host gene induction and transcriptional reprogramming in ... · host gene induction and...

[CANCER RESEARCH 64, 72–84, January 1, 2004]

Host Gene Induction and Transcriptional Reprogramming in Kaposi’s Sarcoma-Associated Herpesvirus (KSHV/HHV-8)-Infected Endothelial, Fibroblast, andB Cells: Insights into Modulation Events Early during Infection

Pramod P. Naranatt,1 Harinivas H. Krishnan,1 Stan R. Svojanovsky,2 Clark Bloomer,3 Sachin Mathur,2 andBala Chandran1

1Department of Microbiology, Molecular Genetics and Immunology, 2Bioinformatics Core, and 3Microarray Core, The University of Kansas Medical Center, Kansas City, Kansas

ABSTRACT

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) is etiologi-cally linked to the endothelial tumor Kaposi’s sarcoma and with twolymphoproliferatve disorders, primary effusion lymphoma and multicen-tric Castleman’s disease. HHV-8 infects a variety of target cells both invivo and in vitro, binds to the in vitro target cells via cell surface heparansulfate, and uses the �3�1 integrin as one of the entry receptors. Withinminutes of infection, HHV-8 induced the integrin-mediated signalingpathways and morphological changes in the target cells (S. M. Akula et al.,Cell, 108: 407–419, 2002; P. P. Naranatt et al., J. Virol., 77: 1524–1539,2003). As an initial step toward understanding the role of host genes inHHV-8 infection and pathogenesis, modulation of host cell gene expressionimmediately after infection was examined. To reflect HHV-8’s broadcellular tropism, mRNAs collected at 2 and 4 h after infection of primaryhuman endothelial [human adult dermal microvascular endothelial cells(HMVECd)] and foreskin fibroblast [human foreskin fibroblast (HFF)]cells and human B cell line (BJAB) were analyzed by oligonucleotide arraywith �22,000 human transcripts. With a criteria of >2-fold gene induc-tion as significant, �1.72% of the genes were differentially expressed, ofwhich, 154 genes were shared by at least two cells and 33 genes shared byall three cells. HHV-8-induced transcriptional profiles in the endothelialand fibroblast cells were closely similar, with substantial differences in theB cells. In contrast to the antiapoptotic regulators induced in HMVECdand HFF cells, proapoptotic regulators were induced in the B cells. Arobust increase in the expression of IFN-induced genes suggestive ofinnate immune response induction was observed in HMVECd and HFFcells, whereas there was a total lack of immunity related protein induc-tions in B cells. These striking cell type-specific behaviors suggest thatHHV-8-induced host cell gene modulation events in B cells may be dif-ferent compared with the adherent endothelial and fibroblast target cells.Functional clustering of modulated genes identified several host moleculeshitherto unknown to HHV-8 infection. These results indicate that earlyduring infection, HHV-8 reprograms the host transcriptional machineryregulating a variety of cellular processes including apoptosis, transcrip-tion, cell cycle regulation, signaling, inflammatory response, and angio-genesis, all of which may play important roles in the biology and patho-genesis of HHV-8.

INTRODUCTION

Kaposi’s sarcoma (KS) is the leading vascular tumor of HIV-infected patients (1). The development of KS in the four epidemio-logically distinct forms (classic, endemic, posttransplant, and AIDS-KS) is associated with the KS-associated herpesvirus or humanherpesvirus 8 (KSHV/HHV-8; Ref. 2). HHV-8 is also etiologicallylinked with two lymphoproliferatve disorders, body cavity-based B-

cell lymphoma or primary effusion lymphoma, and a subset of mul-ticentric Castleman’s disease (2, 3). Although studies have suggesteda decline in the incidence of KS in United States coinciding with theintroduction of highly active antiretroviral therapy, mounting non-compliance failure rates in highly active antiretroviral therapy patientssuggest that KS will represent a major health problem for years tocome (4). In Africa, widespread infection with HIV has resulted in analarming prevalence of HHV-8 infection and the associated diseases(5). Moreover, there is an increasing concern regarding posttransplantKS developing in solid-organ transplant patients, either caused byHHV-8 reactivation or primary infection transmitted from the donor(6).

HHV-8 is in a latent state in the KS endothelial cells and expressesthe genes encoding the latency-associated nuclear antigen (LANA)and a subpopulation of KS lesion inflammatory and spindle cellsdisplays lytic HHV-8 replication (7). In a marked contrast to thewell-established linkage between B-cell lymphomas and latent infec-tion by the related �1-EBV, studies have indicated the important rolesfor both latent and lytic infection in KS pathogenesis (4). KS tumor-igenesis appears to require an ongoing lytic infection because inter-ruption of lytic replication by drugs such as ganciclovir appears toprevent KS development (8). Detection of lytic replication in only asmall percentage of KS cells coupled with the signaling properties ofseveral lytic cycle HHV-8 proteins suggest that products of lyticinfection may act in a paracrine fashion to promote KS tumorigenesis(9). KSHV lytic proteins have been shown to induce growth deregu-lation and angiogenic factors via activation of multiple host cellsignaling cascades, including extracellular signal-regulated kinase(ERK), c-Jun-NH2-terminal kinase, and p38 pathways.

In vitro, HHV-8 has been shown to infect human B, epithelial,endothelial, foreskin fibroblast cells, and keratinocytes (10–12). TheEBV infection of primary B cells results in latent infection, immor-talization of B cells, and the maintenance of latent viral episomesreplicating along with host cell division. Unlike EBV, infection ofprimary B cells by HHV-8 does not result in a sustained latentinfection and immortalization. Our studies have shown that via itsenvelope glycoproteins gpK8.1A and glycoprotein B (gB), HHV-8binds to the ubiquitous cell surface heparan sulfate (HS) molecules(13–15), binds subsequently to the �3�1 integrin via its gB (possess-ing the integrin binding Arginine-Glycine-Aspartic Acid motif), anduses �3�1 integrin as one of the cellular receptors for its entry into thetarget cells (16). Our studies have also shown that within minutes oftarget cell infection, in a gB-integrin �3�1-dependent manner, HHV-8activated the phosphatidylinositol 3�-kinase, protein kinase C-�,mitogen-activated protein/ERK kinase signaling cascade and cyto-skeletal rearrangements (17). Pretreatment of cells with inhibitorsspecific against members of this cascade blocked HHV-8 infectivitysignificantly (17), suggesting that by orchestrating the signal cascade,HHV-8 may create an appropriate intracellular environment to facil-itate the infection.

In addition to the induction of preexisting host cell signal pathways,similar to other viruses, HHV-8’s interactions with host cell surface

Received 9/3/03; revised 10/14/03; accepted 10/20/03.Grant support: USPHS Grants CA 75911 and 82056 (B. C.), a University of Kansas

Medical Center Biomedical Research Training Program Postdoctoral Fellowship(P. P. N.), and USPHS Grant P20 RR16475 (S. R. S.).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Bala Chandran, Department of Microbiology, MolecularGenetics and Immunology, Mail Stop 3029, The University of Kansas Medical Center,3901 Rainbow Boulevard, Kansas City, KS 66160. Phone: (913) 588-7043; Fax:(913) 588-7295; E-mail: [email protected].

72

Research. on July 3, 2021. © 2004 American Association for Cancercancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

molecules may be triggering cellular transcriptional and other re-sponses. The binding of herpes simplex virus type-1 to cell surfacesinduces cellular genes and an antiviral state, which are countered byvirion entry and expression of newly synthesized viral protein(s) (18).The binding of human cytomegalovirus viral gB to an unknown cellsurface receptor induced the IFN-responsive RNAs in the absence ofviral and cellular protein synthesis (19). Integrin interactions withextracellular matrix proteins induce robust host cell gene expressionand mediate a variety of cell functions such as regulation of geneexpression, cell survival, cell cycle progression, cell growth, apopto-sis, and differentiation (20). Because integrin-mediated signaling wasshown to actively contribute to the HHV-8 infectious process (16–17), it can be speculated that target cell gene responses induced duringthe initial stages of infection may have pivotal roles in HHV-8infection. The identification of such cellular signatures might even-tually lead to the development of novel strategies to control HHV-8infection.

As an initial step toward understanding the role of host genespotentially involved in dictating the outcome of HHV-8 infection andpathogenesis, we undertook this study to analyze the cellular tran-scriptional responses in three different target cells in vitro at 2 and 4 hafter HHV-8 infection. Our analysis reveals that HHV-8 triggers arobust modulation of cellular gene expression with cell type-specificand common responses, which may potentially play important roles inthe biology of HHV-8 and pathogenesis.

MATERIALS AND METHODS

Cells. Human adult dermal microvascular endothelial cells (HMVECd),human foreskin fibroblast (HFF), body cavity-based B-cell lymphoma-1 cells(HHV-8-positive and EBV-negative human B cells), recombinant green fluo-rescent HHV-8 (GFP-HHV-8) carrying body cavity-based B-cell lymphoma-1cells (12), and BJAB cells (HHV-8-negative B cells) were grown as perprocedures described before (13).

Virus. HHV-8 for array analysis was purified from GFP-body cavity-basedB-cell lymphoma-1 cells (12) after cells were stimulated with 20 ng/ml12-O-tetradecanoylphorbol-13-acetate (Sigma, St. Louis, MO) for 6 days.GFP-HHV-8 in the spent culture medium was concentrated and gradientpurified by using Nycodenz (Sigma) as described previously (14). Purity of thevirus was judged using general guidelines established in our laboratory (17).

Oligonucleotide Array. Human genome HG-U133A and B (Affymetrix,Santa Clara, CA) are oligonucleotide probe-based gene arrays containing�100,000 unique oligos representing �39,000 transcript variants, which inturn represents �33,000 well-substantiated human genes. The HG-U133B chiprepresents the majority of the expressed sequence tags, which are not wellannotated. Because the HG-U133A chip represents �22,283 most well-char-acterized genes, we used this chip for analysis.

Gene Array Expression Analysis. HMVEC-d, HFF, and BJAB cells werewashed twice in serum-free DMEM or RPMI and incubated in the serum-freemedium for 6–8 h to reduce the effect of the serum or other growth factors inthe analysis. Serum-starved cells were infected with GFP-HHV-8 (at five viralgenome copies/cell), incubated at 37°C for 2 or 4 h. Total RNA from unin-fected (UI) controls and infected cells were isolated using RNeasy minicolumns (Qiagen, Valencia, CA). First strand synthesis was performed using10–15 �g of total RNA, a T7-(dT)24 oligomer and the Superscript ChoiceSystem (Invitrogen, Carlsbad, CA). The T7 promoter introduced during thefirst strand cDNA synthesis was then used to direct the synthesis of cRNA byusing T7 RNA polymerase (Enzo Diagnostics, Farmingdale, NY) and bioti-nylated deoxynucleotide triphosphates. The biotin-labeled cRNA was frag-mented to a mean size of 200 bp before hybridization. Total RNA andbiotin-labeled cRNA were tested for the integrity and size by resolving onAgilent RNA 6000 Nano LabChips (Agilent, Palo Alto, CA). Hybridizationwas performed at 45°C for 16 h (0.1 M MES [4-morpholinepropanesulfonicacid (pH 6.6), 1 M NaCl, 0.02 M EDTA, and 0.01% Tween 20] and washedunder both nonstringent (1 M NaCl, 25°C) and stringent (1 M NaCl, 50°C)conditions. Chips were stained with phycoerythrin-streptavidin (10 �g/ml),

scanned, and analyzed with Microarray Suite 5.0 software (MAS 5.0; Af-fymetrix). Each infection was repeated twice, and each RNA sample washybridized to two chips.

