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Cells+Human CD4Expression and Inhibit HIV Replication in

Decrease CD4FlaviviridaeViruses within the

StapletonChang, Thomas M. Kaufman, Donna Klinzman and Jack T. Jinhua Xiang, James H. McLinden, Robert A. Rydze, Qing

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Viruses within the Flaviviridae Decrease CD4 Expression andInhibit HIV Replication in Human CD4� Cells1

Jinhua Xiang, James H. McLinden, Robert A. Rydze, Qing Chang, Thomas M. Kaufman,Donna Klinzman, and Jack T. Stapleton2

Viral infections alter host cell homeostasis and this may lead to immune evasion and/or interfere with the replication of other microbesin coinfected hosts. Two flaviviruses are associated with a reduction in HIV replication or improved survival in HIV-infected people(dengue virus (DV) and GB virus type C (GBV-C)). GBV-C infection and expression of the GBV-C nonstructural protein 5A (NS5A)and the DV NS5 protein in CD4� T cells inhibit HIV replication in vitro. To determine whether the inhibitory effect on HIV replicationis conserved among other flaviviruses and to characterize mechanism(s) of HIV inhibition, the NS5 proteins of GBV-C, DV, hepatitisC virus, West Nile virus, and yellow fever virus (YFV; vaccine strain 17D) were expressed in CD4� T cells. All NS5 proteins inhibitedHIV replication. This correlated with decreased steady-state CD4 mRNA levels and reduced cell surface CD4 protein expression.Infection of CD4� T cells and macrophages with YFV (17D vaccine strain) also inhibited HIV replication and decreased CD4 geneexpression. In contrast, mumps virus was not inhibited by the expression of flavivirus NS5 protein or by YFV infection, and mumpsinfection did not alter CD4 mRNA or protein levels. In summary, CD4 gene expression is decreased by all human flavivirus NS5 proteinsstudied. CD4 regulation by flaviviruses may interfere with innate and adaptive immunity and contribute to in vitro HIV replicationinhibition. Characterization of the mechanisms by which flaviviruses regulate CD4 expression may lead to novel therapeutic strategiesfor HIV and immunological diseases. The Journal of Immunology, 2009, 183: 0000–0000.

B ased on phylogenetic relationships of viral RNA-depen-dent, RNA polymerase sequences, the family Flaviviri-dae is comprised of three genera and a few unclassified

viruses that include important animal and human pathogens (Fig.1A and reviewed in Ref. 11). Although most of these viruses causetransient, self-limited infection, two may result in persistent humaninfection (hepatitis C virus (HCV)3 and GB virus type C (GBV-C)). Members of the Flaviviridae contain a single-stranded, posi-tive sense RNA genome encoding a polyprotein that is posttrans-lationally cleaved into structural and nonstructural proteins.Although flaviviruses are generally similar in their genome struc-ture and replication strategies, a few important differences exist(2–4). For example, the nonstructural protein 5 (NS5) of pestivi-ruses, hepaciviruses, and the GB viruses are processed into twoproducts termed NS5A and NS5B. NS5A is a multifunctionalphosphoprotein required for infectivity and RNA replication, and

NS5B contains polymerase activity (reviewed in Ref. 5). In con-trast, the NS5 proteins of members of the flavivirus genus are notprocessed and retain polymerase activity (2).

GBV-C is a common human virus with no known disease mani-festations (3, 6). Several studies found an association between GBV-Cviremia and prolonged survival in HIV-infected people (Refs. 7–12and reviewed in Refs. 3 and 13). GBV-C replicates in B and T lym-phocytes including CD4� T cells (14–16), and coinfection of lym-phocytes with GBV-C and HIV results in potent inhibition of bothCCR5- and CXCR4-tropic HIV isolates, including isolates represent-ing clades A through H and group O (17–19). Acute dengue virus(DV) infection is also associated with transient suppression of serumHIV RNA levels and acute, but not convalescent sera inhibits HIVreplication in vitro, suggesting viral interference (20).

Two GBV-C proteins have been shown to interact with CD4� Tcells and inhibit HIV replication in vitro. Expression of the viral en-velope glycoprotein E2 within CD4 cells or addition of recombinantsoluble E2 to CD4� T cells decreases surface expression of CCR5,one of the two major HIV coreceptors, and induces the release ofsoluble factors that inhibit HIV entry in vitro (18, 21, 22). Expressionof the GBV-C nonstructural protein NS5A protein in CD4� T cellsalso inhibits replication of HIV isolates representing three differentclades, independent of entry coreceptor tropism (CCR5 or CXCR4)(23, 24). Both clinical and laboratory-adapted HIV isolates are inhib-ited (17, 23–25). A 16-aa peptide region within GBV-C NS5A issufficient for HIV inhibition and HIV replication is inhibited in cellsincubated in synthetic peptides containing this domain, suggestingthat this molecule has therapeutic potential (25). GBV-C NS5A me-diates HIV inhibition in part by decreasing the surface expression ofCXCR4, the other major HIV coreceptor, and by inducing release ofstromal cell-derived factor 1 (SDF-1; CXCL12), the chemokineligand for CXCR4 (23, 26). Although GBV-C-mediated chemokinemodulation contributes to HIV replication inhibition, neutralization ofSDF-1 did not abolish the inhibition, suggesting that the GBV-CNS5A protein has additional mechanisms by which it inhibits HIV

Department of Internal Medicine, Iowa City Veterans Affairs Medical Center andUniversity of Iowa, Iowa City, IA, 52242

Received for publication July 15, 2009. Accepted for publication October 12, 2009.

