7) 137–149www.elsevier.com/locate/yviro

Virology 366 (200

Functional genomics analyses of differential macaque peripheral bloodmononuclear cell infections by human immunodeficiency virus-1 and

simian immunodeficiency virus

Yu Li, Eric Y. Chan, Michael G. Katze ⁎

Department of Microbiology and Washington National Primate Research Center, University of Washington, Box 358070, Seattle, WA 98195-8070, USA

Received 12 February 2007; returned to author for revision 23 March 2007; accepted 5 April 2007Available online 15 May 2007

Abstract

The pathogenicity of the primate lentiviruses, human, and simian immunodeficiency viruses, is host-specific. Previous studies indicated thatthe highly pathogenic human lentivirus HIV-1 has markedly reduced pathogenicity compared to the pathogenic simian lentivirus SIV in pigtailmacaques (Macaca nemestrina). We therefore hypothesized that the pigtail macaque peripheral blood mononuclear cells (mPBMCs) wouldrespond differently to infections of HIV-1 and pathogenic SIV. To elucidate the cellular responses to the infections of HIV-1 and SIV, we infectedmPBMC with these two viruses. Like infections in vivo, HIV-1 and SIV demonstrated distinct replication kinetics in mPBMCs, with HIV-1replicating at significantly lower levels. Similarly, gene expression profiling facilitated by macaque-specific oligonucleotide microarrays alsorevealed distinct expression patterns of genes between the HIV-1- and SIV-infected mPBMCs; in particular, genes associated with the antigenpresentation, T cell receptor, ERK/MAPK signaling, Wnt/β-catenin signaling, and natural killer cell signaling pathways were differentiallyregulated between these two viruses. Most interestingly, despite the lower levels of replication, HIV-1 triggered a more robust regulation ofimmune response genes early after infection; the converse was true in SIV-infected mPBMCs. Our results therefore suggest that macaques may becontrolling the infection of HIV-1 at an early stage through coordinated regulation of host defense pathways.© 2007 Elsevier Inc. All rights reserved.

Keywords: HIV-1; SIV; Macaque peripheral blood mononuclear cells; Microarray; Gene expression

Introduction

The AIDS/macaque model is the most widely usedexperimental system for studying host responses to the infectionof primate lentiviruses, especially human immunodeficiencyvirus-2 (HIV-2) and simian immunodeficiency virus (SIV)(Kaizu et al., 2006; Lichterfeld et al., 2005). Although humanimmunodeficiency virus-1 (HIV-1) and SIV share limitedsequence identity (Franchini and Bosch, 1989), infection ofSIV in macaques could closely resemble the infection of HIV-1in humans, as characterized by the gradual depletion of CD4+ Tcells symptomatic of AIDS, which causes a dysfunctionalimmune system and eventual death (Mattapallil et al., 2005).Interestingly, HIV-1, the main pathogen causing AIDS in

⁎ Corresponding author. Fax: +1 206 732 6055.E-mail address: honey@u.wahington.edu (M.G. Katze).

0042-6822/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.virol.2007.04.020

human (Simon et al., 2006), demonstrated a minimal pathogenicphenotype in macaques (Agy et al., 1992, 1997; Gartner et al.,1994).

Recent studies suggested that cellular factors may imposerestrictive effects on the HIV-1 replication in macaques (Zhengand Peterlin, 2005). Macaque APOBEC3G/F inhibits HIV-1replication by introducing hypermutations during reversetranscription (Zennou and Bieniasz, 2006). Macaque TRIM5was able to decrease HIV-1 replication also at the step of reversetranscription (Wu et al., 2006). Although these host factors maysignificantly hinder HIV-1 replication in vitro (Hatziioannou etal., 2006; Kamada et al., 2006), whether they are the soledeterminants for the loss of HIV-1 pathogenicity in macaques isstill elusive. Lines of evidence suggest that the dissociation ofthe replicative capacity and the pathogenicity of primatelentiviruses indeed occurred in nonhuman primates (Broussardet al., 2001; Pandrea et al., 2003; Silvestri et al., 2003). More

138 Y. Li et al. / Virology 366 (2007) 137–149

importantly, primate lentiviruses replicating at low levelscan become highly pathogenic during infections in macaques(Beer et al., 2005). Therefore, it would be reasonable tospeculate that the loss of pathogenicity of HIV-1 in macaques isnot solely attributed to its decreased replicative capacity inmacaque cells.

Increasing evidence indicates that the patterns of cellularresponse to the infections of a variety of viruses could wellreflect their pathogenic properties (Kash et al., 2004,2006a,2006b; Kobasa et al., 2007; Ploquin et al., 2006; Wanget al., 2005). Distinct responses by TGF-β signaling wereobserved between infections of pathogenic and nonpathogenicSIV strains in nonhuman primates (Ploquin et al., 2006).Proinflammatory and cell death pathways were found to bedifferentially regulated in mice infected by influenza viruseswith various pathogenic properties (Kash et al., 2004, 2006b).Similarly, the interferon response and immune evasion path-ways were seen to be regulated differently in human as well asmouse cells during the infections by highly pathogenic andlowly pathogenic filoviruses (Kash et al., 2006a) and rabiesviruses (Wang et al., 2005), respectively. Therefore, wehypothesized that the infections of HIV-1 and pathogenic SIVwould induce distinct cellular responses in macaque peripheralblood mononuclear cells (mPBMCs).