Data Analysis and Statistics. The primary data captured using MAS 5.0software resulted in a single raw value for each probe set based on the meanof the differences between the hybridization intensity for the perfect matchfeatures and the mismatch features for a particular transcript (data analysisfundamentals: GeneChip expression analysis can be found online).4 Threetypes of normalizations were applied to this data before additional sorting andanalysis. The hybridization intensities across the treatments of a particular celltype were first normalized by applying the global normalization algorithm thattrims the mean signal intensity of the experiment to the trimmed mean signalof the baseline or control treatment. To facilitate comparison between samplesand experiments, the globally normalized data were further subjected to (a) aper chip normalization to account for chip-wide variations in intensity bydividing each intensity value by the 50th percentile of all values on the chip,and (b) a per gene normalization where each gene is divided by the intensityof that gene in the control sample. To perform the global normalizations withhigh confidence, a regression analysis (Pearson correlation coefficients) wasdone using the raw signal intensities of various treatments.

These files from expression analyses were then exported via MicroDB andData Mining Tool (Affymetrix) for additional filtering and analysis. In theseanalyses, genes designated as significantly changed were those (a) that pos-sessed a reliably detectable signal (absolute call � “Absent” or “Marginal” inthe case of repressions, whereas, in the case of inductions, differentiallyinduced genes were taken into analysis), (b) have detection P � 0.05, and (c)as determined by the statistical algorithm to be changed �2-fold (changecall � “no change” or “marginal”). To additionally increase the significance ofexpression changes, we interrogated our data sets for an increase in averagedifference (intensity) of at least 2-fold at both time points of HHV-8 infection(2 and 4 h) or 3-fold or more at a given time point. The primary oligonucleotidehybridization data as well as all of the tables created after filtering can beobtained by contacting the authors via e-mail ([email protected]).

Clustering and Gene Ontology (GO) Consortium Linking. To classifygene expression profiles into groups according to their behavioral patterns,cluster analysis was conducted. Two separate unsupervised clustering algo-rithms, a hierarchical clustering and a nonhierarchical K-means clustering,were performed (GeneSpring version 5.0; Silicon Genetics, Redwood City,CA). Hierarchical clustering was done using average linkage method andstandard correlation as a similarity measure. K-means clustering was per-formed after subjecting the data sets first for bioscript analysis (GeneSpring)that performs 3, 5, 8, and 10 K-means clusters and returns the classificationwith the highest explained variability. Data were subsequently analyzed fortheir functional affiliations by GO linking (GeneSpring).

Reverse Transcription-PCR (RT-PCR). Total RNAs isolated from theUI or HHV-8-infected cells using RNeasy (Qiagen) were DNase treated(Invitrogen) and subjected to cDNA synthesis using Thermoscript reversetranscriptase and random oligonucleotides (Invitrogen). Primer pairs designedusing Oligo 4.0 (Molecular Biology Insights, Cascade, CO) were used toamplify specific genes from 250 ng of cDNA using HotStar TaqDNA polym-erase (Qiagen). Amplifications were carried out in parallel for both infectedand UI samples. For manual quantitation, successive samples were removedevery three cycles, beginning with cycle 14 and continuing through cycle 44.Progressive PCR samples were resolved on agarose gels, visualized afterethidium bromide staining, and quantitated using AlphaImager 2000 (AlphaInnotech, San Leandro, CA). For all genes, integrated density values corre-sponding to the sum of pixel intensities after background corrections wererecorded for both the UI and infected samples at linear points on the ampli-fication curve and fold changes were created after normalizing to glyceralde-hyde-3-phosphate dehydrogenase (GAPDH). Expression changes of 18 cellu-lar genes were analyzed by RT-PCR using primer sequences that aresummarized in the supplementary data.5

Western Blot Analysis. Activation of p21CIP that was shown to be up-regulated in all of the 3 cell types by the array was confirmed by Western blot

4 Internet address: http://www.affymetrix.com/support/technical/manual/expression_manual.affx.

5 Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org).

73

TRANSCRIPTIONAL REPROGRAMMING BY KSHV/HHV-8



analysis. Target cells were grown to 70–80% confluence or to logarithmicphase by feeding the day before infection, serum-starved, and infected withGFP-HHV-8 at 37°C. At different time points, cells were rinsed with PBS andlysates were prepared (17). Ten �g of lysates were resolved on SDS-12%-PAGE and immunoblotted with antibody detecting p21CIP (Santa Cruz Bio-technology, Santa Cruz, CA) and antimouse-IgG-horseradish peroxidase(KPL, Inc., Gaithersburg, MD). The blots were stripped and reprobed withanti-�-actin antibodies as a loading control (Sigma).

Northern Blot Analysis. The activation of ICAM-1, DUSP5, and integrin�6 genes was confirmed by northern analysis. Ten �g of total RNA fromHHV-8-infected or UI target cells were separated on a 1% agarose-5% form-aldehyde gel in 4-morpholinepropanesulfonic acid buffer [20 mM 4-morpho-linepropanesulfonic acid (pH 7.0), 5 mM sodium acetate, and 1 mM EDTA] andtransferred to positively charged nylon membranes (Sigma) using 20� SSC (3M NaCl and 0.3 M sodium citrate). The membranes were hybridized with aDNA probe specific for ICAM-1, DUSP5, and integrin �6 and prepared asdetailed below. Total RNA isolated from HHV-8-infected BJAB cells (4 h)was converted to cDNA and subjected to PCR using primers specific forICAM-1, DUSP5. or integrin �6. The PCR products were resolved on agarose,purified from the gel, and labeled with [�-32P]dATP using Klenow fragment(Promega, Madison, WI). The membranes were hybridized with 1 � 106 to2 � 106cpm of probes/ml at 65°C overnight in Church buffer [1% BSA, 1 mMEDTA, 0.5 M sodium phosphate (pH 7.2), and 7% SDS]. Internal controlhybridization was performed for GAPDH in parallel. Blots were subjected totwo low stringency washes (2� SSC, 0.1% SDS, 15 min at room temperature)and one high stringency wash (0.5� SSC, 0.1% SDS, 15 min at 65°C) andexposed to a phosphorimager screen for quantitation and subsequently to XARfilm at �70°C.

RESULTS

Induction of Host Gene Expression by HHV-8 and the Validityof Gene Array. We have previously demonstrated that within min-utes of infection, HHV-8 entered the target cells and induced thepreexisting host cell signal pathways (16, 17). Target cells underwentmorphological changes, accumulated actin stress fibers, and inducedthe formation of filopodia and lamellipodia (17). These observationsindicate that virus greatly influences the cellular functions earlyduring infection. To further assess the impact of HHV-8 and itscomponents on the target cells early during infection, comparisons ofglobal gene expression profiles between UI and HHV-8-infected cellswere examined. HMVECd (primary human endothelial cells) andBJAB (human B cell line) represent two major in vivo targets ofHHV-8 infection. HFF (primary human foreskin fibroblast) cells wereincluded because activation of a variety of signaling cascades inresponse to HHV-8 infection was observed in these cells (16, 17). Aninitial time course experiment revealed the induction of a large num-ber of host cell gene expression after 90 min of infection (data notshown). To provide the best snapshot of HHV-8-induced genome-wide changes early during infection, we examined the gene expressionprofiles of cells at 2 and 4 h after infection.

Each RNA samples isolated from different treatments were hybrid-ized with two HG-U133A chips representing �22,283 annotatedtranscripts. Transcription of �50% of the total 22,283 transcripts(�9000) was detected (a present call) at each time point of treatment,which is probably well representative of the total gene expression inthe human cells. The gene expression profiles of HHV-8-infected cellsover the respective UI controls are depicted in the scatter plot shownin Fig. 1. Expression of only a small number of transcripts changed inconsequence to HHV-8 infection at both time points. Expressionratios of most of the genes were close to 1 or changed only �2-foldcutoff line, suggesting that they were not significantly changed inconsequence to HHV-8 infection (Fig. 1). These few changes indicatethat changes in expression unrelated to HHV-8 infection did notpresent a problem in our assays. Comparison of the signal intensities

between the arrays from one cell type revealed a low interarrayvariation (data not shown). Distributions of signal intensities amongtreatments were additionally analyzed by comparing mean signalintensities. This revealed a higher Pearson correlation coefficientbetween 2 and 4 h postinfection samples of one cell type than samplesfrom different cell types (data not shown). This further increases thereliability of the data and validates our gene array results.

HHV-8 Induces Cell Type-Specific as well as Conserved HostCell Gene Expression. Our gene array design included the analysisof HHV-8 interaction with three target cells to reflect the broadcellular tropism shown by this virus (10–12). To obtain a measure ofsimilarity between the three target cells in their capacity to respond toHHV-8 infection and to compare the behavior among the cells,average fold change after 2- and 4 h postinfection for each cell typewas determined, and a correlation matrix with Pearson correlationcoefficient was then calculated between the three cell types. Althoughthe correlation was relatively low as expected, correlation betweenHMVECd and HFF cells was 10 times higher compared with thePearson correlation coefficient of BJAB versus HMVECd and aboutthree times higher than BJAB versus HFF cells (data not shown).

Results of UI controls at two time points revealed high correlation

Fig. 1. Validity of human herpesvirus 8 (HHV-8)-induced host cell gene expressionprofiles. Human adult dermal microvascular endothelial (HMVECd), human foreskinfibroblast (HFF), and BJAB cells were infected with HHV-8 at five genome copies/cell at37°C, and the RNAs isolated at 2 and 4 h after infection were used to generate thebiotin-labeled cRNA probes. These probes were hybridized to two identical HG-U133Achips containing �22,283 human transcripts. The primary gene array data used togenerate the gene expression profiles are depicted by the scatter plot. Each data pointrepresents the relative mean hybridization intensity to one of the 22,283 transcripts inmRNAs purified from 2 or 4 h after infection with HHV-8 (depicted in the y axis), inrelation to the mock-infected controls (x axis). Genes whose expression was unchangedin the infected cells compared with mock are shown in yellow dots. Expression valuesdiffering by �2-fold are indicated by the cutoff line and represented by red (up-regulated)or blue (down-regulated) dots. The color scale at the bottom represents the expressionpattern with yellow representing no change (�1), grades of red representing up-regulation,and grades of blue representing the down-regulated genes.