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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This research was supported in part by a Merit Review grant from the Departmentof Veterans Affairs, Veterans Health Administration, Office of Research and Devel-opment (to J.X. and J.T.S.), a grant from the National Institutes of Health (R01AI-58740; to J.T.S.), and a pilot grant from the University of Iowa National Institutesof Health Clinical & Translational Sciences Program UL1 RR024979 (to J.T.S.).2 Address correspondence and reprint requests to Dr. Jack Stapleton, University ofIowa, SW54, GH, 200 Hawkins Drive, Iowa City, IA 52242. E-mail address:jack-stapleton@uiowa.edu3 Abbreviations used in this paper: HCV, hepatitis C virus; GBV-C, GB virus type C;DV, dengue virus; SDF-1, stem cell-derived factor 1; YFV, yellow fever virus; IRES,internal ribosomal entry site; MDM, monocyte-derived macrophage; WNV, WestNile virus; GT, genotype; Scr, scrambled order; DI, domain I; VC, vector control; FS,frameshift; MOI, multiplicity of infection; MFI, mean fluorescent intensity; EMC,encephalomyocarditis virus.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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replication (23, 26). Expression of DV (serotype 2) NS5 protein inCD4� T cells also inhibits HIV replication and modulates CXCR4and SDF-1 expression (26).

To determine whether HIV inhibition induced by flavivirus NS5protein expression is conserved among additional members of theFlaviviridae, we characterized the effect of NS5 protein expressionof five members of the virus family in CD4� T cells. In addition,we examined yellow fever virus (YFV) infection (17D vaccinestrain) of T lymphocytes and macrophages to determine whetherthe effects of NS5 protein expression were recapitulated duringviral replication. These studies identify a mechanism by whichflavivirus NS5 protein modulates T cell homeostasis and that maycontribute to HIV replication inhibition.

Materials and MethodsViral NS5 protein expression

Viral NS5 protein coding sequences were ligated into a modified pTRE2-Hyg plasmid (BD Clontech) as previously described (23). This plasmidgenerates a bicistronic message encoding the viral sequence upstream of

the encephalomyocarditis virus (EMC) internal ribosomal entry site (IRES)element that directs translation of GFP (23). Jurkat (tet-off) cell lines thatstably expressed the various flavivirus NS5 protein sequences were gen-erated following transfection and selection (23, 25). NS5 inserts and con-trol sequences were confirmed by sequencing plasmid DNA (25) (Univer-sity of Iowa DNA Core Facility; Applied Biosystems automated DNAsequencer 373A).

Protein expression was demonstrated by immunoblot analysis using rab-bit anti-GBV-C NS5A serum, murine anti-HCV NS5A mAb (BioDesign),or a goat polyclonal anti-gp120 mAb (Abcam) (23, 27). For cell linesexpressing NS5 peptides for which no Abs are available, GFP expressionwas demonstrated by flow cytometry and total cellular DNA and RNAwere examined for linkage between NS5A sequences and GFP using PCRor RT-PCR followed by determination of the nucleotide sequence as de-scribed previously (23, 25).

Virus infections

HIV infection of CD4� T cell lines utilized an X4 HIV-1 isolate (clade B,catalog no. 1073; National Institutes of Health AIDS Research and Refer-ence Reagent Program), and infection of primary human PBMCs andmonocyte-derived macrophages (MDMs) utilized this X4 isolate or an R5isolate (clade A; catalog no. 1741) (17, 23). MDMs were prepared as pre-viously described (28) and were infected with R5-tropic HIV 3 days afterdifferentiation (average 10 days in culture). HIV replication was deter-mined by measuring HIV p24 Ag in culture supernatants by ELISA aspreviously described (10, 17). Data presented represent the mean of a min-imum of six independent infections, and error bars represent the SEM.

YFV (17D) and mumps (Jeryl-Lyn) vaccine strain isolates were prop-agated in BHK 21 and Vero cells, respectively (29, 30), and the infectioustiters were determined by terminal dilution (31). YFV and mumps virusinfections were also conducted in CD4� T cell lines, PBMCs, and MDMs.MDM infections were initiated 2 days after differentiation (average, 9days). PBMCs and MDMs were obtained from healthy HIV and HCVAb-negative donors after obtaining written informed consent as describedelsewhere (17). All subjects who donated blood samples provided writteninformed consent, and this project was approved by the University of IowaInstitutional Review Board.

RNA and cell receptor quantification

Total cellular RNA from a minimum of three independent Jurkat cell linecultures was purified (RNeasy kit; Qiagen) and cDNA was synthesized(ReactionReady cDNA synthesis kit; SuperArray Bioscience). RNA con-centrations were determined by real-time RT-PCR as recommended by themanufacturer (SuperArray Bioscience). Fold change mRNA between ex-perimental and vector control (VC) cells was determined using the ��Ctmethod normalized to the average expression of five housekeeping genes invector control cells (�-actin, ribosomal protein L13a, hypoxanthine phos-phoribosyl transferase 1, glyceraldehyde-3-phosphate dehydrogenase, 18Sribosomal RNA) as described by the manufacturer (SuperArray BioscienceCorp). Cell surface receptor protein expression was determined by flowcytometry (FACScan; BD Biosciences) as described previously (10).

Sequence analysis

Nucleotide sequence alignments and predicted protein secondary structureanalyses utilized DNAMan software (Lynnen Biosoft) and a web-basedsequence analysis program, Mega 3.1 (32). RNA-dependent, RNA poly-merase amino acid sequences from representative flaviviruses were alignedas described by Koonin (33). Phylogenetic relationships were constructedusing the neighbor-joining method and trees were generated by Mega 3.1software (32). The viruses used to construct the tree included pestiviruses(bovine viral diarrhea disease virus (GenBank http://www.ncbi.nlm.nih.gov/sites/entrez, accession no. U18059, and classical swine fever virus(NC_002657)), flaviviruses (YFV vaccine strain 17D (X03700), DV(AF359579), and West Nile virus (WNV) strain NY99 (NC_009942)), he-paciviruses (HCV genotypes (GT) GT 1 (M62321), JFH-1 strain GT2(AB047639), GT 3 (D17762), GT 4 (Y11604), GT 5 (Y13184), GT6 (Y12083)), and the unassigned GB viruses (GBV-A (HGU94421),GBV-B (NC001655), a human isolate of GBV-C (AF121950), and a chim-panzee (troglodyte) GBV-C variant (AF070476)).