A comparison of the cellular responses of mPBMCs to theinfections of primate lentiviruses with different pathogenicproperties would greatly improve our understanding of thepathogenesis of primate lentiviruses. To gain further insightsinto the cellular responses to the HIV-1 and pathogenic SIV, weinfected pigtail macaque PBMCs with an HIV-1 strain and ahighly pathogenic SIV strain (Batten et al., 2006). By using

Fig. 1. (A) Indirect Immunofluorescence Assay (IFA) in mPBMCs infected for 6 dayscells in the experiments. On the left are the immunofluorescence analyses of the identof under 1%, while the infection rate for the SIV-infected culture was under 2% on day1-infected and SIV-infected cultures. The data shown here are representative of mPBwith HIV-1 and SIV, along with the mock. The data shown here are a summary of m

macaque specific oligonucleotide microarrays, we were able toelucidate the cellular gene expression changes in mPBMCs inresponse to HIV-1 and SIV infections. Our results suggestedthat an enhanced early immune response distinguished the HIV-1 infection from the SIV infection in mPBMCs and could be amajor contributing factor to reduced HIV-1 pathogenicity inmacaques.

Results and discussion

HIV-1 and SIV demonstrate distinct infection kinetics in thepigtail macaque PBMC cultures

To evaluate the infections of HIV-1 and SIV in mPBMCs,we infected mPBMCs with a nonpathogenic HIV-1 strainalong with a highly pathogenic SIV strain at the same multi-plicity of infection (1 TCID50/cell). Both HIV-1 and SIVstrains used in this study were T-tropic viruses that primarilyinfect CD4+ T cells (Kestler et al., 1990; Peden et al., 1991).We examined the cell distributions of mPBMCs prior to in-fections using flow cytometry. mPBMCs from 3 individualmacaques showed comparable compositions in term of celltypes present (Supplemental Table 1) and were used in theinfection experiments.

During the infections in mPBMCs, HIV-1 and SIV de-monstrated distinct replication kinetics. Although mPBMCscan be infected by both viruses, only a small portion of cellswere actually infected in HIV-1 or SIV cultures (Fig. 1A).Infected cells were not noticeable in either HIV-1- or SIV-infected mPBMC cultures until day 4 post-infection (p.i.) (datanot shown). Even with a higher percentage of infected cells in

with HIV-1and SIV. On the right are phase contrast micrographs of virus-infectedical microscopic field. (B) The HIV-1-infected culture exhibited an infection rate6 p.i. There were approximately the same numbers of cells counted in both HIV-MCs infection from 3 animals. (C) The viabilities of mPBMC cultures infectedPBMCs infections from 3 animals.

139Y. Li et al. / Virology 366 (2007) 137–149

the mPBMC cultures on day 6 p.i., less than 1% of the cellswere estimated to be infected by HIV-1 and under 2% of thecells were infected by SIV (Fig. 1B). Through the course ofinfections, cell viability of neither virus-infected mPBMCcultures differed from mock-infected mPBMC cultures, until asurge in cell death is seen in the SIV-infected cultures on day8 p.i. (Fig. 1C). As a well-accepted parameter for the replicationstatus of HIV/SIV, we measured Gag protein levels in HIV-1-and SIV-infected mPBMCs (Fig. 2A). Given that the SIV Gaglevel was more than 50-fold higher than that of HIV-1 Gag onboth days 4 and 6 p.i., and that there was not a significantlyhigher number of cells infected by SIV than infected by HIV-1in the cultures (Fig. 1), SIV replication appeared to be morerobust than HIV-1 replication in mPBMCs (Kamada et al.,2006). The relative abundance of the viral genome RNAmeasured by real-time PCR assays also indicated that the SIVgenome RNA increased more robustly than the HIV-1 genomeRNA during the infection in mPBMCs (Fig. 2B). Theabundance of the HIV-1 genome temporally increased duringHIV-1 infection and peaked on day 6 p.i., then decreased on day

Fig. 2. (A) Levels of gag protein (Gagp24-HIV-1 and Gagp27-SIV) reflect theprogression of viral infection. The graph tracks the average gag levels forPBMCs from all three infected animals through 8 days post-infection, with theerror bars reflecting individual variations. Both HIV-1- and SIV-infectedcultures showed progressing virus replication in the infected cultures. (B) Levelsof HIV-1 or SIV viral genomic RNA reflect the progression of viral replication.The graph tracks the increase in the quantity the virus RNA genomes relative tothe quantity of the corresponding virus genomes on day 2 p.i. Data shown hereare a summary of mPBMCs infections from 3 animals.

8 p.i. In contrast, the abundance of the SIV genome steadilyincreased through the course of infection. Collectively, our datasuggested that HIV-1 replicated at significantly lower levelscompared to SIV in mPBMCs, consistent with the in vivoresults reported previously (Batten et al., 2006), possibly owingin part to the presence of host restriction factors (Hatziioannouet al., 2006; Kamada et al., 2006; Rodrigo et al., 1997).

HIV-1 and SIV induce cellular gene expression changes viadistinct kinetics

To profile the global cellular gene expression changes inresponse to the infections of HIV-1 and SIV in mPBMCs, weperformed differential gene expression analysis using macaque-specific oligonucleotide microarrays. Taking advantage of themarked specificity of the macaque-specific oligonucleotidemicroarrays, we adopted a 1% false-positive rate (p≤0.01) asthe threshold for selecting differentially expressed genes. Weprofiled HIV-1-infected and SIV-infected cells from two timepoints, days 4 and 6 p.i.; we excluded day 8 p.i. or later due tothe noticeable surge in cell death in the SIV-infected cultureafter day 6 p.i. (Fig. 1C).