74




values, with most of these signals changing �1.5-fold, thus justifyingthe 2-fold threshold filter used for calculating the significant modu-lation (data not shown). To increase the significance further, onlythose genes with �2-fold change in their expression at both timepoints and/or �3-fold at one time point were considered. Such filter-ing revealed that a total of 324, 374, and 175 transcripts changed inHMVECd, HFF, and BJAB cells, respectively. A further breakdownof these results revealed the up-regulation of 215, 243, and 170transcripts, whereas 109, 131, and 5 transcripts were down-regulatedin HMVECd, HFF, and BJAB cells, respectively (data not shown).For delineating the behavioral patterns, this data were then subjectedto hierarchical clustering, and the results obtained for individual cellsare shown Fig. 2A. Overall, more genes were up- or down-regulatedin the HMVECd (Fig. 2A, top panel) and HFF cells (Fig. 2A, middlepanel) than in the BJAB (Fig. 2A, bottom panel) cells. These differ-ential or cell type-specific expression changes were more prominentfor the down-regulated genes (represented in shades of blue) becausevery few genes were repressed in BJAB cells compared with HMV-ECd or HFF cells (Fig. 2A).

Comparison of transcriptional profiling between cells was subse-quently done, which yielded fewer common genes. This is probablybecause as the number of arrays in each data set increases, it becomesless likely for a particular gene to pass such strict filtering criteria.Nevertheless, genes emerge from such comparisons will be of greatinterest for studying their role in the unique biology of HHV-8.Several features were apparent from these analyses: (a) many of thegenes with altered expression were detected in more than one sample,arguing against spurious changes; (b) list generated for dynamicallyexpressed genes is much shorter for BJAB cells; and (c) there wasvirtually nonoverlapping list of genes in all of the three cell typesanalyzed. The Venn diagram shown in Fig. 2B represents the celltype-specific distribution of HHV-8 regulated genes and their overlapin expression. Of the total 686 genes, 33 were regulated across all ofthe cell types, 121 genes were regulated by two of the three cells,whereas the rest did not share the transcriptional responses (Fig. 2B).

Semiquantitative RT-PCR of Selected Genes Confirms theGene Array Results. To confirm the oligonucleotide gene arrayresults by an independent method, semiquantitative RT-PCR analysison UI and HHV-8-infected samples were carried out for 18 selectedgenes. These included 7 genes in which the differential regulation wasseen in all three target cells, 7 in two target cells, and 4 genes that wereuniquely regulated in a single cell type (Table 1). As an example,RT-PCR confirmation of DUSP5 gene that was up-regulated in all ofthe three target cells is shown in Fig. 3. DUSP5 RT-PCR product fromthe infected RNA was readily detectable by sample 8 (cycle 35; Fig.3A, Lane 9), whereas the RT-PCR product from the UI RNA samplewas only detectable by sample 11 (cycle 44; Fig. 3A, Lane 12). Incontrast, the amplification of constitutively expressed GAPDH genereached the linear phase equally in both UI and HHV-8-infected RNAsamples starting from sample 3 (cycle 17; Fig. 3B). Comparison of theintegrated density value scores of the DUSP5 gene from the infectedand UI control RNA is shown in Fig. 3C. These results suggested thatthe DUSP5 gene in the infected RNA underwent linear amplificationnine PCR cycles earlier than did the parallel UI cell RNA.

We carried out the above described comparison for 18 selectedgenes, normalized them to the GAPDH mRNA levels and calculatedthe fold induction. For the 7 genes whose expression changed in all ofthe three target cells, a positive correlation was obtained for all of the7 at both time points in all of the three different target cells. For therest of the genes, RT-PCR was done using RNA from all three targetcells collected 4 h after infection where greater changes was observed(Tables 1 and 2). Larger changes were generally observed by RT-PCR, with the exception of MAP3K8, where lower fold activation

was observed in the RT-PCR reactions (20.11- versus 2.8-fold, Table1). Inhibitor of apoptosis homologue C was up-regulated only in theHMVECd and BJAB by the array; however, by RT-PCR, up-regula-tion was detected in all three infected target cells (Table 1). Similarly,Coxsackie and adenovirus receptor was identified as a down-regulatedgene by the array only in the HMVECd cells. In HFF and BJAB cells,although the results (raw data) showed a down-regulation, the detec-tion Ps were higher (�0.99), and hence, the Coxsackie and adenovirus

Fig. 2. A, hierarchical clustering of human herpesvirus 8 (HHV-8)-induced host cellgene expression infogram. Human adult dermal microvascular endothelial (HMVECd),human foreskin fibroblast (HFF), and BJAB cell genes that were either up- or down-regulated after HHV-8 infection by �2-fold at both time intervals or �3-fold at one timeinterval were analyzed by hierarchical clustering. The data are presented as ratio of themean of average intensities in two hybridizations with RNA from HHV-8-infected cellscompared with the uninfected cells. Each row represents one experimental condition, andeach column represents one transcript. Genes with higher levels of expression (induction)after HHV-8 infection compared with uninfected cells are shown in progressively greatershades of orange/red, and the repressed genes are represented in progressively brightershades of blue (see scale in the bottom). B, cell-type distribution of HHV-8-induceddifferentially expressed host genes: Venn diagram showing the distribution of differen-tially regulated HMVECd, HFF, and BJAB cell genes after 2 and 4 h postinfection withHHV-8 relative to mock infection. Differentially expressed genes were selected fromcombined data of at least two experiments (see “Materials and Methods”). Some differ-entially regulated genes were shared between all of the cell types while others showedunique regulation. Area shaded in white represents the number of genes that were affectedin all three cells. Genes that were differentially regulated in two of the three cells or in asingle-cell type are indicated by the color code at the bottom. Of the total 686 genes, 33were regulated across all of the cell types, 121 genes were regulated by two of the threecells while the rest were not shared transcriptional responses.

75




receptor was considered an outlier and was filtered out in the analysis.Results of RT-PCR analysis showed an up-regulation of Coxsackieand adenovirus receptor in HMVECd-infected cells while no changewas seen in the BJAB and HFF cells (Table 1). The observed differ-ences between the array and RT-PCR assays and the differences in themagnitude of induction detected could be attributable to the increasedsensitivity of RT-PCR and/or the potential for chip saturation. Nev-ertheless, importantly, RT-PCR confirmed the induction or repressionfor the majority of the genes tested in the RNA samples used for arrayanalysis, as well as in the independently derived set of RNA samplesderived from target cells infected with wild-type HHV-8 (data notshown).

Northern and Western Blot Assays for Selected Genes Verifythe Gene Array Data. To obtain additional confirmation of arrayresults (Table 2) by another independent method, Northern blot anal-ysis was performed using RNA preparations tested by gene array.Total RNA from HHV-8-infected cells at 4 h after infection andcontrols were fractionated by gel electrophoresis, blotted, and probedwith 32P-labeled probes. The Northern blots (Fig. 4A) confirmed thegene array results (Table 2) for ICAM-1, DUSP5, and integrin �6genes. A low level of constitutive expression of ICAM-1 and DUSP5was detected in the UI cells (Fig. 4A, top two panels, Lanes 1, 3, and5). Upon HHV-8 infection, ICAM-1 was strongly up-regulated by 22-,20-, and 18-fold in BJAB, HFF, and HMVECd cells, respectively(Fig. 4A, top panel, Lanes 2, 4, and 6). A similar pattern was observedfor the DUSP5 gene (Fig. 4A, panel 2, Lanes 2, 4, and 6). UnlikeICAM-1 and DUSP5 genes, integrin �6 gene expression was detectedonly in the infected BJAB cells by the gene array (data not shown).Northern blot analysis confirmed the array data and a 10-fold induc-tion was observed (Fig. 4A, panel 3, Lanes 1 and 2). Neither anendogenous expression (Fig. 4A, panel 3, Lanes 3 and 5) nor aninduction (Fig. 4A, panel 3, Lanes 5 and 6) was observed for integrin�6 in HMVEC-d and HFF cells. Equal amount of GAPDH wasdetected in all these samples demonstrating the equal loading of RNAin these samples (Fig. 4A, bottom panel).

To correlate the gene array detected transcription changes withprotein levels, we analyzed the expression levels of p21CIP protein by

immunoblot. Compared with UI controls, after HHV-8 infection, genearray detected the consistent induction of p21CIP gene in all threetarget cells at both time points (Table 2). Western blot analysis at 2,3, 4, 6, and 8 h postinfection of all of the target cells showed thatp21CIP was up-regulated with a sustained activation up to 6 h postin-fection (Fig. 4B, Lanes 2–6). Immunoblot for �-actin detected similarexpression in all these samples, demonstrating the equal proteinloading in these experiments (Fig. 4B, bottom panel). In summary, ourdata revealed a good correlation between array and other means oftarget verification, thereby validating our methodology and analysis.

HHV-8-Induced Host Cell Genes Cluster Based on Their Ki-netics of Activation. As our experimental data contains only twoconditions (infection at two time points versus UI), a simple hierar-chical clustering could provide sufficient visual clustering of the dataset. Because a visual inspection might fail to detect any subtle vari-ation patterns hidden in the data set, a more sophisticated K-meansclustering was performed. The initial bioscript analysis on 154 mostinformative genes suggested that K-means clustering into five groupswould represent the highest variability. Thus, in the first stage, thealgorithm was iterated to create five clusters with an additional fiveclusters option for tightening the clustering process. In the secondfine-adjustment phase, the algorithm was iterated for additional clus-ters. However, the results with six or more clusters returned withduplication of kinetic patterns.

An example of the K-means clusters for BJAB cells is shown in Fig.5. Each of the five clusters represented different kinetics of geneactivation that included clusters I and II showing repression kineticsand clusters III–V exhibiting up-regulation (Fig. 5). Cluster I con-tained 29 transcripts (18.83%) that were down-regulated during theinfection. Cluster II genes (15 genes, 9.74%) also were down-regu-lated and is the only cluster that demonstrated a generalized repressionpattern that was maintained from 2–4 h after infection. Cluster IIIcontained 18 transcripts (11.68%), including genes in which inductionpeaked at 2 h after infection and began to return to the basal levelbetween 2 and 4 h after infection. Cluster IV, which represents themaximum number of genes (54 genes, 35.06%), showed a sustainedup-regulation pattern at 2 and 4 h postinfection. Cluster V represented

Table 1 Concordance between gene array and semiquantitative reverse transcription-PCR (RT-PCR) data

To confirm array results by semiquantitative RT-PCR assay, equal amount of RNA isolated from three target cells either mock-infected or human herpes virus 8-infected wereconverted to cDNA using Thermoscript reverse transcriptase and random hexamers at 60°C for 50 min. A total of 250 ng of cDNA was then subjected to PCR for cycles 14–44. Aliquotsof PCR product were resolved on agarose gels, stained with ethidium bromide, and scanned to calculate the IDV values. IDVs at linear points on the amplification curve were comparedbetween infected and mock-treated samples to calculate the fold change in expression. Values in parenthesis represent fold change as detected by RT-PCR assay.

