicate with similar kinetics as

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non-labeled wild-type virus (16).

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Cells were infected with HSV-1 (KOS) or HCMV at an (MOI) of 3 for 1.5 h in the absence of

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serum. The viral inoculum was then removed and replaced with fresh serum-free medium. To

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measure production of infectious viral progeny, infected cells (at 5 dpi unless otherwise

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specified) were scraped, sonicated, centrifuged at 3000 rpm for 5 min and stored in -80 °C 6

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before performing plaque forming assay on MRC-5 human fibroblasts (HCMV), or Vero cells

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(HSV).

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Cloning. Human telomerase (hTERT) cDNA was amplified by PCR from pWZL-Blast-Flag-HA-

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hTERT (addgene #22396) using the following primers: F:

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5’GGAACCAATTCAGTCGACTGGGATCCCGTCCTGCTGCGCACGTG3’;

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R, 5’TTTGTACAAGAAAGCTGGGTTCTAGATCAGTCCAGGATGGTCTTGAAGTCTG3’. hTERT

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cDNA was then cloned via Gibson assembly (17) into the BamHI and XbaI sites of pLenti

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CMV/TO Hygro (Addgene #17484). To generate pLenti6/CMV/PDGFRα, human PDGFRα

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(isoform X1) was amplified from cDNA synthesized with SuperScript II Reverse Transcriptase

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(Invitrogen) from total RNA purified from primary fibroblasts (Trizol, Invitrogen). The following

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primers were used: F:

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5’CATAGAAGACACCGACTCTAGAGGGATCCATGGGGACTTCCCATCCGGC3’;

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R: 5’GAACCGCGGGCCCTCTAGACCTCGAGTTACAGGAAGCTGTCTTCCACCAG3’. The

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PDGFRα cDNA was then cloned into the BamHI and XhoI sites of pLenti6/CMV/V5-D-TOPO

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(Invitrogen) via Gibson assembly. To generate pLenti CMV/TO/MIE, the HCMV major

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immediate early (MIE) gene was amplified by PCR from a BAC plasmid of HCMV TB40/e strain

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using the following primers: F:

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5’GGAACCAATTCAGTCGACTGGGATCCAGAGCTCGTTTAGTGAAC3’;

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R: 5’TTTGTACAAGAAAGCTGGGTTTACTGAGATTTGTTCCTCAGG3’. The MIE gene was

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then cloned into the BamH I/ Xba sites of pLenti CMV/TO Puro vector (a gift from Eric Campeau,

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Addgene # 22262) via Gibson assembly.

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Lentiviral transfection and transduction. 293T cells were seeded at 2x106 cells per 10-cm

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dish and grown for 24 h. For generation of pseudotyped lentivirus, each 10-cm dish of 293T

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cells was transfected with 2.6 µg lentiviral vector, 2.4 µg PAX2, and 0.25 µg VSV-G using

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Fugene 6 (Promega). 24 h later, the medium was removed and replaced with 4 ml fresh

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medium, which was collected after an additional 24 h and filtered through a 0.45-μm filter prior

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to transduction. Fibroblasts were transduced with lentivirus in the presence of 5 µg/ml polybrene

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for overnight incubation. The lentivirus-containing medium was then removed and replaced with

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fresh DMEM medium. 72 h after transduction, cells were placed on selection with antibiotics.

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pLenti CMV/TO/Hygro/hTERT transduced cells were grown in 200 µg/mL hygromycin B

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(Invitrogen) for one week, and the expression of hTERT was confirmed by qPCR. Cells

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transduced with pLenti6/CMV/PDGFRα were selected in 10 µg/mL Blasticidin (Invitrogen) for

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four days. TAg fibroblasts transduced with pLenti CMV/TO/MIE were not drug selected, but

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robust expression of IE2 was verified by western analysis.

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Immunoblotting. For western blotting, cells were washed with PBS and lysed in 1X RIPA buffer

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[50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% Triton X 100, 0.5% sodium deoxycholate and

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cocktail protease inhibitors (cOmplete, EDTA-free, Roche)]. Lysates were sonicated and

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centrifuged at 14,000 x g for 5 min to pellet insoluble material. Protein concentration was

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measured by Bradford (Bio-Rad) protein assay. Supernatants were mixed with 4X loading buffer

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[200 mM Tris (PH 7.0), 8% SDS, 20% 2-mercaptoethanol, and 11% sucrose], boiled, briefly

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centrifuged, run on 8-10% polyacrylamide gel, and transferred to nitrocellulose in Tris-glycine

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transfer buffer. Blots were then stained with Ponceau S to visualize protein bands and ensure

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equal protein loading. The membranes were blocked in 5% milk in Tris-buffered saline-Tween

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20 (TBST), followed by incubation in primary antibody. After subsequent washes, blots were

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treated with secondary antibody and visualized using the enhanced chemiluminescence (ECL) 8

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system (Bio-Rad), imaged using a Molecular Imager® Gel Doc™ XR+ System (Bio-Rad).