The global gene expression patterns of HIV-1- and SIV-infected mPBMCs clearly indicated that despite less robustreplication, the HIV-1 infection induced a stronger cellularresponse than the SIV infection at the earlier time point (Fig.3A). Among ∼1300 cellular genes that had expression changesin 1 out of 4 data points (Supplemental Table 4), 399 genes inHIV-1 d4 and 107 genes in SIV d4 showed expression changes(Fig. 3B), even though the HIV-1 replication was at signi-ficantly lower levels than the SIV replication on day 4 p.i. Onday 6 p.i., the number of changed genes increased to 497 in theSIV-infected mPBMCs but still fewer than the 587 genes inHIV-1-infected mPBMCs. Thus, in spite of more subdued virusreplication, HIV-1 infection appeared to be able to induce asimilar, if not stronger, host response when compared to therelatively more active SIV-1 infection.

Pathways analyses of the cellular gene expression profiles ofmPBMCs infected with HIV-1 and SIV

To characterize the different cellular responses induced bythe infection of HIV-1 or SIV in mPBMCs, we analyzed the listsof differentially expressed genes during the infections of HIV-1and SIV by Ingenuity Pathway Analysis (IPA), a knowledge-based pathway analysis tool used in previous studies (Bowick etal., 2006; Li et al., 2005; Pasieka et al., 2006; Thomas et al.,2006). In brief, functions of cellular genes are mined from peer-reviewed literatures and manually curated into the knowledge-base. A network analysis of the knowledgebase is used toconstruct interaction-based relationships between proteins in theknowledgebase. Furthermore, while our differentially expressedgenes were scattered across most of the networks in theknowledgebase, IPA ranks the networks based on the number ofdifferentially expressed genes in one network as a percentage ofthe total number of genes in the network. In the discussionbelow, we focused on the top-ranking networks, as they

Fig. 4. Pathways that were affected in the HIV-1-infected or SIV-infectedmPBMCs. The Ingenuity Pathway Analysis software was used to generate listsof pathways. The IPA annotation designates the differentially expressed genes inHIV-1-infected or SIV-infected mPBMCs into different pathways. The antigenpresentation, T cell receptor, ERK/MAPK signaling, Wnt/β-catenin signaling,and the natural killer cell signaling pathways were seen to be significantlyimpacted by HIV-1 or SIV infections in mPBMCs. The two histograms representdata from signature genes, defined as having a p≤0.01. The p-value is derivedfrom both the errors in microarray measurements and the estimated varianceamong the 3 animals.

Fig. 3. (A) Hierarchical cluster of global gene expression patterns of mPBMCinfected with HIV-1 and SIV. Each gene in the cluster passed the statistical cutoff(p≤0.01) at least once in 4 data points based on the Rosetta error model inResolver. Every expression value shown in this report is the average expressionvalue from 3 individual animals. Genes having increased expression valuescompared to time matched mock were shown as red while the genes havingdecreased expression values compared to time matched mock were shown asgreen. (B) The number of signature genes that have expression changes(p≤0.01) in HIV-1-infected or SIV-infected mPBMC on days 4 or 6 p.i. The twohistograms represent data from signature genes, defined as having a p≤0.01.The p-value is derived from both the errors in microarray measurements and theestimated variance among the 3 animals.

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represented cellular pathways with an overrepresentation ofdifferentially expressed genes.

In other words, the IPA analyses suggested that thesepathways, as described below, exhibited differential expressionupon HIV-1 and SIV infection in mPBMCs, with some beingunique to HIV-1 or SIV infection (Fig. 4).

Pathway analyses of the cellular gene expression profiles inHIV-1-infected mPBMCs

Antigen presentation pathwayNine genes associated with the antigen presentation pathway

(Table 1A) showed expression changes in the HIV-1-infected

mPBMCs. Among these 9 genes, 6 genes belonged to the MHCclass II family, including CD74, HLA-DMA, HLA-DMB,HLA-DRA, HLA-DRB1, and HLA-DRB5 (Fig. 5A). HLA-DMmolecules (HLA-DMA and HLA-DMB) are known to benecessary for the formation of the antigen presentation complex(Fling et al., 1994) and to enhance the MHC/antigen binding(Sherman et al., 1995). The interaction between HLA-DR(HLA-DRA, HLA-DRB1, and HLA-DRB5) and HLA-DM(HLA-DMA and HLA-DMB) was shown to be crucial inpreventing immune evasion (Zwart et al., 2005). The up-regulation of all these genes suggested that the MHC class IImediated antigen presentation may be enhanced in the HIV-1-infected mPBMCs. Furthermore, an MHC class I gene (HLA-A) was also up-regulated only in the HIV-1-infected mPBMCs.HLA-A presents endogenous antigens to the CD8+ T cells,which in turn play crucial roles in the anti-viral immuneresponses against HIV-1 infection in human cells (Levy, 2003;Lichterfeld et al., 2005). In addition, the interaction betweenMHC I and CD74 play is possibly involved in antigenpresentation (Powis, 2006). By TaqMan qRT-PCR, we alsoconfirmed the up-regulation of CD74 by HIV, but not SIV (Fig.7). Collectively, the up-regulation of MHC-class I/II moleculesmay point to an up-regulation of antigen presentations duringthe HIV-1 infection compared to the SIV infection in macaques,which may in turn enhance immune surveillance and accountfor the subdued pathogenicity of HIV.

T cell receptor pathwayMHC class I/II molecules primarily present antigens to

CD4+ or CD8+ T cells through coupling with the T cell receptorcomplex (TCR/CD3). It may consequently induce the T cell-mediated immune responses (Carreno et al., 2006).