Statistics

Statistics were performed using SigmaStat software version 3.11 (JandelScientific) and SuperArray statistical software. The t tests were used fordirect comparisons of HIV p24 Ag release or mumps virus infectious titer,and �2 analysis was used for cell surface expression measured by flowcytometry. ANOVA was used for multiple comparisons.

FIGURE 1. Phylogenetic relationships of the RNA-dependent, RNA poly-merase sequences of the family Flaviviridae. Representative isolates of viruseswithin the three genera (flavivirus, pestivirus, and hepacivirus) of the Flavi-viridae and the unassigned GB viruses are shown in A. Viruses analyzed in-cluded the vaccine strain (17D) of YFV, DV serotype 2, and WNV, classicalswine fever virus (CSFV), bovine viral diarrhea virus (BVDV), six genotypesof HCV, GB virus A (GBV-A), GB virus B (GBV-B) and the human (hum)and chimpanzee (trog) variants of GBV-C. 0.2 � distance representing 0.2 aasubstitutions per position. B illustrates the NS5 protein coding sequences usedto generate stably expressing Jurkat cell lines. These encode the completeNS5A proteins of GBV-C and HCV and the complete NS5 proteins of DV(serotype 2), WNV, and the YFV(17D). Additional cell lines expressing a16-aa GBV-C NS5A peptide (aa 152–167), the same 16-aa in a Scr, HCV DI(encoding aa 1–215) and a control cell line contains the GBV-C NS5A se-quence that normally encodes aa 126–236 but in which a FS mutation wasinserted (126–236 FS) are also shown.

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ResultsNS5 protein of Flaviviridae expression inhibits HIV replication

CD4� Jurkat T cell lines were previously generated that stablyexpress the DV (serotype 2) NS5 protein, the GBV-C full-lengthNS5A protein, a peptide domain of GBV-C NS5A (aa 152–167),and control cell lines including the VC, GBV-C NS5A 152–167 ina scrambled order (Scr), and a GBV-C NS5A construct in which aframeshift (FS) was introduced (126–236 FS) containing theNS5A RNA but that expresses a 26-aa missense protein (23, 25,26). In addition, new Jurkat cell lines were generated that ex-pressed full-length HCV NS5A, the first 215 aa of HCV NS5A(structural domain I (DI)), and the full-length NS5 proteins WNVand YFV (vaccine strain 17D) using the same bicistronic expres-sion plasmid expressing GFP as a reporter as described and illus-trated in Fig. 1B (23).

GFP expression in these cell lines is shown in Fig. 2A. Cells ex-pressing viral NS5 proteins had lower levels of GFP than did cellsexpressing peptide fragments or the VC, consistent with previousstudies showing that expression levels of downstream open readingframes is inversely related to the size of the upstream open readingframe in bicistronic vectors (34). For the two NS5A proteins forwhich specific Abs are available (GBV-C and HCV), full-length pro-tein was detected in the Jurkat cell lysates by immunoblot (Fig. 2B).No commercial Abs for DV, WNV, or YFV NS5 proteins, theGBV-C NS5A 152–167 peptide, or HCV DI are available. To furthercharacterize the cell lines expressing these proteins (in addition toGFP expression), cellular DNA and RNA for each cell line was ex-tracted and amplified by both PCR and RT-PCR, respectively. Am-plicons included the CMV promoter, the NS5 sequences, and part ofthe EMC IRES as previously described (25). The linkages betweenthe CMV promoter, Kozak sequences, NS5 coding sequences and theEMC IRES, and GFP were confirmed by sequencing. For the RNAextractions, specificity was confirmed as no amplification occurred inthe absence of reverse transcriptase.

HIV replication was inhibited in Jurkat cells expressing the full-length NS5A proteins of HCV and GBV-C and also by the full-length NS5 proteins of DV, WNV, and YFV compared with VCcells (Fig. 3, A and B). Confirming previous studies, expression aa152–167 of GBV-C NS5A was sufficient for HIV inhibition,whereas the scrambled peptide or the FS GBV-C RNA sequencecontrols did not inhibit HIV (25). The GBV-C 152–167 peptidelies within the GBV-C region that shares overall homology with

the structural DI of HCV NS5A (35), although only 3 of the 16 aain the peptide region are identical. Despite the amino acid differ-ences, HIV replication was inhibited in Jurkat cells expressingHCV DI (Fig. 3, A and B; data represent difference in percentreduction in three independent experiments).

HIV inhibition did not appear to be due to cytotoxicity. Al-though expression of the flavivirus NS5 proteins appeared to slowthe doubling time of Jurkat cells, the density of viable cells was notdifferent between the different cell lines and the parent Jurkat cellline following 6 days in culture, and the amount of HIV p24 Agreleased into culture supernatants per cell illustrates that HIV in-hibition does not correlate with the number of viable cells in thedifferent cell lines (supplemental Fig. 1).4 Previous studies of dem-onstrated that vesicular stomatitis virus glycoprotein pseudotypedHIV particles were not inhibited by expression of GBV-C NS5Aprotein or peptides containing the 152–167 region, indicating thatthe inhibition is specific for HIV envelope glycoproteins (25). Inaddition, mumps virus (Jerl-Lyn vaccine strain) replicated as wellin Jurkat cell lines expressing flavivirus NS5 proteins as it did inthe VC cell lines (Fig. 3C). Thus, the antiviral effect of NS5 pro-teins was not indiscriminant. Taken together, these data indicatethat expression of the NS5 protein from all three genera within theFlaviviridae and from the unassigned GB virus group potentlyinhibited HIV replication in a CD4� T cell line.