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Antibodies used were specific for HRas (Santa Cruz Biotechnology Inc.), SV40 ST/LT (Santa

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Cruz Biotechnology Inc.), GAPDH (Cell Signaling Technology), IE1/IE2 (18), UL26(19), PP28

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(20), pp65 (21), UL44 (Virusys) {Strang, 2010 #581} and PDGFRα (Santa Cruz Biotechnology

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Inc.).

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Immunofluorescence. For analysis of pp65 localization, cells were grown on glass coverslips.

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At 4 hpi, cells were washed once with phosphate-buffered saline (PBS), fixed with 2%

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paraformaldehyde in PBS for 20 min, washed three times with PBS, permeabilized with 0.1%

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Triton X-100 and 0.1% SDS for 15 min, and washed twice with PBS containing 0.1% Tween 20.

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Cells were subsequently blocked in PBS containing 2% bovine serum albumin (BSA), 5% goat

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serum, 5% human serum, and 0.3% Triton X-100 for overnight incubation. Cells were then

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incubated with primary antibody to pp65, diluted in 0.05% Tween 20 in PBS for 1 h, washed with

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PBS containing 0.1% Tween 20 three times, incubated with Alexa 488-conjugated anti-mouse

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secondary (Invitrogen) antibody for 1 h, and washed with PBS containing 0.1% Tween 20 three

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times. Coverslips were mounted in SlowFade Gold antifade reagent (Molecular Probes) and

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4′,6′-diamidino-2-phenylindole (DAPI). Confocal images were captured with an FV1000

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Olympus laser scanning confocal microscope. All images were captured under identical

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confocal settings.

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Flow Cytometry. Cells were serum-starved for 4 days and infected with HCMV. At 24 hpi, cells

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were harvested in PBS, fixed in 70% ethanol and stained with 1 µg/ml DAPI in 0.1% TX100.

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Flow cytometric determination of DNA content was conducted using the LSRII Flow Cytometer,

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and data were processed with FlowJo V10 software. 9

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Quantitative PCR. Viral DNA was harvested at 5 dpi post infection in lysis buffer (100 mM NaCl,

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100 mM Tris-HCl, 25 mM, EDTA, 0.5% SDS, 0.1 mg/ml proteinase K and 40 mg/ml RNaseA).

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Quantitative PCR (qPCR) was performed using Fast SYBR green master mix (Applied

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Biosystems), a model 7500 Fast real-time PCR system and Fast 7500 software (Applied

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Biosystems) according to the manufacturer’s instructions. For quantifying viral DNA, a standard

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curve was generated using the BAdwt plasmid (AD169) as template and a primer set targeting

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pp65 (F, 5’CAGGAAGATTTGCTGCCCGTTCAT3’; R, 5’GGCTTTACGGTGTTGTGTCCCAAA3’).

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To measure viral gene expression, relative quantification normalized to GAPDH levels using

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ΔCt method was employed with the following: IE1 (F, 5’CCATGTCCACTCGAACCTTAAT3’; R,

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5’TGAACAAGTGACCGAGGATTG3’), IE2 (F, 5’CCCTTCACGATTCCCAGTATG3’; R.

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5’CTCATGATTGCGGGTGTAGAT3’), UL37 (F, 5’CCGAGTTCTCACCGTCAATTA3’; R,

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6’CTCTCCCGCCTTGGTTAAG3’), and GAPDH (F, 5’GGTGTGAACCATGAGAAGTATGA3’; R,

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5’GAGTCCTTCCACGATACCAAAG3’).

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Statistical Analysis

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For all the bar graphs, the data were represented as means ± standard deviation. Statistical

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analysis was performed using two-sided paired Student’s t-test.

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Results

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Expression of oncogenic proteins inhibits HCMV infection. To gain insight into the

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mechanisms of oncogenic restriction of HCMV infection, we examined the impact of oncogenic

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SV40 TAg and Ras G12V expression on HCMV replication in a previously defined step-wise

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model of transformation (11), consisting of parental human primary foreskin fibroblasts (P),

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telomerase-expressing fibroblasts (P-ht), SV40 TAg-telomerase-expressing fibroblasts (TAg),

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and Ras-transformed fibroblasts (TAgR) (Fig. 1A). Consistent with the ability of HSV-1 to grow

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in a large variety of tumor derived cell lines, expression of the SV40 TAg did not impact HSV-1

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viral titers (Fig. 1B). In contrast, and consistent with the inability of HCMV to productively infect

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most transformed cell lines, HCMV infection was attenuated by about 100-fold in the

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immortalized and the Ras-transformed fibroblasts (Fig. 1C). This attenuation was not a result of

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the expression of human telomerase, which did not affect the production of viral progeny (Fig.

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1D). Given that it has been shown that HCMV cannot replicate well in S phase cells (22), we

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assessed whether differences in the cell cycle could be playing a role in the observed reduction

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in viral production. Towards this end, we synchronized the cell cycle of the primary fibroblasts,

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the TAg cells and the TAgR cells through serum starvation. Despite similar cell cycle

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percentages, the TAg cells and TAgR cells still exhibited a substantial defect in HCMV

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replication relative to the primary fibroblasts (Fig. 1E), suggesting that cell cycle differences

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between the cells was not playing a major role.