Exogenous signals from MHC could be transduced into Tcells via CD247 (CD3-zeta) (Sancho et al., 1992) and CD247-associated molecules, including ZAP-70, LCK, and FYN

Table 1The expression pattern of the genes associated with the pathways affected inHIV-1-infected and SIV-infected mPBMCs

A

Accession # Primary sequencename

HIV-1 SIV

Antigen presentation pathwayNM_004355 CD74 Up-regulated Unch.NM_002116 HLA-A Up-regulated Unch.NM_006120 HLA-DMA Up-regulated Unch.NM_002118 HLA-DMB Up-regulated Unch.NM_019111 HLA-DRA Up-regulated Unch.NM_002124 HLA-DRB1 Up-regulated Unch.NM_002125 HLA-DRB5 Up-regulated Unch.NM_002798 PSMB6 Down-regulated Unch.NM_004159 PSMB8 Down-regulated Down-regulated

T cell receptor pathwayNM_198053 CD247 Up-regulated Unch.NM_001768 CD8A Up-regulated Unch.NM_004931 CD8B Up-regulated Up-regulatedNM_005214 CTLA4 Down-regulated Down-regulatedNM_002037 FYN Up-regulated Unch.NM_005356 LCK Up-regulated Unch.NM_002746 MAPK3 Up-regulated Unch.NM_001079 ZAP70 Up-regulated Unch.

ERK/MAPK signaling pathwayNM_004418 DUSP2 Up-regulated Unch.NM_001946 DUSP6 Up-regulated Unch.NM_002037 FYN Up-regulated Unch.NM_002107 H3F3A Up-regulated Unch.NM_002746 MAPK3 Up-regulated Unch.NM_002661 PLCG2 Up-regulated Unch.NM_005167 PPM1J Up-regulated Up-regulatedNM_001008709 PPP1CA Up-regulated Unch.NM_002730 PRKACA Up-regulated Unch.NM_002732 PRKACG Up-regulated Up-regulatedNM_001010935 RAP1A Down-regulated Unch.NM_001006665 RPS6KA1 Up-regulated Unch.NM_003131 SRF Down-regulated Unch.NM_007315 STAT1 Down-regulated Unch.NM_006289 TLN1 Up-regulated Unch.NM_003405 YWHAH Up-regulated Unch.

B

Accession # Primary sequencename

SIV HIV-1

Wnt/β-catenin signaling pathwayNM_145259 ACVR1C Down-regulated Unch.NM_016269 LEF1 Down-regulated Unch.NM_181674 PPP2R2B Up-regulated Unch.NM_003199 TCF4 Down-regulated Unch.

C

Accession # Primary sequencename

HIV-1 SIV

Natural killer cell pathwayNM_002983 CCL3 Down-regulated Up/Down-regulatedNM_002984 CCL4 Up-regulated Unch.NM_002985 CCL5 Up-regulated Unch.NM_016382 CD244 Up-regulated Up-regulatedNM_000569 FCGR3A Up-regulated Unch.NM_005541 INPP5D Up-regulated Unch.

C

Accession # Primary sequencename

HIV-1 SIV

Natural killer cell pathwayNM_014218 KIR2DL1 Up-regulated Up-regulatedNM_014219 KIR2DL2 Up-regulated Unch.NM_013289 KIR3DL1 Up-regulated Up-regulatedNM_006737 KIR3DL2 Up-regulated Unch.NM_002259 KLRC1 Up-regulated Up-regulatedNM_002260 KLRC2 Up-regulated Up-regulatedNM_002262 KLRD1 Up-regulated Unch.NM_002831 PTPN6 Down-regulated Unch.NM_003177 SYK Up-regulated Up-regulatedNM_003332 TYROBP Up-regulated Up-regulated

(A) The expression pattern of genes associated with the pathways unique in theHIV-1-infected mPBMCs. (B) The expression pattern of genes associated theWnt/β-catenin signaling pathway. (C) The expression pattern of genesassociated with the NK cell signaling pathway in both HIV-1-infected andSIV-infected mPBMCs. The gene expression changes that passed a cutoffp≤0.01 were shown as Up-regulation or Down-regulation. Otherwise, wereshown as Unch. (unchanged). All the gene expression changes were revealed bymacaque-specific oligonucleotide microarrays.

Table 1 (continued)

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(Molina et al., 1992; Sloan-Lancaster et al., 1994; Sugie et al.,2004). In the HIV-1-infected mPBMCs, we observed theexpression changes of 8 genes in the T cell receptor pathway.Notably, the expression of CD247, ZAP-70, LCK, and FYNwas up-regulated in the HIV-1-infected mPBMCs (Fig. 5A,Table 1A); the up-regulation of CD247, LCK, and ZAP70 wasalso confirmed by qRT-PCR (Fig. 7).

The increased presence of T cell receptor associatedmolecules may potentiate T cells for activation or lead toanergy, depending on the availability of co-stimulation from theCD28 pathway (Kane et al., 2000). Although we did notobserve the expression changes of the CD28 signaling pathway,the up-regulation of JUND in the HIV-1-infected mPBMCsuggested that the HIV-1-infected mPBMCs may not be in astate of anergy, the immuno-nonresponsive status (Supplemen-tal Table 2) (Heisel and Keown, 2001). Thus, a heightened stateof T cell activation and the transcription programs it triggeredwould in turn be in agreement with the increased geneexpression regulation exhibited by HIV-1-infected PMBCsrelative to SIV-infected ones, in spite of the lower replicationlevels of HIV-1.

Therefore, our results suggested that T cell activation may beenhanced in HIV-1-infected mPBMCs. Activated Tcells may bemore effective in mediating host responses against HIV-1infection, thereby contributing to the lower pathogenicity ofHIV-1 in macaques.

ERK/MAPK signaling pathwayThe activation of the T cell receptor pathway would in turn

affect a number of down stream signaling pathways includingthe ERK/MAPK pathway (Michie et al., 1999). Interestingly, afew genes associated with the ERK/MAPK pathway weredifferentially expressed only in the HIV-1-infected mPBMCs,including YWHAH (14-3-3), RAP1A, and MAPK3 (ERK1).