Flaviviridae NS5 proteins modulate CD4 expression

To determine potential mechanisms by which NS5 protein ex-pression might inhibit HIV replication, expression of the HIVentry receptors and their natural ligands was examined in Jurkatcells expressing the flavivirus NS5 proteins. Consistent withprevious studies, none of the Jurkat cell lines had detectableCCR5, RANTES (CCL5), MIP-1� (CCL3), or MIP-1� (CCL4)mRNA (23), and CXCR4 mRNA expression was reduced inGBV-C-expressing cells compared with VC cells (�1.32-fold,t test; p � 0.049). However, CXCR4 mRNA reduction was notstatistically different from VC for either HCV or YFV NS5-expressing cells (data not shown). In contrast, steady-state CD4mRNA levels were significantly reduced in all seven of the celllines expressing the full-length NS5 proteins and in the cell line

4 The online version of this article contains supplemental material.

FIGURE 2. Generation of CD4� T cell lines (Jurkat)expressing flavivirus NS5 proteins. Jurkat cell lineswere transfected with the flavivirus NS5 encoding plas-mids and following selection in hygromycin, GFP levelswere determined by flow cytometry (A). Jurkat, Parentalcells without transfection; GBV-C, GBV-C NS5A pro-tein; 152–167, the GBV-C 16-aa peptide; Scr, thescrambled GBV-C peptide; HCV, HCV NS5A protein;DI, HCV DI; DV, DV NS5 protein; YFV, YFV vaccinestrain NS5 protein; and WNV, WNV NS5 protein. �,GFP MFI was less than VC, FS, and Scr (p � 0.001; ttest). †, GFP expression was significantly less than VCand FS controls, but not Scr. GFP expression in all cellscompared with parental Jurkat cells (p � 0.001; A).GBV-C and HCV NS5A protein expression was dem-onstrated in Jurkat cell lysates by immunoblot analysis(B) using rabbit anti-GBV-C NS5A Ab (left panel) or amurine anti-HCV NS5A Ab (right panel). pep, GBV-C152–167.

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expressing GBV-C NS5A aa 152–167 and the HCV DI com-pared with the three control cell lines (Fig. 4A). The effect wasmost pronounced in the cells expressing the GBV-C NS5A pro-tein (85-fold lower than VC; p � 0.0001). The serine at position158 of the GBV-C NS5A is predicted to be phosphorylated, andwe found that mutation of this serine to a phosphomimeticamino acid (glutamic acid) did not effect the HIV inhibition.However, substitution of either alanine or glycine significantlydecreased HIV inhibition compared with the parent, althoughthe effect was not abrogated (25). These data suggest that thestructure and/or phosphorylation of this peptide is critical forthe effect on HIV replication (25). Jurkat cells expressing thesemutant peptides (S158E, S158A, and S158G) were examinedfor CD4 expression and consistent with the HIV inhibitory ef-fect, CD4 down-regulation in cells expressing the S158E mu-tant was similar to the parent peptide. CD4 expression on cellsexpressing the S158A and S158G mutants was significantlyhigher than the parent peptide or the S158E mutant (supple-mental Fig. 2), and CD4 down-regulation was significantly lessthan control Jurkat cells (supplemental Fig. 2).

FIGURE 3. The NS5 proteins of Flaviviridae inhibit HIV but notmumps virus in Jurkat cells. HIV replication 4 days after infection wassignificantly reduced in Jurkat cell lines selected for expression of full-length GBV-C and HCV NS5A proteins, GBV-C NS5A aa 152–167(152–167), HCV DI, and NS5 proteins of DV serotype 2, WNV, andYFV NS5 proteins compared with three control cell lines: VC, GBV-CNS5A FS sequences, and GBV-C NS5A aa 152–167 in a Scr (A). HIVp24 Ag released into culture supernatants was below the limit of de-tection in all cell lines except the three control cell lines (VC, FS, Scr).The value shown (5 pg/ml) is one-half the lower limit of detection (10pg/ml) and the p value was �0.001 for all seven cell lines comparedwith each of the three control cell lines. The kinetics of HIV replicationin the cell lines is shown in B. HIV replication was significantly higherin the three control cell lines (VC, FS, Scr) compared with the NS5-expressing cell lines on days 3 and thereafter (B; p � 0.001 on each dayfor all). Mumps virus replication in these Jurkat cell lines was notreduced in any of the NS5A-expressing cell lines compared with the VCor FS, respectively, 4 days after infection (C).

FIGURE 4. Flaviviridae NS5 proteins decrease CD4 expression.Steady-state mRNA levels for CD4 were significantly reduced in Jurkatcells expressing the GBV-C, HCV, DV serotype 2, WNV, YFV 17Dvaccine strain NS5 proteins, and the GBV-C peptide (aa 152–167) com-pared with cells containing the VC (A; �, p � 0.03 for all). CD4 mRNAexpression was not altered in Jurkat cells containing the GBV-C NS5AFS or scrambled 152–167 GBV-C peptide sequence (Scr) comparedwith VC (A). Cell surface CD4 levels (MFI) were significantly reducedin the same cells in which mRNA levels were reduced (B; �, p � 0.001).

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Consistent with the reduction in CD4 mRNA levels, CD4 re-ceptor surface expression was significantly reduced on Jurkat celllines expressing full-length flavivirus NS5 proteins, the GBV-CNS5A aa 152–167 peptide, and HCV DI compared with the threecontrol cell lines (Fig. 4B). Total cellular CD4 was also reduced byNS5 protein expression as determined by immunoblot analysis,indicating that the down-regulation was not due to impaired re-ceptor cycling (data not shown). The finding that CD4 expressionwas reduced by GBV-C NS5A was unexpected, as we did notobserve this in a previous study (23). Upon reviewing the CD4expression data related to GBV-C NS5A expression, we confirmedthat the cells examined in that study had no change in CD4 ex-pression compared with control cells (Fig. 5A). However, thesecells had been passed in cell culture �40 times and a later passageof these same cells had been frozen in liquid nitrogen. The latepassage cells were thawed and re-evaluated on two separate occa-sions. These high passage cell lines no longer expressed NS5A

protein and in these the surface expression of CD4 was not dif-ferent from that of controls (Fig. 5A). To further assess this, Jurkatcells frozen at an earlier passage were also thawed and examinedfor CD4 expression by flow cytometry. These cells expressedGBV-C NS5A protein by immunoblot analysis, and CD4 surfaceexpression was significantly lower than that of control cells (Fig.5B; p � 0.001, t test comparing CD4 mean fluorescent intensity(MFI) on NS5A-expressing cells with VC cells). The reasons forloss of GBV-C NS5A expression in the one cell line are unclear,but subsequently we have examined cells maintained in culture for�50 passages and NS5A expression was maintained.