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We next set out to investigate whether the expression of these oncogenic alleles affects

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HCMV gene expression. Compared to the primary fibroblasts, the accumulation of a variety of

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HCMV proteins was substantially reduced over the course of infection. Viral genes from the

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various kinetic classes of infection, including the IE1 immediate early gene, the UL26 early gene,

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and the pp28 late gene, all failed to accumulate in TAg or TAgR cells from 4 hpi to 72 hpi (Fig.

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1F). Together, these results indicate that the expression of SV40 TAg inhibits HCMV infection at

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a very early stage of infection.

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Given that HCMV IE1 expression was inhibited at 4 hpi in TAg cells and TAgR cells (Fig.

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1D), we hypothesized that HCMV viral entry may not occur properly in these cells. Immediately

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upon successful envelope fusion, the viral nucleocapsid, together with specific tegument

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proteins, is transported to the nucleus where the expression of immediate early genes begins.

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The pp65 tegument protein is delivered to the nucleus upon initial viral envelope fusion. After

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examining the localization of pp65 and viral genomes at 4 hpi, we found that pp65 and viral

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genomes were localized to the nuclei in the primary fibroblasts, but not the nuclei of TAg or

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TAgR fibroblasts (Fig. 2 A&C). As quantified in Fig. 2 B&D, about 70% of the primary fibroblasts

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exhibited nucleic pp65 and viral genome upon infection. In contrast, no viral genome/pp65-

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specific staining was evident in the nuclei of cells expressing TAg. We also quantified the

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abundance of viral genomes in purified cellular cytosolic and nuclear fractions. As shown in Fig.

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2 E&F, the majority of viral genomes were localized in the nuclei of the primary fibroblasts. In

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comparison, there was a substantial decrease of viral genomes in the nuclei of TAg or TAgR

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fibroblasts. Together, these results suggest that the entry of HCMV is blocked in T antigen-

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expressing fibroblasts.

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PDGFRα expression increases HCMV entry in TAg-expressing fibroblasts. A number of

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different growth factor receptors have been reported to be important for HCMV entry, including

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PDGFRα and EGFR (12, 23). Given the observed defect in viral entry, we sought to determine

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whether PDGFRα levels were impacted by TAg expression. In comparison to the primary cells,

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both the protein (Fig. 3A) and mRNA levels (Fig. 3B) of PDGFRα were markedly reduced in TAg 12

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and TAgR fibroblasts. As the decreased levels of PDGFRα could attenuate viral entry in the

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TAg-transformed fibroblasts, we examined whether PDGFRα over-expression could increase

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HCMV entry into these cells. Primary, TAg and TAgR fibroblasts were transduced with a control,

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or PDGFRα-expressing lentiviral vector (Fig. 3C). PDGFRα over-expression significantly

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increased pp65 nuclear localization in TAg (~55 %) and TAgR fibroblasts (~20 %) relative to a

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control vector, where no detectable nuclear pp65 was evident (Fig. 3D&E). pp65 was primarily

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localized in the nuclei of primary fibroblasts, irrespective of whether they were transduced with a

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control or PDGFRα-expression vector (Fig. 3D&E). PDGFRα transduction also increased the

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expression of genes encoded by the viral genome, enhancing virally-encoded EGFP expression

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(Fig. 3F). These results indicate that PDGFRα over-expression can substantially rescue HCMV

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entry in TAg-immortalized fibroblasts.

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PDGFRα over-expression partially rescues HCMV gene expression, and DNA replication

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but not virion production in TAg-expressing fibroblasts. Given that PDGFRα over-

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expression significantly increased HCMV entry in TAg-expressing fibroblasts, we tested whether

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PDGFRα over-expression rescued viral gene expression, DNA replication and the production of

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infectious progeny. As shown in Fig. 4A, at 4 hpi, the IE1 protein accumulated to a lesser extent

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in TAg and TAgR cells than in primary fibroblasts (Fig. 4A). However, by 24 hpi PDGFRα-

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transduction substantially increased IE1 expression in TAg and TAgR fibroblasts to levels

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comparable to primary fibroblasts (Fig. 4A). However, the levels of IE2, the major viral

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transactivator were rescued to a lesser extent. Substantially reduced levels of IE2 accumulated

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in PDGFRα-expressing TAg and TAgR fibroblasts at all times post infection (Fig 4A). The

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accumulation of downstream viral proteins such as UL26 and pp28, were partially rescued by

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PDGFRα over-expression, but only to a small extent, consistent with the observed reduction in

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IE2 levels. In addition, PDGFRa over-expression only partially rescued viral DNA replication in 13

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TAg-expressing cells (Fig. 4B), and did not rescue virion production in TAg-expressing cells (Fig.