Fig. 5. (A) Networks of pathways unique in the HIV-1 regulated mPBMCs. The networks showing the relationship of genes in the antigen presentation, the T cellreceptor, and the ERK/MAPK pathways are shown. (B) Relationship networks of genes associated with the NK cell signaling pathway in both HIV-1- and SIV-infectedmPBMCs. The networks were built by Ingenuity Pathway Analysis. Each dot represents a gene, with green indicating down-regulation of the gene, and red indicatingup-regulation of the gene. Each line connecting two genes indicates a relationship between the gene products as reported in literature.

142 Y. Li et al. / Virology 366 (2007) 137–149

The 14-3-3 protein plays an inhibitory role in apoptosis bydifferentially regulating the activities of the MAPK pathways(Xing et al., 2000). An up-regulation of YWHAH may conferprotective effects on the immune cells during the HIV-1infection in macaques. The RAP1A protein is important inmediating T cell anergy by blocking the TCR signals to ERKs(Boussiotis et al., 1997; Carey et al., 2000). The down-regulation of RAP1A supported that the HIV-1-infectedmPBMC may not be in an anergy status. Furthermore, theMAPK3 gene, known to play important roles in T celldevelopment (Fischer et al., 2005), was up-regulated only inHIV-1-infected mPBMCs and as confirmed by qRT-PCR (Fig.7); this up-regulation could be the direct result of the activationof the T cell receptor pathway (Haks et al., 2005). Given thatERK pathway plays a critical role during the development of Tcells and the immune selection (Alberola-Ila and Hernandez-Hoyos, 2003), up-regulation of the ERK signaling pathwayearly on during the HIV-1 infection in macaques may enhancethe functions of T cells and consequently induce stronger anti-viral responses, in turn accounting for the subdued pathogeni-city of the virus in comparison to SIV.

Taken together, our data suggested that the signaling cascadefrom the MHC/TCR interaction to the downstream ERK/MAPK signaling pathway was up-regulated in the HIV-1-infected mPBMCs compared to SIV-infected ones. Theheightened state of immune activation may be an indicationof the host's success in warding off HIV-1 infection, resulting in

reduced pathogenicity in macaques. However, many genes inthe T cell receptor and the ERK/MAPK pathways, for exampleCD247 (CD3-zeta), PLCG2 and MAPK3 (ERK1) itself, are notexclusively expressed in T cells but also in other cell types suchas B cells and NK cells (Arase et al., 2003; Chiu et al., 2005; Yuet al., 2000).

Therefore, it is possible that other cell populations in themPBMCs, besides T cells, were activated during the HIV-1infections as well. Given the dominance of immune cells inPBMC cultures, our results implicated the activation of differentsubsets of immune cells.

The activation of these immune cells may be capable ofmediating stronger antiviral immune responses during the HIV-1 infection compared to the SIV infection in macaques.

Pathway analyses of cellular gene expression profiles inSIV-infected mPBMCs

Wnt/β-catenin signaling pathwayThe Wnt/β-catenin signaling pathway plays important roles

in the survival of T cells (Goux et al., 2005). Our resultsindicated that the Wnt/β-catenin signaling pathway may berepressed in the SIV-infected mPBMCs, as characterized by theexpression changes of 3 key genes in the Wnt/β-cateninsignaling pathway, TCF-4, LEF-1, and PPP2R2B (Hovanes etal., 2001; Li et al., 2001; Miller and Moon, 1996) (Table 1B).TCF-4 was shown to have repressive effects on the HIV-1

Fig. 6. HIV-1 infection regulates the expression of a large number of immuneresponse genes at day 4 p.i. The immune response genes were defined by theknowledge-based pathway analysis tool, Ingenuity Pathway Analysis. Theexpression changes were revealed by macaque-specific oligonucleotidemicroarrays. (□) indicates the down-regulated genes and ( ) indicates theup-regulated genes. Each gene expression changes passed the statistical cutoff(p≤0.01). The two histograms represent data from signature genes, defined ashaving a p≤0.01. The p-value is derived from both the errors in microarraymeasurements and the estimated variance among the 3 animals.

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replication in human cells (Wortman et al., 2002). A down-regulation of TCF-4 in the SIV-infected mPBMC, as confirmedby qRT-PCR (Fig. 7), may contribute to a more robustreplication of SIV than HIV-1 in mPBMCs. LEF-1, shown toplay a critical role in the survival of T cells (Ioannidis et al.,2001), was also down-regulated in the SIV-infected mPBMCs.Given that a down-regulation of LEF-1 may impair the CD8+ Tcell development and its functions (Okamura et al., 1998), SIVmay be able to compromise the antiviral functions of the CD8+

T cells by down-regulating the expression of LEF-1 during theinfection in macaques.

Moreover, PPP2R2B (PP2A), known to inhibit the Wnt/β-catenin pathway (Li et al., 2001), was up-regulated only inthe SIV-infected mPBMCs. Our data therefore suggested thatWnt/β-catenin signaling pathway may be repressed in theSIV-infected mPBMCs. Given that the pathogenic SIVinfection in macaques was characterized by increased T celldeath (Batten et al., 2006), the concerted down-regulation ofWnt/β-catenin signaling pathway, characterized by the down-regulation of LEF-1 and TCF-4 and the up-regulation ofPPP2R2B, deals a double blow to T cell functions andsurvival and may contribute to the pathogenicity of SIV inmacaques.