Gene name

Fold change in expression

Human adult dermal microvascularendothelial cells Human foreskin fibroblast cells BJAB cells

2 h 4 h 2 h 4 h 2 h 4 h

Dual specificity phosphatase 5 6.32 (9.5) 19.06 (8.95) 8.85 (9.51) 8.85 (15.8) 5.22 (15) 2.5 (11)Immediate early response 3 3.34 (7) 2.79 (3.8) 5.77 (6.9) 5.64 (8.3) 6.28 (10) 1.67 (2.8)Sprouty (Drosophila) homologue 2 2.3 (5.4) 2.49 (4.35) 4.27 (4.8) 4.69 (4.9) 3.67 (3.0) 4.44 (4.5)ICAM1 6.59 (8.2) 12.02 (12.7) 23.1 (16.84) 17.8 (14.8) 1.2 (4.22) 3.1 (3.76)B-cell lymphoma 6 5.54 (6.42) 4.61 (4.8) 3.47 (2.99) 3.18 (4.34) 1.81 (3.7) 3.71 (6.76)u-Plasminogen activator receptor 2.22 (4.0) 5.46 (6.1) 8.78 (9.99) 11.82 (14) 4.62 (6.0) 5.21 (8.2)PIM1 (protein kinase related) 2.95 (8.0) 2.96 (6.1) 9.6 (16) � (3.0) 2.34 (5.0) 2.99 (4.0)Human IAP homologue C 13.74 (20) 4.06 (10) � (5.0) � (4.0) 2.26 (9.0) 7.87 (11.0)Protein kinase C, � �a (n/d)b � (�) 3.98 (n/d) 2.99 (3.53) 2.14 (n/d) 2.9 (3.6)Potential tumor suppressor (ST7) � (n/d) � (�) 1.87 (n/d) 3.78 (16) 1.67 (n/d) 4.26 (6.0)Matrix metalloproteinase 1 1.58 (n/d) 19.06 (20.0) � (n/d) � (�) �1.16 (n/d) 5.21 (7.5)Mx resistance 1 2.6 (n/d) 2.29 (10) 21.59 (n/d) 19.5 (20) � (n/d) � (�)MAP kinase kinase kinase 8 � (n/d) � (�) 10.57 (n/d) 20.11 (2.8) � (n/d) � (�)Coxsackie and adenovirus receptor �1.5 �13.19 (15) � (n/d) � (�) � (n/d) � (�)Down in ovarian cancer 1 �1.33 �7.24 (�10) � (n/d) � (�) � (n/d) � (�)Regulator of G-protein signaling 16 � (n/d) � (�) 4.65 (n/d) 10.19 (15) 9.46 (n/d) 2.16 (7.5)Phospholipid scramblase 4 �2.88 (n/d) �3.25 (�7) �2.78 (n/d) �2.57 (�5) � (n/d) � (�)Sorting nexin 2 � (n/d) � (�) � (n/d) � (�) �1.04 (n/d) �4.71 (�6)

a , negative in array with change in expression �1.5 fold; (�), negative in RT-PCR.b , n/d, not done by RT-PCR.

76




by 36 genes showed up-regulation that was maintained at both 2 and4 h after infection. Although these clusters included genes involved ina variety of functions, some general patterns were prominent. ClusterI represented the majority of cytokines such as interleukin (IL)-1�,IL-6, chemokine (CXC) ligand 2,5, CXCL10 (IP10), cytokine Gro-�,�, and �, and the IFN-stimulated genes such as 2�5�OAS, MxA, Mr15,000 protein, T-cell � chemoattractant precursor, and guanylate

binding protein 1. The tables listing members of different clusters I–Vare presented in the supplementary data.5

Although similar kinetic patterns were observed for the differen-tially expressed HFF and HMVECd genes, the following differenceswere prominent: (a) variation in the number of genes in each cluster;and (b) variation in the functional groups to which genes in eachcluster belonged. For example, a majority of the IFN-stimulated genesshowed the repression pattern and belonged to gene cluster I in theBJAB cells. In contrast, in HFF and HMVECd cells, IFN-responsivegenes and other cytokine genes belonged to cluster IV with a sus-tained up-regulation at 2 and 4 h postinfection (data not shown), apattern similar to clusters IV and V of the BJAB cells. Another cleardifference was the observation that majority of the up-regulatedangiogenic factors such as (vascular endothelial growth factor-A(VEGF-A), angiopoietin-related protein-4, stanniocalcin, and endo-thelial differentiation gene-1 in the HFF and HMVECd cells weredown-regulated in the BJAB cells. The lack of functional correlationsin these cells was additionally supportive of our initial observation oflow correlation between the BJAB and other two primary adherentcells. These results suggested that HHV-8-induced modulation ofgene expression in B cells was different compared with the adherentcells.

Cell Type-Specific Gene Activations Early during HHV-8 In-fection. To further investigate the HHV-8 induced cell type-specificgene modulations, the uniquely regulated genes in BJAB (n � 102),HFF (n � 239), and HMVECd (n � 191) cells that were not sharedwith other cells (Fig. 2B) were analyzed for their biological functions.The GO building primarily identified GO terms involved in variousbiological processes, cell components, and molecular functions.Nodes for molecular functions were more elaborate and appropriate,and hence, we focused more on the GO molecular functions. NotableGO molecular functions that associated with all of the cells includemajor processes such as apoptosis, cell cycle, cancer, microtubulardynamics, structural proteins, transport, and signal transduction (Fig.6). The top-ranked biological process also reflected the same molec-ular function, i.e., cell signaling and cell-cell communication ac-counted for �90% of the responses (data not shown). Such mappingof biological functions again showed the following behavioral differ-ence in BJAB cells compared with the two primary adherent targetcells: (a) despite the smallest number of uniquely regulated genes inBJAB, it included a large number of apoptosis regulators; (b) majorityof apoptotic regulators in B cells were proapoptotic unlike the anti-apoptotic mediators induced in the HFF and HMVECd cells; and (c)there was a total lack of immunity-related proteins in BJAB cells. Thestriking cell type-specific behaviors additionally supported the notionthat at least in the initial stages of infection, HHV-8-induced host cellgene modulation events in B cells were different compared with theadherent endothelial and fibroblast target cells.

Biological Implications of Host Cell Genes Modulated duringthe Early Phases of HHV-8 Infection. To explore the relevance ofHHV-8-modulated genes in its biology, an interactive exercise involv-ing several databases such as Unigene, LocusLink, Gene Cards,PubMed, and extensive review of literature was conducted. For thisanalysis, only the list of 154 genes shared by at least two cell typeswas used. Of these dynamically regulated 154 transcripts, functions of13 sequences were not known. The remaining gene products wereclassified according to known functions in signal induction, apoptosis,transcription, host defense, inflammatory responses, angiogenesis,tumorigenesis, cell and tissue structural dynamics, metabolic path-ways, and various other functions (Table 2). These analyses revealedthe modulation of several genes not previously implicated in KS orlinked to HHV-8 biology. With a specific focus on the most inform-

Fig. 3. Semiquantitative reverse transcription-PCR (RT-PCR) confirmation of DUSP5gene up-regulation detected by gene array. A, DNase-treated total RNAs isolated from theuninfected or human herpesvirus 8 (HHV-8)-infected cells were subjected to RT-PCRusing specific primers. Successive samples removed from every three cycles (14–44) andresolved on agarose gel, and fold changes were calculated after normalizing to glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) gene. Ethidium bromide-stained RT-PCRamplified DUSP5 gene products after agarose gel electrophoresis in uninfected or HHV-8-infected BJAB, human foreskin fibroblast (HFF), and human adult dermal microvas-cular endothelial (HMVECd) cells are shown. Lane 1 shows the 100-bp marker and Lanes2–12 show the resolved RT-PCR products from cycles 14–44. B, representative ampli-fication of GAPDH gene as control using the same uninfected and HHV-8-infected BJABcell RNA samples used in Fig. 4A. Lane 1 shows 100-bp ladder and Lanes 2–8 representRT-PCR products obtained from cycles 14–32. C, representative histogram depicting theintegrated density value (IDV) quantitation of differential amplification of DUSP5 gene inmock- and HHV-8-infected BJAB cell RNA samples.

77




Table 2 Dynamically regulated host genes of target cells early during human herpes virus 8 infectiona

Gene name and function Accession no.

Fold change at h postinfection

Human adult dermalmicrovascular

endothelial cellsHuman foreskinfibroblast cells BJAB

2 h 4 h 2 h 4 h 2 h 4 h

Signal transductionDual specificity phosphatase 5 U16996 6.32 19.06 8.85 8.85 5.22 2.5Sprouty (Drosophila) homologue 4 W48843 4.08 10.17 5.33 5.24 3.85 4Immediate early response 3 NM_003897 3.34 2.79 5.77 5.64 6.28 1.67Sprouty (Drosophila) homologue 2 NM_005842 2.3 2.49 4.27 4.69 3.67 4.44Phospholipid scramblase 1 NM_021105 2.07 2.45 1.45 3.82 2.49 3.92Regulator of G-protein signaling 16 U94829.1 4.65 10.19 9.46 2.16Preprourokinase K03226.1 7.27 9.15 3.67 9.38Dual specificity phosphatase 6 BC005047 4.36 9.96 7.22 8.04Dual specificity phosphatase 1 NM_004417 5.32 4.74 2.4 4.37Phosphoprotein regulated by MAPK pathways NM_025195 6.05 9.22 7.75 4.1 14.95 5.03Protein kinase C, � NM_006254 3.98 2.99 2.14 2.9Phospholipase A2, group IVA M68874 1.59 5.04 1.11 14.45ADP-ribosylation factor-like 7 BG435404 1.25 4.67 2.65 4.07Mitogen-activated protein kinase kinase 3 AA780381 1.54 3.99 3.66 2.36A kinase (cAMP) anchor protein 13 AK022014 9.01 14.95 2.06 3.38Type II serinethreonine kinase receptor U20165 2.22 2.09 2.98 2.95Serum-inducible kinase NM_006622 �1.91 �3.17 �2.13 �2.32Diphtheria toxin receptor NM_001945 1.02 3.25 94.82 15.58 12.67 8.38Heparin-binding EGF-like growth factor M60278 1.1 4.61 18.09 3.84 6.46 4.26SMAD7 NM_005904 12.85 1.69 5.5 4.48 3.12 2.8Inositol 1,4,5-triphosphate receptor, type 1 NM_002222 3.36 4.56 4.26 3.49