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4C). Notably, the expression of PDGFRα reduced virion production in primary fibroblasts, and

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TAgR fibroblasts (Fig. 4C), suggesting that its constitutive expression may inhibit the production

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of infectious virus. Host proteins that are involved in viral entry could potentially complicate viral

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egress strategies, e.g. through interactions with viral receptors prior to egress, which could

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block subsequent infection. A viral strategy to prevent this would be to reduce the expression of

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the host proteins involved in entry. Consistent with this possibility, we find that PDGFRα

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expression is acutely down-regulated by HCMV infection (Fig. 4D&E). Combined, our results

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indicate that while PDGFRα expression can substantially rescue HCMV entry and IE1

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expression in TAg-expressing fibroblasts, its expression restores neither wildtype levels of viral

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gene expression, nor the wild-type production of viral progeny.

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PDGFRα and TAg expression in fibroblasts result in differential accumulation of major

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immediate early transcripts. The observation that PDGFRα expression largely rescued IE1

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but not IE2 levels suggests that TAg expression may differentially impact the accumulation of

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immediate early transcripts. To explore this issue, we employed gene specific qPCR primers to

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examine various immediate early transcripts during infection, including IE1, IE2 and UL37. At 4

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hpi, PDGFRα expression increased IE1 specific mRNA abundance by over 200-fold in TAg cells

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and 180-fold in TAgR cells (Fig. 5A and Table 1). At 24 hpi, PDGFRα expression increased IE1-

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mRNA abundance by 119-fold and 63-fold in TAg and TAgR cells, respectively (Fig. 5A and

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Table 1). IE2-specific mRNA was also induced upon PDGFRα expression, with ~30-fold and 40-

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fold increases in PDGFRα-expressing TAg and TAgR cells at 4 hpi, and ~50-fold increases in

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PDGFRα-expressing TAg and TAgR cells at 24 hpi (Fig. 5B and Table 1). We also examined

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whether PDGFRα expression could rescue the expression of another immediate early gene

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UL37. As shown in Fig. 5C, PDGFRα transduction increased UL37 mRNA expression at both 4 14

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hpi and 24 hpi in TAg-expressing cells. As viral entry was not completely rescued by PDGFRα

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transduction in TAg and TAgR cells (Fig 3E), it follows that the expression of these immediate

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early genes would not be fully rescued to levels typical of primary fibroblast infection.

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Collectively, these results suggest that PDGFRα transduction substantially increases the

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expression of various viral immediate early genes.

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IE1 and IE2 are both expressed from the major immediate early (MIE) promoter, and

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their respective proteins arise from differential splicing of common transcripts (1, 25). In primary

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fibroblasts, the relative proportion of IE2-specific message relative to the sum of IE1 and IE2,

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increased from 30% at 4 hpi to ~50% at 24 hpi (Fig. 5D&E). Notably, this transition did not occur

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in TAg-expressing fibroblasts. The IE2 transcript fraction dropped from 20-30% at 4 hpi to

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below 10% at 24 hpi in TAg-expressing cells (Fig. 5D&E). These results suggest that that TAg

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expression prevents a shift that favors IE2 mRNA accumulation over IE1. This defect likely

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results in the reduced IE2 protein accumulation observed in Fig. 4, and potentially contributes to

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the defects in viral gene expression associated with TAg expression.

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The impact of IE2 over-expression on HCMV infection of TAg-expressing fibroblasts.

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Given the essential role that IE2 plays in the HCMV life cycle, we hypothesized that decreased

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IE2 levels may be responsible for attenuated HCMV virion production in PDGFRα-transduced

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TAg-expressing cells. To test this hypothesis, we sought to examine whether IE2 over-

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expression could rescue viral infection in TAg-expressing cells. Transduction with HCMV MIE in

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TAg-expressing fibroblasts resulted in IE2 expression levels that were similar to those of

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infected primary fibroblasts (Fig. 6A). MIE transduction also increased the accumulation of the

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viral early proteins UL26 and UL44, as well as the true late protein pp28 (Fig. 6B). However, 15

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while viral DNA replication increased by ~2-fold, it still accumulated to less than 10% of the viral

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DNA present in primary fibroblasts (Fig. 6C). Further, MIE transduction did not rescue

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infectious virion production in TAg-expressing cells (Fig. 6D). Combined, these data indicate

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that while IE2 over-expression can rescue the gene expression of the HCMV genes analyzed,

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IE2 over-expression is not sufficient to rescue infectious virion production.

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PDGFRα over-expression increases HCMV gene expression and DNA replication but not

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virion production in HeLa cells. To explore whether the observed restrictions in the

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genetically defined fibroblast model of oncogenesis were similar in cancer-derived cells, we

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examined how PDGFRα over-expression affects HCMV replication in HeLa cells. Lentiviral

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transduction increased PDGFRα expression in HeLa cells (Fig. 7A). As shown in Fig. 7B,

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PDGFRα over-expression markedly increased virally encoded EGFP expression upon infection.

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Further, the expression of viral genes from the various kinetic classes of infection, immediate

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early genes IE1 and IE2, early genes UL26 and UL44, and late genes pp28 and pp65, were all

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increased substantially by PDGFRα over-expression in HeLa cells (Fig. 7C). The expression

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levels of IE1, IE2, UL26, and UL44 in PDGFRα-expressing HeLa cells were comparable to

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those in the primary fibroblasts (Fig. 7C). However, the late proteins pp28 and pp65 were

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rescued to a lesser extent (Fig. 7C). Given that these true late genes require DNA replication,

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we sought to examine how PDGFRα over-expression affects viral DNA replication in HeLa cells.