Natural killer cell signaling pathwayNatural killer (NK) cells play important roles in restraining

the infection of HIV-1 in humans (Fauci et al., 2005; Mavilio etal., 2003). By extension, we speculated that the NK cellsignaling may be different in the HIV-1-infected and SIV-infected mPBMCs. Interestingly, we observed the regulation ofthe NK signaling pathway in both HIV-1-infected and SIV-infected mPBMCs, albeit with different patterns (Fig. 5B andTable 1C). A few NK cell signaling-associated genes werecommonly regulated in both HIV-1 and SIV-infected mPBMCs.CD244 (2B4), a NK cell receptor that stimulate NK cellcytotoxicity (Messmer et al., 2006), and SYK, a key gene to NKcell functions (Jiang et al., 2002), were up-regulated in bothHIV-1- and SIV-infected mPBMCs; the up-regulation of CD244by both viruses was confirmed by qRT-PCR (Fig. 7). However,the NK cell marker FCGR3A (CD16), CCL4 and CCL5 (Fig. 7)were up-regulated in mPBMCs infected with HIV-1, but notwith SIV, suggesting that NK cells may be at a heightenedactivation status in the HIV-1-infected mPBMCs (Arase et al.,2003; Dorner et al., 2004).

Reportedly, NK cells are able to repress the HIV-1 infectionin human cells by secreting large amounts of CCL4 and CCL5to block the entry of HIV-1 (Fehniger et al., 1998). However,given that CCL5 only blocks the infection of the M-tropicHIV-1 but not the T-tropic HIV-1 used in this study, blockingthe entry of HIV-1 by CCL5 may not entirely account for thelow pathogenicity of this specific HIV-1 strain in macaques.Recent studies suggested that NK cells play crucial roles inconnecting the innate and adaptive immune responses (Cooperet al., 2004; Degli-Esposti and Smyth, 2005; Moretta, 2002).Therefore, activated NK cells may play roles in coordinatingother immune cells to effectively repress the HIV-1 infection inmacaques and contribute to the reduced HIV-1 pathogenicity

indirectly (Gerosa et al., 2002; Groot et al., 2006; Piccioli etal., 2002).

HIV-1 infection induces earlier and stronger immune responsethan the SIV infection in mPBMCs

The pathway analyses described above revealed that theinfections of HIV-1 and SIV induced changes in largely distinctsets of pathways. It should be noted that the expression changesof these pathways could take place in more than one cellpopulation. For instance, the antigen presentation pathwayscould appear in different types of dendritic cells (DCs).

The T cell receptor pathway may be affected in CD4+ T cellsand/or CD8+ T cells. Furthermore, the intracellular signalingpathways such as the ERK and the Wnt/β-catenin signalingpathways are functional not only in T cells (Tan et al., 2006) butalso in B cells and other immune cells (Guo and Rothstein,2005; Yu et al., 2000). By design, gene expression profilingdoes not single out individual cell populations that are affectedby the expression of these pathways; instead, the analysisreveals changes in the transcriptional program associated withthe virus infections. Notably, using IPA, we observed differ-ential expression predominantly among genes involved inimmune response. Thus, we attempted to examine the expres-sion pattern of the immune response genes as a whole betweenthe HIV-1-infected and SIV-infected mPBMCs.

Interestingly, our results suggested that HIV-1 infectioninduced expression changes of immune response genes earlieron and to a greater extent than SIV infection in mPBMCs (Fig.6). Based on annotations in the IPA knowledgebase, weidentified 187 immune response genes (Supplemental Table 2)from our list of ∼1300 differentially expressed genes in HIV-1-infected and SIV-infected mPBMCs (Supplemental Table 4).Overall, immune response genes had expression changes in theHIV-1-infected mPBMCs, in contrast to 91 immune responsegenes in the SIV-infected cells. Interestingly, 95 genes wereregulated by HIV-1 infection on day 4 p.i., only 25 immune

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genes were changed in SIV-infected mPBMCs at the same timepoint. In other words, the HIV-1 infection induced about 4 timesas many immune response genes as the SIV infection did on day4 p.i. On day 6 p.i., HIV-1 and SIV induced expression changesof 71 and 76 immune response genes, respectively. Given thatthe HIV-1 replication is significantly lower than that of SIV onboth days 4 and 6 p.i., our expression profiling results ofimmune response genes therefore suggested that the HIV-1infection induced a more robust and prompt immune responsethan the SIV infection.

Previous studies have suggested that chronic immuneactivation is a major characteristic of the pathogenic HIV-1and SIV infections in vivo (Folks et al., 1997; George et al.,2006; Giorgi et al., 1999; Onanga et al., 2006). However, ourmolecular profiling data from the HIV-1-infected mPBMCsdescribed a different scenario by suggesting that the HIV-1infection may induce a better controlled immune activation inmacaques. HIV-1 infection in mPBMCs transiently induced theexpression changes of a few key genes that are crucial incontrolling the activation of immune cells. CD247, ZAP70, andLCK, known to be expressed in multiple immune cells, were

Fig. 7. qRT-PCR confirmation of the microarray results. The expression changes of sethe virus-infected cells relative to the time-matched mock-infected cells were calcul

up-regulated only at day 4 p.i. but not at the later time point(Supplemental Table 2); on the other hand, CTLA-4, a repressorof T cell activation critical to regulating immune cell activities(Schneider et al., 2006) and the overall immune response (Ruddand Schneider, 2003), was down-regulated on day 4 but not onday 6 p.i. in the HIV-1-infected mPBMCs, as confirmed byqRT-PCR (Fig. 7). The transient down-regulation of CTLA-4 inthe HIV-1-infected mPBMCs at the early time point is in linewith the enhanced T cell receptor signaling we observed at thesame time point and provides further evidence for T cellactivation at this earlier time point. On the other hand, therecovery of CTLA-4 expression on day 6 p.i. and the concurrentdown-350 regulation of ICOS, an inducer of T cell activation(Nurieva et al., 2003), indicate that activation of these immunecells is not persistent. In contrast, SIV-infected mPBMCs didnot exhibit comparable decrease in T cell activation, as therepressor CTLA-4 stayed down-regulated on both days 4 and6 p.i., with the inducer ICOS unaffected by SIV infection.