AntiapoptoticBasic helix-loop-helix domain containing, class B, 2 BG326045 4.04 7.01 2.16 4.96 3.8 2.63B-cell lymphoma 6 NM_001706 5.54 4.61 3.47 3.18 1.81 3.71Inhibitor of DNA binding 2 NM_002166 6.2 1.25 3.35 1.42 2.63 2.57Leukemia inhibitory factor NM_002309 92.76 43.83 4.22 1.69Fas-interacting serinethreonine kinase 3 AF305239.1 2.65 2.56 2.68 2.48Human IAP homologue C U37546.1 13.74 4.06 2.26 7.87BCL2-related protein A1 NM_004049 2.5 4.17 2.89 10.48TNF�-induced protein 3 NM_006290 6.08 4.79 6.28 4.24TNF-induced protein (GG2-1) BC005352 3.18 1.53 4.63 3.85BAG-family molecular chaperone regulator-2 AF095192.1 �4.74 �1.77 �2.94 �2.94Phospholipid scramblase 4 NM_020353 �2.88 �3.25 �2.78 �2.57Cyclin-dependent kinase inhibitor 1A NM_000389 2.72 2.39 2.61 2.19 3.84 3.72Myeloid cell leukemia sequence 1 AI275690 2.32 2.78 2.68 2.94

Transcription factors/proto-oncogenesjun B NM_002229 12.18 12.18 10.51 5.16 4.7 5.25Early growth response 3 NM_004430 6.14 11.23 10.17 1.31 12.05 2.3B-cell CLL lymphoma 3 AI829875 5.75 4.19 20.23 3.7 1.88 3.2Nf B enhancer in B-cells inhibitor, � AI078167 3 1.51 18.04 10.26 2.33 3.49HIV-1 enhancer-binding protein 2 AL023584 7.3 4.08 2.07 3.54 3.07 2.95FOS-like antigen 2 NM_024530 3.4 2.15 6.64 2.62 2.5 2.87TGFB-inducible early growth response NM_005655 2.9 2.42 4.76 2.05 4.49 1.49Pleckstrin homology-like domain, family A,1 NM_007350 4.65 5.8 3.33 3.1DNA-binding zinc finger AB017493 2.71 5.14 5.16 3.08Zinc finger protein homologous to Zfp-36 NM_003407 2.8 2.05 3.84 3.02NFB enhancer in B cells 1 (p105) M55643.1 2.4 3.64 2.53 2.99CCAAT enhancer binding protein, � AL564683 2.2 2.22 2.41 2.98Early growth response 1 NM_001964 6.52 13.68 4.45 3.24p54 (EGR binding protein 1) AF045451.1 4.48 3.05 5.1 2.36NFB enhancer in B-cells 2 (p49p100) NM_002502 2.31 2.88 1.7 6.1v-maf, oncogene family, protein F AL021977 2.53 3.25 3.41 3.31Forkhead box O1A NM_002015 2.98 2.22 2.78 3.87�-Glucocorticoid receptor X03348.1 2.84 2.92 1.61 3.05Glucocorticoid receptor �-2 U01351.1 3.26 1.54 2.63 2.55Transcription factor 8 (represses IL2 expression) NM_030751 3.14 3.83 9.55 3.54CCAAT enhancer binding protein (CEBP), � NM_005195 7.68 3.92 2.63 2.42v-ets homologue 2 NM_005239 1.49 5.56 3.85 4.2Cas-Br-M, retroviral transforming sequence-b U26710.1 1.75 6.4 1.42 3.76Nuclear factor, interleukin 3 regulated NM_005384 2.75 2.64 4.36 3.6FOS-like antigen-1 BG251266 2.53 3.81 2.92 3.05ELL-related RNA pol II elongation factor NM_012081 2.72 2.59 1.14 3.27Transcriptional coactivator with PDZ-binding motif AA081084 2.2 2.81 1.39 3.24Estrogen-responsive B box protein NM_006470 �4.19 �1.19 �2.73 �2.88Nuclear receptor subfamily 2, group F, member 2 AL554245 �1.52 �3.78 �2.61 �3.69

IFN responsiveInterferon regulatory factor 7, transcript variant c NM_004030 3.24 3.28 3.99 3.4IFN-inducible, 67kD guanylate binding protein 1 BC002666 3.03 2.12 3.61 1.89Mx resistance 1, IFN-inducible protein p78 NM_002462 2.6 2.29 21.59 19.5IFN-stimulated T-cell � chemoattractant AF030514 15.09 14.46 2.19 2.95Interferon regulatory factor 1 NM_002198 5.88 2.25 9.98 1.622-5oligoadenylate synthetase 2 (OAS2) NM_016817 3.55 3.4 3.53 4.71Interferon-stimulated protein, 15 kDa NM_005101 3.06 3.21 2.13 3.4ISGF-3 M97935 2.27 2.01 2.82 2.19

78




Table 2 Continued





2 h 4 h 2 h 4 h 2 h 4 h

CytokinesInterleukin 4 receptor NM_000418 2.4 3.02 1.82 2.29GRO3 oncogene (GRO �) NM_002090 63.69 3.42 30.64 33.9Interleukin 6 (interferon, � 2) NM_000600 34.51 38.4 13.71 35.23Interleukin 1 � M15330 3.48 12.37 1.96 12.52GRO1 oncogene NM_001511 12.96 1.16 5.41 5.9Monocyte chemotactic protein MCP1 S69738.1 4.59 1.09 8.91 2.43STAT-induced STAT inhibitor 3 NM_003955 16.63 35.81 18.49 10.89 2.56 2.22Prostate differentiation factor AF003934 1.64 3.13 3.35 2.68Cytokine gro-� M57731 88.87 5.62 8.43 4.78Pre-B-cell colony-enhancing factor BF575514 1.29 3.06 3.5 4.58Cytokine, Cys-X-Cys, member 10 NM_001565 2.64 2.7 2.42 2.41Macrophage-specific CSF-1 M37435.1 2.53 2.58 5.14 2.47

AngiogenesisIL-8 C termmal variant AF043337 11.26 5.28 39.4 64.39 2.93 3.27ICAM1 AI608725 6.59 12.02 23.1 17.87 1.2 3.1Tissue inhibitor of metalloproteinase 1 NM_003254 2.44 3.39 2.59 2.41Microvascular endothelial differentiation gene 1 NM_012328 2.24 3.13 1.07 4.01Inhibitor of DNA binding 3 NM_002167 3.44 1.49 23.1 59.36Thrombomodulin NM_000361 3.84 3.84 6.16 52.49PPAR (�) angiopoietin related protein 4 NM_016109 3.69 1.9 31.17 18.33Plasminogen activator inhibitor 2 NM_002575 1.8 5.35 8.23 22.73Urokinase plasminogen activator receptor precursor AY029180.1 2.86 10.24 20.02 23.98 3.93 5.02Urokinase-type plasminogen activator receptor U08839.1 2.22 5.46 8.78 11.82 4.62 5.21Matrix metalloproteinase 1 NM_002421 1.58 19.06 �1.16 5.21Plasminogen activator, tissue NM_000930 1.43 3.26 1.78 4.5Stanniocalcin AI300520 2.28 35.36 12.41 49.58VEGF AF022375.1 2.92 2.8 6.8 5.13fms-related tyrosine kinase AA058828 1.55 8.19 1.35 3.26Vascular permeability factor VEGF (VEGFA) M27281.1 2.59 2.62 6.67 5.46Vascular endothelial growth factor C U58111.1 3.4 2.15 6.64 2.62 2.5 2.87

Cancer signaturesUridine phosphorylase NM_003364 3.75 8.77 8.51 14.9Potential tumor suppressor (ST7) NM_013437 1.87 3.78 1.67 4.26N-myc downstream regulated NM_006096 1.95 4.22 2.33 3.02Snail 1 (Drosophila homologue) NM_005985 4.84 5.55 5.65 2.86Nucleoside phosphorylase NM_000270 2.93 6.55 2.92 6.25Insulin-induced gene 1 NM_005542 2.94 2.25 3.01 1.42Absent in melanoma 1 U83115.1 �2.73 �4.04 �2.41 �2.65

Cell and structural dynamicsProtein kinase-related oncogene (PIM1) M24779.1 2.95 2.96 9.6 1 2.34 2.99Integrin, � 2 NM_002203 1.61 10.08 2.1 13.14Cytovillin 2 (ezrin) J05021.1 �1.74 �3.41 �2.31 �1.18Plectin, intermediate filament binding protein Z54367 �1.47 �3.45 �1.29 �3.39Nephropontin M83248.1 1.68 2.34 2.16 12.65

Small molecular/vesicle transportAquaporin N74607 3.57 4.03 5.26 10.55 4.94 6.3Solute carrier family 2 NM_006931 7.3 17.5 3.82 2.64 4.06 4.13Importin � 3 U93240.1 2.87 2.43 2.61 3.55Solute carrier family 20, member 1 NM_005415 4.14 2.9 3.1 2.96RAN binding protein 2-like 1 AL043571 3.68 1.7 2.89 2.31RAB31, member RAS oncogene family BE789881 2.96 2.66 1.35 3.45Ral guanine nucleotide dissociation stimulator AI421559 2.89 1.85 4.96 2.67Niemann-Pick disease, type C1 NM_000271 1.58 4.94 1.66 3.75Solute carrier family 4, NaHC03 cotransporter 3 AF047033.1 1.2 4.6 2.64 2.61

Proteolysis/protein processingUbiquitin-conjugating enzyme E2D 1 AL545760 2.29 6.7 2.64 3.08Secretory granule, neuroendocrine protein 1 NM_003020 1.31 3.44 1.4 5.34

Molecular chaperonsSuperoxide dismutase 2 X15132.1 2.44 9.6 1.7 4.24DnaJ-like heat shock protein 40 NM_007034 �3.78 �1.22 �3.44 �2.1Zinc finger protein 238 AJ223321 �2.16 �3.78 �2.48 �2.2Zinc finger protein 133 U09366 �2.93 �2.13 �2.12 �2.49

Metabolism6-phosphofructo-2-kinasefructose-2,6-biphosphatase NM_004566 4.32 6.3 3.05 2.17 2.39 2.27Prostaglandin-endoperoxide synthase 2 (Cox2) NM_000963 84.65 44.79 11.16 9.55GTP cyclohydrolase 1 NM_000161 5.13 4.86 7.05 6.28Glutamine-fructose-6-phosphate transaminase 2 NM_005110 1.25 3.29 2.92 5Tryptophanyl-tRNA synthetase NM_004184 1.91 3.28 2.27 3.66Carnitine octanoyltransferase NM_021151 �2.57 �2.76 �2.46 �2.66

OthersNotch homologue AC005390 4.83 5.52 2.27 3.67 2.49 3.14Hypothetical protein FLJ23306 N36408 4.1 2.99 6.57 1.68 4.43 2.08Transmembrane protein 2 NM_013390 2.02 4.15 2.84 2.38 2.03 2.97Hypothetical protein FLJ12929 NM_024937 1.42 3.76 1.74 4.14Hypothetical FLJ21897 AW138902 2.25 2.39 2 2.29Hypothetical DKFZp586J0720 AI613483 1.35 2.91 1.99 2.03

79




ative genes, here, we discuss some of these gene products with respectto their role in the biology of HHV-8 and potential involvement in KS.