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As shown in Fig. 7D, PDGFRα over-expression partially rescued viral DNA replication, but only

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to about 12% of the primary fibroblast level. Further, PDGFRα over-expression did not increase

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virion production in HeLa cells (Fig. 7E). As AD169 is defective of replicating in epithelial cells,

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we sought to investigate whether PDGFRα over-expression would influence the replication of an

361

HCMV clinical strain (TB40/e) in HeLa cells. As shown in Fig. 7F, PDGFRα over-expression

362

increased virally-encoded EGFP expression in HeLa cells. However, PDGFRα over-expression 16

363

failed to rescue TB40/e virion production in HeLa cells (Fig. 7G). With respect to PDGFRα

364

expression, these results largely recapitulate the previous results in TAg-expressing fibroblasts.

365

Collectively, our data suggest that PDGFRα over-expression can largely rescue HCMV entry

366

and gene expression, but only partly rescue DNA replication in tumor-derived cell lines.

367

17

368

Discussion. In contrast to many herpesviruses, HCMV cannot productively infect most cancer-

369

derived cell lines (1, 5). The mechanisms of this restriction are not clear. Here we show that the

370

expression of the SV40 TAg blocks HCMV but not HSV replication in human fibroblasts.

371

Specifically, TAg expression inhibited HCMV tegument protein delivery, indicative of a block of

372

virion fusion. Further, TAg expression down-regulated PDGFRα expression. This reduction in

373

PDGFRα was found to be functionally relevant, as PDGFRα transduction partly rescued viral

374

entry and gene expression, although not the production of infectious viral progeny. TAg

375

expression induced further downstream restrictions including attenuating the accumulation of

376

IE2 and viral DNA replication. Transduction of IE2 further restored HCMV gene expression, and

377

increased viral DNA replication, but it did not increase production of infectious progeny.

378

Collectively, our results indicate that oncogenic TAg expression induces multiple blocks to

379

successful HCMV infection.

380 381

HCMV phosphorylates and activates PDGFRα, which is required for HCMV entry and

382

gene expression in human embryonic lung fibroblasts (12). Specifically, HCMV gB protein

383

physically interacts with and activates PDGFRα in human fibroblasts (12). It was later reported

384

that PDGFRα is not the physical receptor for HCMV entry in endothelial and epithelial cells, but

385

instead that its increased expression promotes entry of HCMV strains that lack gH/gL/UL128-

386

131, key viral determinants of tropism in endothelial/epithelial cells, via aberrant pathways

387

involving endocytosis and endosome fusion (26). Our results show that SV40 TAg expression

388

down-regulates the accumulation of PDGFRα in human foreskin fibroblasts. This is in

389

agreement with reports indicating that the SV40 early region reduces the mRNA abundance of

390

growth factor receptors in fibroblasts (27-28). The expression of PDGFRα appears to generally

391

be important for HCMV entry into transformed cells, as we found that PDGFRα over-expression

18

392

substantially increases HCMV AD169 entry, gene expression and DNA replication in TAg-

393

expressing fibroblasts as well as HeLa cells.

394 395

Despite promoting entry, expression of PDGFRα did not rescue production of infectious

396

HCMV. Notably, PDGFRα over-expression decreased production of infectious virions in primary

397

fibroblasts (Fig. 4C). During HCMV infection in primary fibroblasts, HCMV was found to acutely

398

down-regulate PDGFRα expression as early as 24 hpi (Fig. 4D&E). Similarly, another

399

postulated HCMV receptor EGFR is reportedly down-regulated transcriptionally upon HCMV

400

infection in human fetal lung fibroblasts (29). Collectively, these data suggest that down-

401

regulation of host proteins involved in entry at the later times of infection may be important for

402

proper production of viral progeny. Such scenarios have been proposed for other viruses, for

403

example, the Duck hepatitis B virus that induces the degradation of its receptor, gp180 (30).

404 405

We find that in cells expressing TAg and PDGFRα, the balance of IE1 and IE2

406

expression is altered resulting in substantially less IE2 expression relative to IE1. Given the role

407

of IE2 as the major viral transcriptional activator, the viral gene expression defects associated

408

with HCMV infection of PDGFRα–expressing TAg fibroblasts are likely a result of this reduced

409

IE2 expression. This hypothesis is supported by the data showing that transduction of IE2

410

rescues viral gene expression. However, the findings that IE2 transduction did not rescue the

411

production of viral progeny and did not substantially rescue viral DNA replication, suggests an

412

additional restriction(s) that inhibits viral DNA replication.

413

19

414

Our results indicate that in primary cells, the processing of MIE transcripts that encode

415

IE2 and IE1 shifts from a 35:65 ratio of IE2:IE1 at 4 hpi to an approximate 50:50 ratio at 24 hpi.