Taken together, our data indicated that HIV-1 and SIVinfections were associated with distinct T cell activation states:HIV-1 appears to first induce and then suppress Tcell activation,

lected genes were also quantified by qRT-PCR assay. The expression changes inated.

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which is in line with the heightened immune response on day4 p.i.; HIV-1 replication was subsequently subdued at latertime points, as T cell activation is required for efficient HIVreplication (Gowda et al., 1989); in contrast, SIV-1 does notappear to effect a similar suppression on T cells and thereforemay support more robust virus replication.

The distinct pathogenic properties of HIV-1 and SIV inmacaques are likely to be caused by multiple factors. Persistentactivation and the activation induced cell death (AICD) ofimmune cells have been reported extensively as a key to theHIV-1 pathogenesis in humans (Grossman et al., 2006). Unlikethe pathogenic infection in humans, HIV-1 infection may onlytemporally induce the activation of immune cells in macaques.The temporal immune activation could be robust enough tohinder the progression of infection by antiviral immuneresponses but not enough to induce massive cell death inmacaques. The other reason could be a failure of the macaquesto mount an effective immune response in spite of robust SIVreplication at the early stage of infection. Unlike the SIVinfection, a low level replication of HIV-1 would be able totrigger a robust immune response in macaque. It is conceivablethat the immune activation can be triggered by both direct andindirect mechanisms. Specifically, we speculate that thetemporal surge in the release of cytokines by the HIV-infectedcells (Bahbouhi et al., 2004), albeit in the minority, can signalgene expression changes in bystander cells in a paracrinefashion. Evading the immune surveillance is one well adoptedstrategy of primate lentiviruses during pathogenic infections(Evans and Desrosiers, 2001). One well studied example is nef,a gene encoded by all primate lentiviruses. Nef plays a centralrole in the evasion of host immune surveillance (Fackler andBaur, 2002). The pathogenicity of SIV with defects in the nefgene is significantly compromised in macaques (Kestler et al.,1990). A recent study suggested that, unlike SIV Nef, HIV-1Nef does not suppress the immune cell activation (Schindler etal., 2006). Given that the Nef is dominantly expressed at thevery early stage during the SIV infection, the repressive role ofNef in the immune activation could be crucial to thepathogenicity of SIV in macaques. Therefore, a failure ofsuppressing the immune cell activation by HIV-1 Nef mayallow for rapid antiviral immune response to take effect duringthe HIV-1 infection in macaques.

In conclusion, our results indicated that the infection of HIV-1 and SIV in mPBMCs induced distinct pattern of cellular geneexpression changes. While HIV-1 and SIV exhibited similarreplication rates up until day 4 p.i., noticeable differences inhost gene expression observed from that point on suggested thatthe pathogenic potential of the viruses could be reflected in thetranscriptional programs triggered by the infections. Specifi-cally, HIV-1 infection transiently triggered enhanced antigenpresentation, as well as signaling and activation of T cells andNK cells at the early time point. By comparison, SIV infectionwas associated repressed T cell activation via Wnt/β-cateninsignaling. HIV-1 infection induced more robust cellular res-ponse, possibly able to keep HIV-1 replication in check earlieron. However, as the infection progresses, the immune responseis modulated, as seen in the lessened T cell activation at the late

time point, which may render the susceptible cells less permi-ssive to virus replication. The failure to sustain robust virusproduction caused by the host antiviral immune responses maypartially explain the low pathogenicity of HIV-1 in macaques;unlike HIV-1, SIV is likely to have acquired the necessaryfitness through evolution to sustain replication via the moreexpedient repression of host immune response. To reveal therestrained pathogenicity of HIV-1 in macaques mechanistically,further studies should focus on the investigation of the roles ofindividual cell populations during the HIV-1 infection in themPBMCs.

Material and methods

PBMC preparation

Peripheral blood was collected from three healthy pigtailmacaques at Washington National Primate Research Center.Macaque PBMCs were recovered by standard Ficoll density-gradient centrifugation. The recovered cells were then washed,counted, and placed at a density of 2×106 cells/ml in completeRPMI 1640 growth medium containing 10% fetal bovine serumand 5% lectin-purified IL-2 (Roche Diagnostics). Phytohemag-glutinin (Sigma Chemical) was then added at a concentration of5 g/ml, and after a 24 h incubation period, the medium wasreplaced with fresh complete medium and the culture wasincubated at 37 °C for 4 days prior to infections.

Virus

The SIV strain, SIVmac239, containing an open readingframe of the nef gene was produced from two plasmidsp239SpSp5′ and p239SpE3′Nef open (The NIH AIDS Researchand Reference Reagent Program) as described previously (Gibbset al., 1994). The HIV-1 strain, HIV-1 Lai, was the lab stockoriginally obtained from the Institute Pasteur, France (Peden etal., 1991). The SIV stock was grown in CEMx174 cells and theHIV-1 stock grown in CEMss cells. Both virus stocks werefiltered through 0.22 m filters to remove cell derbies prior to theinfections. The infectious titers of HIV-1 and SIV weredetermined in CEMx174 cells by the end-point dilution assays(Rodrigo et al., 1997).