CD4 mRNA down-regulation did not occur as a result of globaltranscriptional inhibition, as the CD4 mRNA levels in the Jurkatcells expressing the GBV-C FS and scrambled NS5A peptide werenot significantly different from those of VC cells, and severalmRNAs highly expressed in Jurkat cells (CREBBP, CXCR3,MAPK8, TLR6) were the same in the NS5-expressing cells com-pared with the VC. In addition, NS5A expression did not decreasethe expression of all cell surface receptors. For example, CD81surface expression was not altered in Jurkat cells expressingGBV-C or HCV NS5A compared with the VC or FS (Fig. 6A anddata not shown). To determine whether NS5A down-regulatedCD4 in primary human CD4 cells, transient expression of GBV-Cand HCV NS5A demonstrated that CD4 surface expression wasreduced compared with the FS control-transfected cells (Fig. 6B).Similar to that observed in previous studies, CXCR4 was down-regulated by NS5 protein (supplemental Fig. 3A), and CCR5 wasnot detected on any of the Jurkat cell lines studied (data notshown).

FIGURE 5. GBV-C NS5A expression is required for CD4 modula-tion. A illustrates CD4 surface expression on Jurkat cells stably trans-fected with the VC (green), two late passes of a cell line (light blue andyellow) GBV-C NS5A, FS (black), and Jurkat cells without any trans-fection (purple). Cells previously shown to express GBV-C NS5A hadbeen passed �40 times before measuring CD4 expression and NS5Awas no longer detected in these cells. Mean CD4 fluorescent intensityis shown in parentheses (all five cell lines were within 8 of 174). Billustrates CD4 expression from low passage Jurkat cells (�15 pas-sages) expressing GBV-C NS5A (black) compared with Jurkat cellswith the VC, NS5A 1–152, 1–109, FS, or nontransfected Jurkat cells(dark blue, purple, light blue, green, and yellow, respectively). Jurkatcells incubated with isotype control are shown in red. CD4 surfaceexpression was significantly lower in the GBV-C NS5A-expressingcells compared with all of the control cell lines. Mean CD4 fluorescenceintensity is shown in parentheses.

FIGURE 6. GBV-C and HCV NS5A effects on cell surface receptorexpression. GBV-C and HCV NS5A proteins did not decrease the ex-pression of another cell surface receptor (CD81) on Jurkat cells com-pared with the VC (MFI in parentheses; A). However, CD4 expressionwas decreased in primary human CD4-enriched T cells transiently ex-pressing these NS5A proteins (B). Data represent CD4 MFI 48 h aftertransfection compared with VC cells (B; �, p � 0.001; †, p � 0.014).

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YFV inhibits HIV replication and down-regulates CD4expression

Jurkat and MT-2 (CD4� T cell) cell lines, primary humanCD4� T cells, and macrophages were infected with YFV withor without HIV superinfection. YFV replication in these cellswas confirmed by measuring YFV RNA and by the release ofinfectious particles into culture supernatants. HIV replicationwas completely blocked by YFV infection in both T cell lines

(Jurkat cell data shown in Fig. 7A). This was confirmed in MT-2cells by a block of HIV-induced syncytia induction (Fig. 7B)and by measuring HIV protein synthesis in cells with or withoutYFV infection. YFV infection was initiated 24 h before HIVinoculation (Fig. 7C).

YFV infection of primary human PBMCs also resulted in theinhibition of both CCR5- and CXCR4-tropic HIV isolates (Fig. 8,A and B). The timing of YFV and HIV infection was important,

FIGURE 7. YFV (vaccine strain) infection inhibits HIV replication in a human CD4� T cell line. YFV was applied to Jurkat cells 24 h before theaddition of HIV and HIV replication was assessed by HIV p24 Ag release into culture medium (A). The MOI of YFV is shown (mock infection � 0, YFVrange � 0.01–1.0 infectious units/cell). YFV infection (MOI, 1.0) of MT-2 cells 24 h before HIV infection blocked HIV-induced syncytia formation (B).NC, Negative control (YFV MOI, 0). HIV envelope protein expression (gp120, gp160) was reduced by YFV infection as measured by immunoblot analysis(C). MOI, 0.01, 0.1, and 1.

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since YFV infection did not inhibit HIV replication if HIVinfection was initiated 24 h before YFV, and more limited inhibi-tion occurred when YFV and HIV infection were initiated simul-taneously compared with when YFV preceded HIV infection by24 h (Fig. 8C). YFV infection did not inhibit mumps virus infec-tion, even when mumps virus inoculation was initiated 24 h afterYFV in either MT-2 cells or PBMCs (data not shown). YFV rep-lication was required for the effect, since UV-inactivated YFVfailed to inhibit HIV replication (data not shown).

YFV replication reduced steady-state CD4 mRNA levels (datanot shown), and CD4 surface expression on MT-2 cells (Fig. 9A)and in primary human CD4� T cells measured 4 days after infec-tion (Fig. 9B). CD4 modulation was specific for YFV infection, asmumps virus infection did not reduce CD4 expression on MT-2cells (Fig. 9C). YFV infection resulted in decreased surface ex-pression of CXCR4 on MT-2 cells, primary PHA-IL-2-stimulatedCD4� T cells, and MDMs compared with uninfected control cells(supplemental Fig. 3B). Mumps virus infection did not down-reg-ulate CXCR4 in MT-2 cells, and YFV infection decreased CCR5surface expression on PHA-IL2-stimulated primary CD4� T cells(supplemental Fig. 3B). YFV infection did not decrease cell via-bility of primary or transformed CD4 cells compared with unin-fected control cells. Unexpectedly, YFV infection induced prolif-eration of CD4 cells cultured without PHA and IL-2 and did not

FIGURE 8. YFV inhibits HIV in primary human T cells. YFV (vaccinestrain) was used to infect primary human PBMCs (MOI, 0 (mock infectioncontrol), 0.1, and 1) 24 h before infection with CCR5-tropic (R5; A) orCXCR4-tropic (X4; B) HIV isolates and HIV replication was measured byrelease of p24 Ag into culture supernatant. Inhibition of an X4 HIV isolateoccurred when YFV was applied 24 h before (YF24 � HIV) or simulta-neously with HIV (HIV � YF), but not with HIV alone (HIV) or if HIVinfection preceded YFV by 24 h (HIV24 � YF; C). YF, YFV infectiononly, no HIV infection.