416

In contrast, in TAg-expressing fibroblasts, this ratio starts at about 20:80 at 4 hpi and shifts to a

417

less than 10:90 ratio of IE2:IE1. It is currently unclear how the production, splicing or processing

418

of these transcripts may differ in primary versus TAg-expressing fibroblasts. Notably, a similar

419

switch favoring IE1 expression at the expense of IE2 expression was observed upon inhibition

420

of Cox-2, which blocks HCMV infection (31). While it is unclear if there is any link between our

421

observations and those made upon Cox-2 inhibition, given the essential nature of IE2

422

expression for successful HCMV infection, the mechanisms that govern these events are of

423

particular interest, and warrant future study.

424 425

HCMV has been increasingly implicated as an etiological agent in oncogenesis. HCMV

426

genes have also been found in a variety of human carcinomas, but not in matched tissue

427

controls (32-35). HCMV nucleic acids and proteins have also been found to be associated with

428

a high percentage of malignant gliomas and meduloblastomas(36-40). Further many HCMV

429

gene products have also been shown to induce oncogenic activities, including activation/

430

deregulation of cell-growth pathways, and inhibition of tumor suppressor pathways (reviewed in

431

(41, 42)). Consistent with the activation of these oncogenic events, HCMV gene products have

432

been found to transform rodent fibroblasts (43) and to stimulate oncogenesis and cooperate

433

with other oncogenic mutations in in vivo models of tumorigenesis (39, 44, 45). While HCMV is

434

clearly not sufficient to induce tumor formation in people, a hit and run contribution has been

435

proposed (46). Collectively, these results suggest the possibility that abortive infection of an

436

immortalized cell, in a manner analogous to our observations, could potentially provide pre-

437

cancerous cells with viral factors/ functions that could stimulate the oncogenic process. Such a

438

scenario would be consistent with the findings that cells derived from meduloblastoma clinical 20

439

samples were found to express high levels of functional viral gene products (40). Further study

440

is necessary to explore the potential links between HCMV and oncogenesis.

441 442

In summary, we find that SV40 TAg expression induces multiple restrictions to HCMV

443

infection: 1) TAg expression blocks HCMV entry by inhibiting the expression of the entry protein

444

PDGFRα; 2) TAg expression inhibits viral gene expression by preferentially down-regulating IE2

445

expression; 3) TAg expression inhibits viral DNA replication upon IE2 restoration. Most viruses

446

that are capable of in vitro propagation grow to substantially higher titers in cancer-derived cell

447

lines. The inability to do so makes HCMV somewhat unique in relation to most viruses studied.

448

At first pass, the failure of HCMV to replicate in transformed cells appears to have limited

449

relevance to HCMV-associated disease. However, understanding the mechanisms through

450

which specific oncogenic signaling events can limit HCMV infection is broadly informative, and

451

the information gained can be applied to other infectious contexts. For example, the

452

mechanisms responsible for the observed modulation of IE1 versus IE2 levels could be

453

important in other types of HCMV infection, e.g. controlling viral gene expression during latency

454

or reactivation. Once fully elucidated, these mechanisms could potentially be therapeutically

455

targeted to modulate infectious outcomes.

456 457

21

References

458 459

1.

Howley PM (ed), Fields Virology. Lippincott-Williams and Wilkins, New York.

460 461

2.

3.

4.

5.

6.

7.

Wang D, Shenk T. 2005. Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism. Proc Natl Acad Sci U S A 102:18153-18158.

472 473

Wang D, Shenk T. 2005. Human cytomegalovirus UL131 open reading frame is required for epithelial cell tropism. J Virol 79:10330-10338.

470 471

Smith JD. 1986. Human cytomegalovirus: demonstration of permissive epithelial cells and nonpermissive fibroblastic cells in a survey of human cell lines. J Virol 60:583-588.

468 469

Hahn G, Jores R, Mocarski ES. 1998. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc Natl Acad Sci U S A 95:3937-3942.

466 467

Cannon MJ. 2009. Congenital cytomegalovirus (CMV) epidemiology and awareness. J Clin Virol 46 Suppl 4:S6-10.

464 465

Gerna G, Baldanti F, Revello MG. 2004. Pathogenesis of human cytomegalovirus infection and cellular targets. Hum Immunol 65:381-386.

462 463

Mocarski ES, Shenk T, Pass RF. 2006. Cytomegaloviruses, p 2701-2757. In Knipe DM,

8.

Ryckman BJ, Chase MC, Johnson DC. 2008. HCMV gH/gL/UL128-131 interferes with

474

virus entry into epithelial cells: evidence for cell type-specific receptors. Proc Natl Acad

475

Sci U S A 105:14118-14123.

476

9.

Ryckman BJ, Rainish BL, Chase MC, Borton JA, Nelson JA, Jarvis MA, Johnson

477

DC. 2008. Characterization of the human cytomegalovirus gH/gL/UL128-131 complex

478

that mediates entry into epithelial and endothelial cells. J Virol 82:60-70.

479

10.

Ryckman BJ, Jarvis MA, Drummond DD, Nelson JA, Johnson DC. 2006. Human

480

cytomegalovirus entry into epithelial and endothelial cells depends on genes UL128 to

481

UL150 and occurs by endocytosis and low-pH fusion. J Virol 80:710-722.

22

482

11.

Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA.