Up-Regulation of Genes Regulating the Signaling Networksduring the Early Phases of HHV-8 Infection. HHV-8 activates avariety of cellular signaling molecules early during infection (16, 17).

Inducible signaling agonists and antagonists play a vital role inregulating the strength, duration, and range of action of cellularsignals. Our array results demonstrated the modulation of a number ofmolecules involved in the regulation of signaling cascades. Thisincluded IER3/IEX-1, which is a new type of ERK substrate (does notinteract with c-Jun NH2-terminal kinase or p38). IEX-1 has a dual rolein ERK signaling by acting both as an ERK downstream effectermediating survival and as a regulator of ERK (21). HHV-8 alsoactivated other regulatory molecules of cell signaling like sproutyhomologues and DUSP5 genes. Human sprouty 1–4 are orthologs ofDrosophila sprouty, which is a general intracellular tyrosine kinasesignaling inhibitor (22). DUSPs inactivate kinases by dephosphory-lating both the phospho-serine/threonine and phospho-tyrosine resi-dues. They negatively regulate the members of the mitogen-activatedprotein kinase superfamily and DUSP5 has maximal activity towardERK (23). HHV-8 induced significant activation of ERK in HMVECdand HFF cells early during infection, and such activations weresustained for the first 30 min, after which, the activation lowered tothe background level (17). Activation of Sprouty 2 and 4 and DUSP1, 5, and 6 observed during HHV-8 infection (Table 2) may constitutea significant feedback inhibitory mechanism for deactivating ERK1/2,thereby restoring these signaling pathways to their virus sensitivepreinfection state.

Up-Regulation of Genes Encoding Antiapoptotic Proteins dur-ing the Early Phases of HHV-8 Infection. HHV-8 encodes andexpresses several antiapoptotic proteins during its latency (v-FLIP)and lytic cycle (v-BCl-2, K7, and K15; Ref. 4). However, HHV-8 alsomust have developed additional mechanisms early during infection toblock apoptosis that is probably triggered by virus binding and entryinto the target cells (Table 2). These factors mediate both transient andsustained protection against various apoptotic stimuli. Our resultsshow that HHV-8 induces the inhibitor of apoptosis in HMVECd cellsby �14- and 4-fold at 2 and 4 h after infection, respectively, and by�2- (2 h) and 8 (4 h)-fold in BJAB cells (Table 2). The inhibitor ofapoptosis family of proteins prevents cell death by binding to andinhibiting active caspases 3 and 9 (24). HHV-8 induced the bcl-2-related protein A1 in HMVECd and BJAB cells (Table 2). Bcl-2-related protein A1 does not block proapoptotic caspases but is be-lieved to be a temporary protection mechanism against apoptoticstimuli (25). Myeloid cell leukemia-1 was another antiapoptotic Bcl-2family member that was up-regulated by HHV-8 (Table 2). Similar toother Bcl-2 family members, myeloid cell leukemia-1 localizes in themitochondria and can associate with other proapoptotic family mem-

Fig. 4. Validation of gene array data by Northern and Western blots. A, total RNA (10�g) from the uninfected (UI) (Lanes 1, 3, and 5) and BJAB, human foreskin fibroblast(HFF), and human adult dermal microvascular endothelial (HMVECd) cells infected withHHV-8 for 4 h (Lanes 2, 4, and 6, respectively) were resolved on 1% denaturing agarosegels and transferred to nylon membrane for 20 h using 20� SSC. Membranes werehybridized overnight with the [32P]-labeled probes for indicated genes. B, changes in theexpression of p21CIP after HHV-8 infection. Serum-starved target cells (Lane 1) orHHV-8-infected cells at different time points (Lanes 2–6) were normalized for equalprotein loading and were resolved on a SDS-12%-PAGE. To monitor the p21CIP induc-tion, blots were reacted with anti-p21CIP antibodies (top three panels) or with anti-�-actinantibodies (bottom panel).

Table 2 Continued





2 h 4 h 2 h 4 h 2 h 4 h

Hypothetical protein FLJ10669 NM_018174 1.21 6.36 2.85 2.25Hypothetical MGC:5618 BF575213 3.48 9.76 3.24 4.82KIAA0247 protein AA521267 1.57 5.13 4.03 6.4Hypothetical protein FLJ20285 NM_017745 7.08 2.02 4.83 2.21Hypothetical protein FLJ22393 NM_025106 2.36 2.32 5.92 3.61Tetraspan NET-6 protein D50911.2 3.46 2.81 1.33 7.96Hypothetical FLJ22893 AK026546.1 1.87 5.09 1.96 4.32ATP-binding cassette, subfamily B, member 2 NM_000593 2.44 3.36 3.73 3.71Hypothetical protein FLJ20898 NM_024600 2.41 6.26 2.05 2.34cDNA DKFZp564D042 AL049983.1 2.22 2.04 2.86 2.01Retinal degeneration B � NM_012417 1.45 3.3 1.04 3.19IFN-induced transmembrane protein 1 AA749101 2.9 2.45 2.81 2.68Dickkopf (Xenopus laevis) homologue 1 NM_012242 �4.76 �1.28 �4.92 �3.6Up-regulated by 1,25-dihydroxyvitamin D-3 NM_006472 �2.06 �3.87 �3.41 �5.03Progesterone membrane binding protein NM_006320 �3.39 �3.5 �2.37 �1.45

80




bers (25). The only virus infection that is known to induce bcl-2-related protein A1 and myeloid cell leukemia-1 is EBV mediated byits latent membrane protein-1 (26). Neither of the proteins was shownto be induced in gene array experiments with cells latently infectedwith HHV-8 (27, 28). The antiapoptotic proteins induced by HHV-8such as bcl-2-related protein A1 can promote viability on a rapidshort-term basis early during infection and thus allowing the recruit-ment of other Bcl-2 family members for further cell fate decisions.

Up-Regulation of Genes Encoding Angiogenic Signatures dur-ing the Early Phases of HHV-8 Infection. KS lesions are charac-terized by extensive neoangiogenesis (29). A striking finding of ourarray results is the induction of several genes involved in the controlof vascular remodeling and angiogenesis. HHV-8 induced VEGF,thrombomodulin, urokinase-type plasminogen activator receptor, ma-trix metalloproteinase (MMP-1), tissue inhibitor of matrix metallo-proteinase-1, and angiopoietin-related protein 4 (Table 2). Duringangiogenesis, local coagulation and fibrinolysis must be modulated ina controlled fashion. The urokinase-type plasminogen activator sys-tem is one of the most efficient proteolytic systems active in theextracellular environment (30). Our finding of significant induction ofurokinase-type plasminogen activator receptor by HHV-8 is excitingand consistent with a role of urokinase-type plasminogen activatorreceptor for the metastatic phenotype (31) because 51.6% of the KStissues were positive for urokinase type plasminogen activator recep-tor immunostaining (31). MMPs are believed to be pivotal enzymes ininvasion and angiogenesis, whereas tissue inhibitors of metallopro-teinase-1 are antagonists to a number of MMPs and reduce theneovascularization process (32). HHV-8 induced a robust activation

of MMP-1 and tissue inhibitors of metalloproteinase-1, and by 4 h, theup-regulation of MMP-1 was stronger than tissue inhibitors of met-alloproteinase-1 (2–6-fold above, Table 2), suggesting the inductionof a powerful angiogenic signal early during infection.

Among the several positive regulators of angiogenesis, two familiesof growth factors are largely specific for vascular endothelium byvirtue of having receptors that are mostly restricted to endothelialcells, namely VEGF and the more recently discovered angiopoietins(33). These two families seem to work in complementary and coor-dinated fashion during vascular development. We observed a strongactivation of VEGF-A, C, and angiopoietin like-4 genes by HHV-8(Table 2). VEGFs have been shown to be up-regulated by severalHHV-8 proteins such as v-IL6, vGCR, vMIP-I, and vMIP-II (4).Though angiopoietins display strong angiogenic activity independentof VEGF, their role in KS is not yet defined.

KS lesions are composed of a complex mixture of different celltypes with a prominent infiltrate of extravasated erythrocytes andlymphocytes. During tissue inflammation, normal endothelial cellscan be induced to become adhesive for circulating blood cells and tosupport their transmigration into inflamed tissue. Galea et al. (34)have shown that the lymphocyte function-associated antigen-1-ICAM-1 interaction is the primary one involved in the adhesion ofperipheral blood lymphocytes to KSY1 cells, a KS cell line. HHV-8-encoded ORF74 up-regulates the expression of VCAM-1, ICAM-1,and E-selectin, whereas HHV-8-K5 is known to down-regulate it (35).The strong induction of ICAM-1 gene during the early stages ofinfection in the HMVECd and HFF cells (Table 2) is suggestive of itspotentially important role in KS pathogenesis and in the activation ofinflammatory responses.

Differential Regulation of Host Cell Defense Genes during theEarly Phases of HHV-8 Infection. Although no direct activation ofIFN-�, �, or � or its receptors was observed in the gene array,up-regulation of IFN-regulated genes was observed (Table 2). Thisdifferential regulation of IFN-regulated genes was not observed in theB-cell line, BJAB. Among IFN-regulated genes activated by HHV-8,OAS2, MxA, and guanylate-binding protein-1 have been shown torestrict the growth of certain viruses (36). HHV-8-induced IFN-stimulated gene 15 is a cytokine responsible for augmenting andamplifying the immunomodulatory effects of IFN-� or IFN-�. HHV-8infection also revealed the activation of IFN regulatory factors 1 and

Fig. 5. Expression patterns of HHV-8-regulated genes in BJAB cells: 154 genes inwhich the expression was changed in at least two of the three cells infected with HHV-8were clustered by K-means algorithm according to their expression profiles into fivegroups (I–V). The number of genes related to each cluster is indicated above. The x axisrepresents the mock and 2- and 4-h HHV-8 infection experimental points. The y axisrepresents normalized log-transformed gene expression values. The maximum represen-tation was for cluster IV that showed sustained gene activation at both time pointsexamined.

Fig. 6. GO mapping of HHV-8-induced cell type-specific regulated host cell genes.Genes that were significantly but uniquely regulated in one cell type only [191 genes inhuman adult dermal microvascular endothelial cells (HMVECd), 239 in human foreskinfibroblast (HFF) cells, and 102 genes in BJAB cells] were subjected to grouping usingtheir biological functions revealed by simplified GO building. The y axis represents thevarious biological processes, and x axis represents the percentage of genes associated witheach of such nodes.