FIGURE 9. YFV infection down-regulates CD4 expression on hu-man CD4� T cells. YFV infection decreased the CD4 surface density onMT-2 cells (A) and primary human peripheral blood T cells (B) 48 hafter infection in a dose-dependent manner. The MFI for each experi-ment is shown in parentheses. NC, Negative control. In contrast, mumpsvirus infection did not alter CD4 surface density 48 h after infection inMT-2 cells (C).

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promote CD4 cell death (supplemental Fig. 4). Thus, the effects ofYFV infection on HIV replication did not appear to be due tocellular toxicity.

Human MDMs express CD4 and CCR5 and are permissive forR5-tropic HIV infection (36). In addition, macrophages are re-ported to be permissive for YFV infection (37), as well as DV (38),WNV (39), and HCV (40). YFV infection of primary humanMDMs 24 h before HIV infection significantly and potently in-hibited HIV replication ( p � 0.001; Fig. 10A) and led to signifi-cant down-regulation of CD4 surface expression ( p � 0.021; Fig.10B), indicating that the effect on CD4 expression is not limited toT cells. YFV replication was documented in the MDMs by infec-tivity and viral RNA determination in culture supernatants (datanot shown). Similar to YFV effects on PBMCs, YFV infection ofmacrophages did not result in a reduction in viable MT-2 cellsor MDMs.

DiscussionExpression of the NS5 protein of five different members of theFlaviviridae decreased CD4 expression in a CD4� T cell line andin primary CD4� T cells with resultant inhibition of HIV replica-tion (Figs. 2–3). Although CD4� T cells are not the primary targetfor DV, WNV, YFV, or HCV, evidence suggests that all of theseviruses infect these cells (14–16, 41–46). In contrast, human T andB cells are permissive for GBV-C and appear to be the primary siteof viral replication (16). The mechanism of flavivirus NS5 protein-

mediated CD4 regulation appears to be at the transcriptional level,as NS5 protein expression in T cells reduces steady-state CD4mRNA levels compared with the VC cells. CD4 levels were alsoreduced during YFV infection in lymphocytes, even when infec-tion was performed at a low multiplicity of infection (MOI).Monocytes and MDMs are thought to be a primary target of YFVinfection, and these cells are permissive for YFV (47), DV (38),WNV (39), and HCV (40) in vitro. We found that YFV infectionled to decreased CD4 expression and inhibited HIV replication inmacrophages as well as in CD4� T cells (Fig. 10). Since YFV hada profound effect on HIV replication even at a low MOI, it sug-gests that the effects of YFV infection extend beyond CD4 mod-ulation and studies are underway to further characterize the kinet-ics and extent of these interactions. As predicted, down-regulationof CD4 neither inhibited mumps virus infection, nor did mumpsvirus infection modulate CD4 expression (Figs. 3C and 9C).

CD4 is a type I transmembrane glycoprotein expressed on asubset of T lymphocytes and on cells of the macrophage/monocytelineage, and it is the binding receptor for the HIV envelope proteingp120 (48–50). CD4-bearing T cells recognize Ags presented byMHC class II molecules on APCs, triggering immune responsesincluding the activation of B cells and recruitment of phagocytes(48). CD4 also serves as a coreceptor for the TCR, amplifying theTCR signal by recruiting the tyrosine kinase lck, which is an es-sential step in the signaling cascades of activated T cells and aprerequisite for T cell proliferation (48). Thus, in addition to de-creasing HIV binding and entry, down-regulation of CD4 will im-pair helper T cell function and potentially delay or prevent thedevelopment of adaptive immune responses. These effects poten-tially contribute to persistent infection for HCV and GBV-C.

HIV replication is highly dependent upon T cell proliferationand activation and CD4 activation is a key factor in HIV patho-genesis (reviewed in Refs. 51, 52). Down-regulation of CD4 byflaviviruses may also attenuate HIV pathogenesis by decreasingeither the TCR signal required for activation or by lowering thethreshold of the TCR signal. Consistent with the latter hypothesis,a previous study found decreased surface expression of cell surfaceactivation markers (CD38 and CCR5) on CD4 and CD8 T cells inGBV-C and HIV-coinfected individuals compared with people in-fected with HIV alone (21), and GBV-C infected individuals haveblunted CD4 proliferation in response to IL-2 therapy (53).

In addition to flaviviruses, infection with HIV (54), human her-pesvirus 7 (55), and a rabbit pox virus (56) decreases CD4 surfaceexpression on lymphocytes. Although the mechanisms of down-regulation are incompletely understood, specific HIV proteins re-duce CD4 expression (Nef, vpu, gp120) and similar to our findingswith flavivirus NS5 protein, HIV gp120 reduces steady-state CD4mRNA levels (54). CD4 down-modulation has not previously beendescribed among the Flaviviridae, yet the NS5 protein of all fivehuman flaviviruses studied significantly decreased CD4 mRNAand protein expression. The finding that flaviviruses modulate CD4expression suggests that this may be a mechanism by which theseviruses evade host immune recognition. In addition, this effect maycontribute to viral persistence for HCV and GBV-C.