483

1999. Creation of human tumour cells with defined genetic elements. Nature 400:464-

484

468.

485

12.

receptor activation is required for human cytomegalovirus infection. Nature 455:391-395.

486 487

Soroceanu L, Akhavan A, Cobbs CS. 2008. Platelet-derived growth factor-alpha

13.

Yu D, Smith GA, Enquist LW, Shenk T. 2002. Construction of a self-excisable bacterial

488

artificial chromosome containing the human cytomegalovirus genome and mutagenesis

489

of the diploid TRL/IRL13 gene. J Virol 76:2316-2328.

490

14.

specific RANTES decoy receptor. Proc Natl Acad Sci U S A 101:16642-16647.

491 492

Wang D, Bresnahan W, Shenk T. 2004. Human cytomegalovirus encodes a highly

15.

O'Connor CM, Murphy EA. 2012. A myeloid progenitor cell line capable of supporting

493

human cytomegalovirus latency and reactivation, resulting in infectious progeny. J Virol

494

86:9854-9865.

495

16.

Rosenke K, Fortunato EA. 2004. Bromodeoxyuridine-labeled viral particles as a tool for

496

visualization of the immediate-early events of human cytomegalovirus infection. Journal

497

of Virology 78:7818-7822.

498

17.

Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA, 3rd, Smith HO. 2009.

499

Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods

500

6:343-345.

501

18.

apoptosis. J Virol 69:7960-7970.

502 503

Zhu H, Shen Y, Shenk T. 1995. Human cytomegalovirus IE1 and IE2 proteins block

19.

Munger J, Yu D, Shenk T. 2006. UL26-deficient human cytomegalovirus produces

504

virions with hypophosphorylated pp28 tegument protein that is unstable within newly

505

infected cells. J Virol 80:3541-3548.

23

506

20.

Silva MC, Yu QC, Enquist L, Shenk T. 2003. Human cytomegalovirus UL99-encoded

507

pp28 is required for the cytoplasmic envelopment of tegument-associated capsids. J

508

Virol 77:10594-10605.

509

21.

Browne EP, Shenk T. 2003. Human cytomegalovirus UL83-coded pp65 virion protein

510

inhibits antiviral gene expression in infected cells. Proc Natl Acad Sci U S A 100:11439-

511

11444.

512

22.

Fortunato EA, Sanchez V, Yen JY, Spector DH. 2002. Infection of cells with human

513

cytomegalovirus during S phase results in a blockade to immediate-early gene

514

expression that can be overcome by inhibition of the proteasome. Journal of Virology

515

76:5369-5379.

516

23.

factor receptor is a cellular receptor for human cytomegalovirus. Nature 424:456-461.

517 518

Wang X, Huong SM, Chiu ML, Raab-Traub N, Huang ES. 2003. Epidermal growth

24.

Garrett JS, Coughlin SR, Niman HL, Tremble PM, Giels GM, Williams LT. 1984.

519

Blockade of autocrine stimulation in simian sarcoma virus-transformed cells reverses

520

down-regulation of platelet-derived growth factor receptors. Proc Natl Acad Sci U S A

521

81:7466-7470.

522

25.

IE86 protein. Curr Top Microbiol Immunol 325:133-152.

523 524

Stinski MF, Petrik DT. 2008. Functional roles of the human cytomegalovirus essential

26.

Vanarsdall AL, Wisner TW, Lei H, Kazlauskas A, Johnson DC. 2012. PDGF receptor-

525

alpha does not promote HCMV entry into epithelial and endothelial cells but increased

526

quantities stimulate entry by an abnormal pathway. PLoS Pathog 8:e1002905.

527

27.

Bae VL, Jackson‐Cook CK, Brothman AR, Maygardens SJ, Ware JL. 1994.

528

Tumorigenicity of SV40 T antigen immortalized human prostate epithelial cells:

529

association with decreased epidermal growth factor receptor (EGFR) expression.

530

International journal of cancer 58:721-729.

24

531

28.

Wang JL, Nister M, Bongcam-Rudloff E, Ponten J, Westermark B. 1996.

532

Suppression of platelet-derived growth factor alpha- and beta-receptor mRNA levels in

533

human fibroblasts by SV40 T/t antigen. J Cell Physiol 166:12-21.

534

29.

Beutler T, Hoflich C, Stevens PA, Kruger DH, Prosch S. 2003. Downregulation of the

535

epidermal growth factor receptor by human cytomegalovirus infection in human fetal

536

lung fibroblasts. Am J Respir Cell Mol Biol 28:86-94.

537

30.

regulation of hepatitis B virus receptor in infected hepatocytes. J Virol 75:143-150.

538 539

31.

Zhu H, Cong JP, Yu D, Bresnahan WA, Shenk TE. 2002. Inhibition of cyclooxygenase 2 blocks human cytomegalovirus replication. Proc Natl Acad Sci U S A 99:3932-3937.

540 541

Breiner KM, Urban S, Glass B, Schaller H. 2001. Envelope protein-mediated down-

32.

Samanta M, Harkins L, Klemm K, Britt WJ, Cobbs CS. 2003. High prevalence of

542

human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma. J

543

Urol 170:998-1002.