81




7, the actions of which are known to be down-regulated by HHV-8-encoded proteins vIRF1 and ORF 45, respectively (4).

Modulation of Cytokine Genes during the Early Phases ofHHV-8 Infection. KS is a multifocal angiogenic tumor consisting ofcharacteristic spindle cells and infiltrating leukocytes. Unlike mostcancers, KS does not appear to be caused by clonal expansion of atransformed cell. Instead, it appears to be a hyperplastic disordercaused, in part, by local production of inflammatory cytokines such asIL-1, IL-6, IFN-�, and tumor necrosis factor �, and growth factorssuch as basic fibroblast growth factor and VEGF. This is supported bythe fact that infiltration of inflammatory cells in KS lesions, includingCD8 T cells, monocytes, macrophages, and dendritic cells, precedesthe proliferation of the spindle-shaped endothelial cells. Infiltratingcells systematically produce inflammatory cytokines that are likelyresponsible for the activation of vessels and endothelial cells, increaseof adhesiveness with extravasation, and recruitment of lymphocytesand monocytes. In total agreement with this, our experiments haveshown that HHV-8 induces a variety of cytokines, including IL-8 andGro1, Gro2, and Gro3 at early time points of infection.

Modulation of Stress Response Genes during the Early Phasesof HHV-8 Infection. Many inhibitors of stress responses are knownto inhibit virus infection (37). Notable among the HHV-8-inducedstress response genes are manganese superoxide distmutase and cy-clooxygenase-2 (Cox-2). Cox-2 is a proinflammatory stress com-pound whose expression was the most strongly up-regulated gene (by84.65-fold at 2 h and 44.79-fold at 4 h) in HHV-8-infected HMVECdcells (Table 2). Cox-2 is believed to promote viral infection byinhibiting the target cell nitric acid synthesis that function to induceantiviral status (37). Human cytomegalovirus induces Cox-2, andinhibition of Cox-2 by specific inhibitors significantly reduced theyield of human cytomegalovirus (37). Identification and abrogation ofsuch stress responses offer clues to gene expression that could be arate-limiting step in the efficient establishment of virus infection.Studies using specific Cox-2 inhibitors to determine the role of Cox-2in HHV-8 infection are in progress.

DISCUSSION

Our studies describe a comprehensive picture of global genechanges soon after HHV-8 infection of three susceptible cell types.Our experimental design of analyzing the host cell transcriptionalchanges immediately after infection is quite distinct from the previousgene induction studies that were carried out at later time points ofvirus infection. Data presented here provide the framework and start-ing point for the further detailed analysis of the induced factors’ rolesin the biology of HHV-8 early during infection.

As with other viruses, several events occur during the early stagesof target cell infection by HHV-8 that must be playing active roles indeciding the outcome of infection. For better conceptual purposes, wehave divided these early events into six overlapping dynamic phases.Phase I involves the binding of virus to cell surface via its interactionswith HS (13–15), integrins (16), and possibly to other yet to beidentified molecule(s). This is followed by virus entry into the targetcells (Phase II; Refs. 16, 17), probably overlapping with the inductionof host cell signal pathways during Phase I (17) that facilitate theentry. In Phase III, the viral capsid/tegument moves in the cytoplasmfacilitated by the induced signal pathways and probably overlaps withPhase IV host cell gene transcription and expression. In Phase V, viralDNA enters into the nucleus followed by viral gene expression (latentand/or lytic), which is greatly influenced by the HHV-8-inducedsignal pathways and expressed host cell genes. Phase VI involves theoverlapping viral gene-induced host cell gene expression, which mayexert an influence on success of viral infection. As part of understand-

ing the early events of HHV-8 infection, this study was designed toanalyze the Phase IV host cell gene expression immediately afterinfection.

Several evidences presented in this article such as the changes inonly a small number of transcripts at both time points, gene expressiondifferences in the three cell types, close similarity of gene expressionin the adherent cells, RT-PCR and Northern blot confirmation of arraydata with RNA samples derived from different set of experiments, aswell as the Western blot assays, asserted that the observed host genemodulation was caused by HHV-8 infection. The impact of HHV-8binding and entry into the target cells (Phases I–III) upon the host cellgene expression was not analyzed here because of the followingreasons. The binding and entry processes of herpesviruses are com-plex events involving multiple cell surface receptors. AlthoughHHV-8 binding to cell surface HS and �3�1 integrin has been dem-onstrated, it must be also interacting with other yet to be unidentifiedmolecule(s). We did not use virus pretreated with heparin in ourstudies to study the impact of binding on host cell gene expressionbecause soluble heparin though lowered the binding of radiolabeledHHV-8 to the target cells significantly, certain percentage of virus stillbind and entered the cells resulting in low level of infection (13).Similarly, infectivity neutralizing HHV-8 anti-gB, gpK8.1A, gH, andgL antibodies, Arginine-Glycine-Aspartic Acid peptides, anti-integrinantibodies, and soluble integrins did not prevent the binding ofHHV-8 to the target cells (14, 16). Hence, we could not use thesetreatments in the array analyses. Moreover, although these treatmentsblocked infection at a post-HS binding stage of infection, whethervirus still enters the target cells is not known at present and is underinvestigation. Similarly, UV-inactivated herpesviruses have beenshown to enter the target cells. Our recent studies show that HHV-8uses multiple integrin molecules during the binding and entry process(F. Z. Wang, P. P. Naranatt, N. S. Walia, H. H. Krishnan, L. Zeng, andB. Chandran, unpublished observations), making the choice of inte-grin molecule harder to block HHV-8 entry. Currently, there are nocharacterized HHV-8 mutants that cannot bind or enter the targetcells. The complex dynamics of HHV-8-receptor(s) interaction is atthe very early stage of understanding, and it is not known whether HSand integrin interaction occur simultaneously or sequentially. Hence,in the present set of experiments, we set out to study the transcrip-tional reprogramming early during infectivity rather than delineatingthe responses that are HHV-8 binding/entry specific (Phases I–III).

Previous studies of analyzing host cell gene modulation by viruseshave been limited to the expression of one or limited numbers ofgenes after infection. However, gene array technology makes it pos-sible to analyze the induction of multiple target genes at a genome-wide scale at a given time point. This technology has been used toanalyze the effects of a number of human virus infections on cellphysiology beginning with human cytomegalovirus (38). A commonfinding in many of these investigations is the up-regulation of genesinvolved in the inflammatory response (39). Beyond a common innateimmune response to infection, different cells may have characteristicsignatures to different pathogens, often as a result of the highlyspecific activities of a particular pathogen’s virulence determinants.Thus, a number of molecules with potential usefulness in controllingvirus infections have emerged from such analyses (27, 37).

Our array data revealed a number of unique observations. Forexample, HHV-8 infection has a major impact on the expressionpattern of cellular genes that exhibited cell-type specificity. Thedifferentially expressed genes belonged to a variety of cellular path-ways. The striking cell type-specific behaviors suggest that at least inthe initial stages of infection, HHV-8-induced host cell gene modu-lation events in B cells may be different compared with those in theadherent endothelial and fibroblast cells. The differences in B cells are

82




very interesting and may potentially reflect the biology and differ-ences in the outcome of HHV-8 infection in vivo. Human B cells area reservoir of latently infected HHV-8. HHV-8-associated primaryeffusion lymphoma and multicentric Castleman’s disease differ fromKS in many respects, most notably in the expressed viral genes. Inaddition to the latency-associated ORF 73, ORF 72, K13, and K12genes that are expressed in the KS endothelial cells, HHV-8 alsoexpresses the ORF 10.5 (LANA2/vIRF3) and K2 (vIL-6) in primaryeffusion lymphoma cells, both in vivo and in vitro, and additional lyticcycle genes in the multicentric Castleman’s disease cells (2). Al-though the BJAB cells that we analyzed have been used by othersextensively as representative of B cells (10, 40), it may be importantto confirm these observations using other B-cell lines and mostimportantly using primary B cells as characterization of HHV-8infection in these cells becomes established. Nevertheless, this is aninteresting observation, and additional work is needed to correlate therelevance of B-cell-specific changes induced by HHV-8 with itsB-cell pathogenesis.

Why should HHV-8 need to modulate the host cell transcriptionalmachinery during the initial phases of infection? Early during infec-tion of target cells, HHV-8 has to overcome several host-mediatedobstacles. For example, HHV-8 has to (a) block the apoptosis of hostcells triggered by the virus binding and entry processes, (b) modulatehost cell transcription to overcome the restriction on virus genetranscription, (c) block the activation and effects of innate immuneresponses, and (d) evade the elimination by the constant surveillancepressure from the host immune system such as IFNs, tumor necrosisfactor �, complement, antibodies, ADCC, natural killer cells, CTL,and phagocytic cells. To establish a successful infection, HHV-8 musthave developed many ways to manipulate and overcome these obsta-cles, using both viral and host proteins. Our observation of modulationof host genes that govern vital cellular processes such as apoptosis,transcription, host defense and inflammation, extracellular matrixremodeling, angiogenesis, and protein processing during the earlycourse of infection is exciting because these cellular transcriptionalreprogramming probably be serving vital roles in overcoming theabove-mentioned obstacles and establish a successful infection.

Poole et al. (28) have used a different primary effusion lymphomacell line (JSC1) to produce the virus that was used to infect primaryendothelial cells for a period of 3–5 weeks, and gene array experi-ments were done when almost all of the cells changed from typicalcobblestone to spindle-shaped morphology and were positive forLANA. Moses et al. (27) infected endothelial cells previously immor-talized by retroviral expression of human papillomavirus E6 and E7genes, and arrays were done with after �4 weeks of HHV-8 infectionwhen �90% of the cells were LANA positive and showed the spindlecell phenotype. Mikovits et al. (41) reported the expression changesafter bone marrow-derived primary CD34 cells were infected withHHV-8 and maintained for 30 days before the analysis. These threestudies were done after at least several days after infection, wherelatency had been established and with cells appearing to resemble atransformed phenotype. In contrast, our objective was to analyze thehost gene expression changes at very early time points during theprimary HHV-8 infection of target cells. In the examination of hostgenes modulated early during infection in the present study, a strongup-regulation of IFN-responsive genes such as IRF 7, Mx1, IFN-inducible transmembrane protein 1, OAS, IFN-stimulated protein 15,IRF-1, and IFN-inducible Mr 67,000 guanylate binding protein 1 wasobserved. Studies by Poole et al. (28) showed the up-regulation ofother IFN-responsive genes such as IFN-induced transmembrane pro-tein 3, Mx R2, IFN-�-inducible protein (IFI-6–16), and IFN-inducibleprotein 56. Thus, although both of these studies showed similarinduction of IFN-responsive genes, the effect on specific genes

seemed to vary. This is probably expected because the Poole et al.(28) array was done after the virus had established latency, duringwhich time the effect on some of the IFN-responsive genes may havebeen reduced. Moreover, it is known that several viruses like herpessimplex virus type-1 (18) and vesicular stomatitis virus (42) areknown to first induce an antiviral status and disarm this at later timepoints of infection to favor the virus infection. Other common genesthat were shared between our early time points of infection and theprevious latently infected later time point analyses included SSI-3,vEts transcription factor, tissue plasminogen activator, IL-8, BCl-3,nucleoside phosphorylase, and tissue inhibitors of metalloprotein-ase-1. Thus, 10 of the genes that were up-regulated in our arrayanalysis were also up-regulated in those Poole et al. (28). Comparisonof transcriptional profiles between Moses et al. (27) and Pool et al.(28) showed that only 7 of 124 induced and 3 of 60 repressed geneswere common (27). Authors argued that such low correlation wasbecause 117 of 124 of the induced genes were not spotted in botharrays. Our comparison between the elements used for HG-U133Aand Human UniGem V2.0 microarrays, used by Poole et al. (28),revealed that nearly one-third of the most informative genes detectedby us were absent in the cDNA arrays used by Poole et al. (28). This,together with the differences in the time points of analysis, might haveprofoundly influenced the transcriptional changes observed betweenour study and previous studies (27, 28, 41).