CD4 modulation appeared to be the most significant for GBV-CNS5A; however, this may reflect differences in NS5A expressionlevels, as quantitative protein expression levels cannot be directlycompared for the different constructs. Nevertheless, CD4 down-regulation required only 16 aa within the GBV-C NS5A protein(Fig. 4) and previous studies found that incubation of cells with theGBV-C peptide inhibits HIV replication (25). Since there is �30%amino acid sequence identity between the GBV-C NS5A peptideand the HCV DI or DV, WNV, or YFV NS5 sequences, it appearsthat the HIV CD4 modulatory effect is mediated by a structural

FIGURE 10. YFV inhibits HIV and down-regulates CD4 expression inhuman MDMs. YFV infection of primary human MDMs was initiated 2days after differentiation (MOI, 0 (mock control), 0.1, and 1) and HIVinfection was initiated 1 day later. HIV release into culture supernatants isshown in A. CD4 expression on MDMs was determined 7 days after YFVor mock-control infection by flow cytometry (B). The MFI for each ex-periment is shown in parentheses. Background fluorescence of YFV-in-fected cells using an isotype control Ab is shown.

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element that is conserved among these viruses. Studies are under-way to further characterize the peptide domains that down-regulateCD4 expression in these viruses and to identify the intracellularbinding partners of these proteins. Generation of structural mimicsof these peptides may lead to novel, cellular-based therapeuticsdesigned to modulate CD4 expression, which may have a role in avariety of infectious and immune-based diseases.

AcknowledgmentsWe thank Drs. Steven Polyak (University of Washington, Seattle, WA),Alan Rothman (University of Massachusetts, Worcester, MA), and AndrewDayton (U.S. Food and Drug Administration, Bethesda, MD) and CharlesRice (The Rockefeller University, New York, NY) and Catherine Patterson(Case Western University, Cleveland, OH), respectively, for providingcDNA containing HCV, DV (serotype 2), YFV (17D), and WNV NS5coding sequences. We also thank Dr. Peter Simmonds (University of Ed-inburgh, Edinburgh, U.K.) for advice related to sequence alignments andDrs. William Nauseef and Stanley Perlman (University of Iowa, Iowa City,IA) for critical review of this manuscript. The University of Iowa FlowCytometry and DNA Core Facilities were used for flow cytometry andDNA sequence analyses.

DisclosuresDrs. J. Xiang and J. T. Stapleton hold a patent (U.S. Patent No. 7,291,723)on the use of GBV-C as a therapeutic modality for HIV and Drs. J. Xiang,J. M. McLinden, and J. T. Stapleton have a patent application pendingrelated to the use of flavivirus NS5 protein as an anti-HIV therapy.

References1. Calisher, C. H., and E. A. Gould. 2003. Taxonomy of the virus family Flaviviri-

dae. Adv. Virus Res. 59: 1–19.2. Lindenbach, B. D., and C. M. Rice. 2003. Molecular biology of flaviviruses. Adv.

Virus Res. 59: 23–61.3. Stapleton, J. T. 2003. GB virus type C/hepatitis G virus. Semin. Liver Dis. 23:

137–148.4. Moradpour, D., F. Penin, and C. M. Rice. 2007. Replication of hepatitis C virus.

Nat. Rev. Mircrobiol. 5: 453–463.5. Macdonald, A., and M. Harris. 2004. Hepatitis C virus NS5A: tales of a promis-

cuous protein. J. Gen. Virol. 85: 2485–2502.6. Alter, H. J. 1997. G-pers creepers, where’d you get those papers? A reassessment

of the literature on the hepatitis G virus. Transfusion 37: 569–572.7. Heringlake, S., J. Ockenga, H. L. Tillmann, C. Trautwein, D. Meissner, M. Stoll,

J. Hunt, C. Jou, N. Solomon, R. E. Schmidt, and M. P. Manns. 1998. GB VirusC/hepatitis G virus infection: a favorable prognostic factor in human immuno-deficiency virus-infected patients? J. Infect. Dis. 177: 1723–1726.

8. Toyoda, H., Y. Fukuda, T. Hayakawa, J. Takamatsu, and H. Saito. 1998. Effectof GB virus C/hepatitis G virus coinfection on the course of HIV infection inhemophilia patients in Japan. J. Acquir. Immune Defic. Syndr. 17: 209–213.

9. Tillmann, H. L., H. Heiken, A. Knapik-Botor, S. Heringlake, J. Ockenga,J. C. Wilber, B. Goergen, J. Detmer, M. P. Manns, M. Stoll, R. E. Schmidt, andM. P. Manns. 2001. Infection with GB virus C and reduced mortality amongHIV-infected patients. N. Engl. J. Med. 345: 715–724.

10. Xiang, J., S. Wunschmann, D. J. Diekema, D. Klinzman, K. D. Patrick,S. L. George, and J. T. Stapleton. 2001. Effect of coinfection with GB Virus C(hepatitis G virus) on survival among patients with HIV infection. N. Engl.J. Med. 345: 707–714.

11. Williams, C. F., D. Klinzman, T. E. Yamashita, J. Xiang, P. M. Polgreen,C. Rinaldo, C. Liu, J. Phair, J. B. Margolick, D. Zdunek, G. Hess, andJ. T. Stapleton. 2004. Persistent GB virus C infection and survival in HIV-in-fected men. N. Engl. J. Med. 350: 981–990.

12. Van der Bij, A. K., N. Kloosterboer, M. Prins, B. Boeser-Nunnink, R. B. Geskus,J. M. A. Lange, R. A. Coutinho, and H. Schuitemaker. 2005. GB Virus C coin-fection and HIV-1 disease progression: The Amsterdam cohort study. J. Infect.Dis. 191: 678–685.

13. Zhang, W., K. Chaloner, H. L. Tillmann, C. F. Williams, and J. T. Stapleton.2006. Effect of early and late GBV-C viremia on survival of HIV infected indi-viduals: A meta-analysis. HIV Med. 7: 173–180.

14. Fogeda, M., S. Navas, J. Martin, M. Casqueiro, E. Rodriguez, C. Arocena, andV. Carreno. 1999. In vitro infection of human peripheral blood mononuclear cellsby GB virus C/hepatitis G virus. J. Virol. 73: 4052–4061.