544

33.

Harkins L, Volk AL, Samanta M, Mikolaenko I, Britt WJ, Bland KI, Cobbs CS. 2002.

545

Specific localisation of human cytomegalovirus nucleic acids and proteins in human

546

colorectal cancer. Lancet 360:1557-1563.

547

34.

El-Shinawi M, Mohamed HT, El-Ghonaimy EA, Tantawy M, Younis A, Schneider RJ,

548

Mohamed MM. 2013. Human cytomegalovirus infection enhances NF-kappaB/p65

549

signaling in inflammatory breast cancer patients. PLoS One 8:e55755.

550

35.

Taher C, de Boniface J, Mohammad AA, Religa P, Hartman J, Yaiw KC, Frisell J,

551

Rahbar A, Soderberg-Naucler C. 2013. High prevalence of human cytomegalovirus

552

proteins and nucleic acids in primary breast cancer and metastatic sentinel lymph nodes.

553

PLoS One 8:e56795.

554

36.

Cobbs CS, Harkins L, Samanta M, Gillespie GY, Bharara S, King PH, Nabors LB,

555

Cobbs CG, Britt WJ. 2002. Human cytomegalovirus infection and expression in human

556

malignant glioma. Cancer Res 62:3347-3350. 25

557

37.

Cobbs CS, Soroceanu L, Denham S, Zhang W, Kraus MH. 2008. Modulation of

558

oncogenic phenotype in human glioma cells by cytomegalovirus IE1-mediated

559

mitogenicity. Cancer Res 68:724-730.

560

38.

Ranganathan P, Clark PA, Kuo JS, Salamat MS, Kalejta RF. 2012. Significant

561

association of multiple human cytomegalovirus genomic Loci with glioblastoma

562

multiforme samples. J Virol 86:854-864.

563

39.

Soroceanu L, Matlaf L, Bezrookove V, Harkins L, Martinez R, Greene M,

564

Soteropoulos P, Cobbs CS. 2011. Human cytomegalovirus US28 found in

565

glioblastoma promotes an invasive and angiogenic phenotype. Cancer Res 71:6643-

566

6653.

567

40.

Baryawno N, Rahbar A, Wolmer-Solberg N, Taher C, Odeberg J, Darabi A, Khan Z,

568

Sveinbjornsson B, Fuskevag OM, Segerstrom L, Nordenskjold M, Siesjo P, Kogner

569

P, Johnsen JI, Soderberg-Naucler C. 2011. Detection of human cytomegalovirus in

570

medulloblastomas reveals a potential therapeutic target. J Clin Invest 121:4043-4055.

571

41.

Microbiol Immunol 325:243-262.

572 573

42.

43.

Doniger J, Muralidhar S, Rosenthal LJ. 1999. Human cytomegalovirus and human herpesvirus 6 genes that transform and transactivate. Clin Microbiol Rev 12:367-382.

576 577

Yurochko AD. 2008. Human cytomegalovirus modulation of signal transduction. Curr Top Microbiol Immunol 325:205-220.

574 575

Sanchez V, Spector DH. 2008. Subversion of cell cycle regulatory pathways. Curr Top

44.

Price RL, Song J, Bingmer K, Kim TH, Yi JY, Nowicki MO, Mo X, Hollon T, Murnan

578

E, Alvarez-Breckenridge C, Fernandez S, Kaur B, Rivera A, Oglesbee M, Cook C,

579

Chiocca EA, Kwon CH. 2013. Cytomegalovirus contributes to glioblastoma in the

580

context of tumor suppressor mutations. Cancer Res 73:3441-3450.

26

581

45.

Price RL, Bingmer K, Harkins L, Iwenofu OH, Kwon CH, Cook C, Pelloski C,

582

Chiocca EA. 2012. Cytomegalovirus infection leads to pleomorphic

583

rhabdomyosarcomas in Trp53+/- mice. Cancer Res 72:5669-5674.

584

46.

Shen Y, Zhu H, Shenk T. 1997. Human cytomagalovirus IE1 and IE2 proteins are

585

mutagenic and mediate "hit-and-run" oncogenic transformation in cooperation with the

586

adenovirus E1A proteins. Proc Natl Acad Sci U S A 94:3341-3345.

587 588 589

27

Figure legends

590 591 592

Figure 1. The expression of SV40 TAg blocks HCMV replication. A. The protein levels of

593

SV40 large T antigen (LT) and HRas were measured by western blot in primary fibroblasts (P)

594

or their transduced derivatives. These fibroblast lines include those expressing telomerase

595

alone (P-ht), those expressing telomerase plus the SV40 early region (TAg), or those

596

expressing the combination of telomerase, the SV40 early region, and HRasV12 (TAgR). B-D.

597

The cells in (A) were infected with human simplex virus (B), or HCMV AD169 (C and D) at an

598

MOI = 3. Production of infectious virions at 1 dpi (B), 3-7 dpi (C) and 5 dpi (D) was measured by

599

plaque assay. Asterisks indicate significant differences between values for primary cells and

600

TAg cells (* significant at p