Another important observation in our study is the repression ofmRNA for a subset of genes in the infected cells. Repression does notappear to be a general degradation phenomenon because we did notobserve a great reduction in the number of transcripts. BecauseHHV-8 infection can affect both the synthesis and the stability ofcellular mRNAs, the data obtained here probably reflect the interfer-ence of HHV-8 at multiple steps in host gene expression. Among thegenes that were down-regulated at least in two cells, dickkopf homo-logue 1 was of particular interest because this molecule is known to beone of the secreted inhibitors of Wnt signaling. Wnt signaling is ahighly conserved developmental pathway in which �-catenin medi-ates changes in gene expression (43). HHV-8-encoded LANA hasbeen shown to stabilize �-catenin by binding to its negative regulatorglycogen synthase kinase-3� and inducing its nuclear accumulation(44). Our results indicated the down-regulation of one of the negativeregulators of Wnt signaling, thereby suggesting that additional mech-anisms may be operative as positive feedback of Wnt signaling.Among other genes that were down-regulated, the majority belong to themolecular chaperons (BCL2-associated athanogene family member-2,nuclear receptor subfamily, and Zinc finger proteins 133 and 238) andtumor suppressors (absent in melanoma 1 and Thioredoxin-interactingprotein). Although the biological significance of down-regulating chap-erons is not known, deregulations of tumor suppressors may be beneficialto HHV-8 and associated oncogeneic process.

HHV-8’s exploitation of cell cycle regulatory �3�1 integrin mole-cule for entry into the target cells and the induction of integrin-mediated mitogenic signaling pathways may have important implica-tions in the unique biology of KS lesions and HHV-8’s role in KSpathogenesis. Besides the delivery of viral DNA into the cells, HHV-8interactions with the host cell receptor(s) induced signal pathways,and the observed host cell gene expression such as the induction ofgenes encoding angiogenic signatures, antiapoptosis, cytokines, andstress response genes may bring about important biological conse-quences and play a significant role in KS pathogenesis. In addition toidentifying specific host response genes within each functional groupthat are already known to be important for HHV-8 infection, ourfindings identify novel candidate genes that are not yet known toHHV-8 biology. For example, up-regulated stress gene Cox-2 andsignaling regulatory Heparin binding-epidermal growth factor and

83




repressed tumor suppressor absent in melanoma 1 and thioredoxin-interacting protein. The roles of these molecules in HHV-8 infectionand KS development remain to be studied. The close similarity ofRT-PCR and Northern and Western blot assays with the array data interms of similar magnitudes and direction of changes in the genesidentified suggests that the array data may serve as a guide post forevaluating interesting genes.

Similar to HHV-8 interactions with UI endothelial cells, interac-tions with latently infected cells may also stimulate the production ofcytokines/growth factors, which in turn may stimulate the endothelialgrowth. A synergism may exist between latently infected endothelialcells and incoming infection that may control endothelial cell growthby an autocrine-parocrine loop. Additional studies are needed toexamine the consequences of HHV-8-induced signaling pathways andtranscriptional reprogramming in the regulation of virus gene expres-sion during a primary infection of endothelial and B cells and on cellsthat are already programmed by the cell growth-modulating HHV-8latency-associated proteins and whether such interactions play essen-tial roles in the establishment and/or maintenance of latent infectionand/or cellular proliferation of latently infected endothelial cellsand/or B cells. Additional studies with virus strains, mutants, viralproteins, and host cell types such as primary B cells will be requiredto obtain a more complete picture of host cell gene expression andbiological effects after HHV-8 infection. A greater understanding ofhost cell gene reprogramming induced by HHV-8 may eventually leadto the development of novel therapies to control KS lesions.

REFERENCES

1. Scadden, D. T. AIDS related malignancies. Ann. Rev. Med., 54: 285–303, 2003.2. Antman, K., and Chang, Y. Kaposi’s sarcoma. N. Engl. J. Med., 342: 1027–1038,

2000.3. Ganem, D. Human herpesvirus 8 and its role in the genesis of Kaposi’s sarcoma. Curr.

Clin. Top. Infect. Dis., 18: 237–251, 1998.4. Dourmishev, L. A., Dourmishev, A. L., Palmeri, D., Schwartz, R. A., and Lucac,

D. M. Molecular genetics of Kaposi’s sarcoma-associated herpesvirus (human her-pesvirus 8) epidemiology and pathogenesis. Microbiol. Mol. Rev., 67: 175–212,2003.

5. Dedicoat, M., and Newton, R. Review of the distribution of Kaposi’s sarcoma-associated herpesvirus (KSHV) in Africa in relation to the incidence of Kaposi’ssarcoma. Br. J. Cancer, 88: 1–3, 2003.

6. Luppi, M., Barozzi, P., Schulz, T. F., Trovato, R., Donelli, A., Narni, F., Sheldon, J.,Marasca, R., and Torelli, G. Molecular evidence of organ-related transmission ofKaposi’s sarcoma-associated herpesvirus or human herpesvirus-8 in transplant pa-tients. Blood, 96: 3279–3281, 2000.

7. Staskus, K. A., Sun, R., Miller, G., Racz, P., Jaslowski, A., Metroka, C., Brett-Smith,H., and Haase, A. T. Cellular tropism and viral interleukin-6 expression distinguishhuman herpesvirus 8 involvement in Kaposi’s sarcoma, primary effusion lymphoma,and multicentric Castleman’s disease. J. Virol., 73: 4181–4187, 1999.

8. Martin, D. F., Kuppermann, B. D., Wolitz, R. A., Palestine, A. G., Li, H., andRobinson, C. A. Oral ganciclovir for patients with cytomegalovirus retinitis treatedwith a ganciclovir implant. Roche Ganciclovir Study Group. N. Engl. J. Med., 340:1063–1070, 1999.

9. Ganem, D. KSHV and Kaposi’s sarcoma: the end of the beginning? Cell, 91:157–160, 1997.

10. Bechtel, J. T., Liang, Y., Hvidding, J., and Ganem, D. Host range of kaposi’s sarcomaassociated herpesvirus in cultered cells. J. Virol., 77: 6474–6481, 2003.

11. Cerimele, F., Curreli, F., Ely, E., Friedman-Kien, A. E., Cesarman, E., and Flore, O.Kaposi’s sarcoma associated herpesvirus can productively infect primary humankeratinocytes and alter their growth properties. J. Virol., 75: 2435–2443, 2001.

12. Vieira, J., O’Hearn, P., Kimball, L., Chandran, B., and Corey, L. Activation of KSHV(HHV-8) lytic replication by human cytomegalovirus. J. Virol., 75: 1378–1386,2001.

13. Akula, S. M., Wang, F. Z., Vieira, J., and Chandran, B. Human herpesvirus 8interaction with target cells involves heparan sulfate. Virology, 282: 245–255, 2001.

14. Akula, S. M., Pramod, N. P., Wang, F. Z., and Chandran, B. Human herpesvirus 8envelope-associated glycoprotein B interacts with Heparan sulfate-like moieties.Virology, 284: 235–249, 2001.

15. Wang, F. Z., Akula, S. M, Pramod, N. P., Zeng, L., and Chandran, B. Humanherpesvirus 8 envelope glycoprotein K8.1A interaction with the target cells involvesheparan sulfate. J. Virol., 75: 7517–7527, 2001.

16. Akula, S. M., Pramod, N. P., Wang, F. Z., and Chandran, B. Integrin �3�1(CD49c/28)is a cellular receptor for Kaposi’s sarcoma associated herpesvirus (KSHV/HHV-8)entry into the target cells. Cell, 108: 407–419, 2002.

17. Naranatt, P. P., Akula, S. M., Zien, C. A., Krishnan, H. H., and Chandran, B. Kaposi’ssarcoma-associated herpesvirus induces the phosphatidylinositol 3-kinase-PKC-�-MEK-ERK signaling pathway in target cells early during infection: implications forinfectivity. J. Virol., 77: 1524–1539, 2003.

18. Mossman, K. L., Macgregor, P. F., Rozmus, J. J., Goryachev, A. B., Edwards, A. M.,and Smiley, J. R. Herpes simplex virus triggers and then disarms a host antiviralresponse. J. Virol., 75: 750–758, 2001.

19. Boyle, K. A., Pietropaolo, R. L., and Compton, T. Engagement of the cellular receptorfor glycoprotein B of human cytomegalovirus activates the interferon-responsivepathway. Mol. Cell. Biol., 19: 3607–3613, 1999.

20. Giancotti, F. G., and Ruoslahti, E. Integrin signaling. Science (Wash. DC), 285:1028–1032, 1999.

21. Garcia, J., Ye, Y., Arranz, V., Letourneux, C., Pezeron, G., and Porteu, F. IEX-1: anew ERK substrate involved in both ERK survival activity and ERK activation.EMBO J., 21: 5151–5163, 2002.

22. Casci, T., Vinós, J., and Freeman, M. Sprouty, an intracellular inhibitor of rassignaling. Cell, 96: 655–665, 1999.

23. Ishibashi, T., Bottaro, D. P., Michieli, P., Kelley, C. A., and Aaronson, S. A. A noveldual specificity phosphatase induced by serum stimulation and heat shock. J. Biol.Chem., 269: 29897–29902, 1994.

24. Verhagen, A. M., Coulson, E. J., and Vaux, D. L. Inhibitor of apoptosis proteins andtheir relatives: IAPs and other BIRPs. Genome Biol., 2: Reviews 3009.1–3009.10,

host gene induction and transcriptional reprogramming in ... · host gene induction and...

Documents