Virus infection

Unfractionated mPBMCs (10×106 cells) from 3 pigtailmacaques showed comparable compositions in terms of the celltypes present (Supplemental Table 1). The mPBMCs from eachindividual animal were infected with HIV-1 or SIV at amultiplicity of 1 TCID50/cell or mock-infected with a volume ofculture supernatant from uninfected cells equal to the volume ofvirus stocks. After a 120 min incubation period at 37 °C,unattached virus was removed by 3 washes with phosphate-buffered saline. Mock-infected cells were washed in the samemanner. The cells were then divided in 24-well tissue cultureplates at 0.5×106 cells/well in 2 ml of complete medium perwell per animal. For each animal, 2 wells of cells infected with

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each virus or mock were harvested on days 2, 4, 6, and 8 p.i.Cells were counted at harvesting and the percentage of viablecells was determined by trypan blue dye exclusion. The cellswere then collected by centrifugation and lysed in buffer RLTfrom RNeasy RNA isolation kit (Qiagen, CA). Culture super-natants were monitored for p27 or p24 levels by antigen-captureELISA (ZeptoMetrix Corp. NY). The percentage of infectedcells was estimated by indirect immunofluorescence assay (IFA)using pooled sera from SIV-infected macaques or HIV-1-infected humans as described previously (Agy et al., 1991).

Microarray analyses

For oligonucleotide array analyses, total RNA was isolatedby RNeasy RNA isolation kit (Qiagen, CA). Equal amount ofRNA isolated from 2 duplicate wells was combined before thecomplimentary RNA (cRNA) amplification. The cRNA wasthen amplified using the Agilent low RNA input linear ampli-fication kit (Agilent Technologies, CA). The quantity andquality of cRNA were evaluated by capillary electrophoresisusing an Agilent Technologies 2100 Bioanalyzer. Probe label-ing and microarray hybridizations were performed as describedin the Agilent 60-mer oligo microarray processing protocol(Agilent Technologies, CA). Labeled probes derived from SIV-infected cells, HIV-1-infected cells, and their time matchedmock-infected controls were co-hybridized against the probesderived from a common reference RNA sample on the Agilentmacaque oligonucleotide microarrays (Version 2) (AgilentTechnologies, CA). The common reference RNA was poolingRNA isolated from mPBMCs of 10 uninfected pigtail macaquesexcluding the animals used in this study and the commonreference probes were produced in the same fashion as the otherprobes. Slides were scanned with an Agilent microarray scannerand image analysis was performed using Agilent Feature Ex-tractor Software. Each microarray experiment was done using adye-swapping technique, thereby providing 2 technical repli-cates for each measurement (Kerr and Churchill, 2001).

All data were entered into a custom-designed database,Expression Array Manager, and then uploaded into Resolver5.0 (Rosetta Biosoftware, WA) for analyses. The expressionvalue of each individual gene was the average of 3 expressionvalues from 3 animals calculated by the Resolver softwareusing the Rosetta error model (Weng et al., 2006). Fordetermining differential expression, the p-value for any givengene represents contributions from both the variance amongmeasurements from the 3 animals, as well as the estimatederrors in microarray signal measurements. Initially, genes wereselected as differentially expressed based on a criterion of agreater than 99% probability of being differentially expressed(p≤0.01). Biological gene sets (referred to as Biosets) werecompiled for various cellular processes by selecting genes ofinterest that were both represented on the microarray andwhich had Gene Ontology (GO) annotation (Gene OntologyConsortium, 2004). Once compiled, Biosets were used tocharacterize and compare gene expression patterns betweenexperiments. These Biosets were also analyzed using theIngenuity Pathway Analysis (IPA) Software (www.ingenuity.

com, Ingenuity Systems, CA) to assign functional informationand biological relevance to the groups of genes beingregulated. In accordance with proposed standards (Brazma etal., 2001), all data described in this report, including sampleinformation, intensity measurements, gene lists, error analysis,microarray content, and slide hybridization conditions, areavailable in the public domain through Expression ArrayManager at http://www.expression.washington.edu/public.

qRT-PCR

Quantitative real-time PCR (rtPCR) was used to verify thegene expression changes. Total RNA samples were treated withDNase using DNA-free DNase Treatment and RemovalReagents (Ambion Inc. TX). Reverse Transcription wasperformed using TaqMan Reverse Transcription Reagents(Applied Biosystems, CA). Primer and probe sets for each ofthe target sequences were designed using macaque ESTsequences available from Macaque.org (Supplemental Table3) and mapping them to the M. mulatta genome available fromBaylor College of Medicine. rtPCR was performed on the ABI7500 Real-time PCR System using TaqMan chemistry (AppliedBiosystems, CA). Each target was run in quadruplicate, with20 μL reaction volumes of TaqMan 2× PCR Universal MasterMix (Applied Biosystems, CA). 18S RNA was chosen asendogenous control to normalize quantification of the targets.

The relative quantification of the viral genome RNA wasperformed by using the SIV-3′LTR primer/probe set and theHIV-1-3′LTR primer/probe set (Supplemental Table 3). Therelative quantification of SIV or HIV-1 genome RNA of eachtime point was calculated by using the quantity of the cor-responding virus genome RNA from day 2 p.i. as the base line.

Acknowledgments

The authors gratefully thank Drs. Satya Dandekar andMichael B. Agy for their helpful comments. This work wassupported by the NIH National Center for Research Resources(grants RR016354 and RR00166 to M.G.K.) and the NationalInstitute on Drug Abuse (grant 1P30DA01562501 to M.G.K.).Y.L. and E.Y.C. are supported by the Rosetta InpharmaticsFellowship in Molecular Profiling.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.virol.2007.04.020.

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