15. Xiang, J., S. Wunschmann, W. N. Schmidt, J. Shao, and J. T. Stapleton. 2000.Full-length GB virus C (hepatitis G virus) RNA transcripts are infectious inprimary CD4-positive T cells. J. Virol. 74: 9125–9133.

16. George, S. L., D. Varmaz, and J. T. Stapleton. 2006. GB virus C replicates inprimary T and B lymphocytes. J. Infect. Dis. 193: 451–454.

17. Xiang, J., S. L. George, S. Wunschmann, Q. Chang, D. Klinzman, andJ. T. Stapleton. 2004. Inhibition of HIV-1 replication by GB virus C infectionthrough increases in RANTES, MIP-1�, MIP-1�, and SDF-1. Lancet 363:2040–2046.

18. Jung, S., O. Knauer, N. Donhauser, M. Eichenmuller, M. Helm, B. Fleckenstein,and H. Reil. 2005. Inhibition of HIV strains by GB virus C in cell culture can bemediated by CD4 and CD8 T-lymphocyte derived soluble factors. AIDS 19:1267–1272.

19. Xiang, J., M. A. Sathar, J. H. McLinden, D. Klinzman, Q. Chang, andJ. T. Stapleton. 2005. South African GB virus C isolates: interactions betweengenotypes 1 and 5 GBV-C isolates and the human immunodeficiency virus. J. In-fect. Dis. 192: 2147–2151.

20. Watt, G., P. Kantipong, and K. Jongsakul. 2003. Decrease in human immuno-deficiency virus type 1 load during acute dengue fever. Clin. Infect. Dis. 36:1067–1069.

21. Nattermann, J., H. D. Nischalke, B. Kupfer, J. Rockstroh, L. Hess, T. Sauerbruch,and U. Spengler. 2003. Regulation of CC chemokine receptor 5 in hepatitis Gvirus infection. AIDS 17: 1457–1462.

22. Jung, S., M. Eichenmueller, N. Donhauser, F. Neipel, A. M. Engel, G. Hess,B. Fleckenstein, and H. Reil. 2007. HIV entry inhibition by the envelope 2 gly-coprotein of GB virus C. AIDS 21: 645–647.

23. Xiang, J., J. H. McLinden, Q. Chang, T. M. Kaufman, and J. T. Stapleton. 2006.An 85 amino acid segment of the GB virus type C NS5A phosphoprotein inhibitsHIV-1 replication in CD4� Jurkat T-cells. Proc. Natl. Acad. Sci. USA 103:15570–15575.

24. Chang, Q., J. H. McLinden, J. T. Stapleton, M. A. Sathar, and J. Xiang. 2007. GBvirus C NS5A protein from genotypes 1, 2, 3, and 5 and a 30 amino acid NS5Afragment expression inhibit HIV-1 replication in a CD4� T-lymphocytes. J. Gen.Virol. 88: 3341–3346.

25. Xiang, J., J. H. McLinden, Q. Chang, E. L. Jordan, and J. T. Stapleton. 2008.Characterization of a peptide domain within the GB virus C NS5A phosphopro-tein that inhibits HIV replication. PLoS One 3: e2580.

26. McLinden, J. H., J. T. Stapleton, Q. Chang, and J. Xiang. 2008. Expression of thedengue virus type 2 NS5 protein in a CD4� T cell line inhibits HIV replication.J. Infect. Dis. 198: 860–863.

27. Xiang, J., C. Martinez-Smith, M. Jr. Gale, D. R. LaBrecque, W. N. Schmidt, andJ. T. Stapleton. 2005. GB virus C NS5A sequence polymorphisms: associationwith interferon susceptibility and inhibition of PKR-mediated eIF-2� phosphor-ylation. J. Interferon Cytokine Res. 25: 261–270.

28. Schelsinger, L. S., S. R. Hurr, and T. M. Kaufman. 1994. Binding of the terminalmannosyl units of lipoarabinomannan from a virulent strain of Mycobacteriumtuberculosis to human macrophages. J. Immunol. 152: 4070–4079.

29. Sakata, H., M. Hishiyama, and A. Sugiura. 1984. Enzyme-linked immunosorbentassay compared with neutralization tests for evaluation of live mumps vaccines.J. Clin. Microbiol. 19: 21–25.

30. Litwin, J. 1964. Growth of 17D yellow fever virus and factors influencing itstransmission within cell cultures in vitro. Acta Pathol. Microbiol. Scand. 61:605–618.

31. Cook, R. T., J. T. Stapleton, D. Klinzman, and Z. K. Ballas. 1997. Effect of asingle ethanol ingestion on lymphocyte subsets and in vitro HIV replication.J. Invest. Med. 45: 265–271.

32. Kumar, S., D. Tan, and M. Nei. 2004. MEGA3: Integrated software for molecularevolutionary genetics analysis and sequence alignment. Briefings Bioinform. 5:150–163.

33. Koonin, E. V. 1991. The phylogeny of RNA-dependent RNA polymerases ofpositive-strand RNA viruses. J. Gen. Virol. 72: 2197–2206.

34. Yu, Y., A. J. Annala, J. R. Barrios, T. Toyokuni, N. Satyamurthy, M. Namavari,S. R. Cherry, M. E. Phelps, H. R. Herschman, and S. S. Gambhir. 2008. Quan-tification of target gene expression by imaging reporter gene expression in livinganimals. Nat. Med. 6: 933–937.

35. Tellinghuisen, T. L., J. Marcotrigiano, A. E. Gorbalenya, and C. M. Rice. 2004.The NS5A protein of hepatitis C virus is a zinc metalloprotein. J. Biol. Chem.279: 48576–48687.

36. Gartner, S., P. Markovits, D. M. Markovits, M. H. Kaplan, R. C. Gallo, andM. Popovic. 1986. The role of mononuclear phagocytes in HTLV-III/LAV in-fection. Science 233: 215–219.

37. Berges, B. K., W. H. Wheat, B. E. Palmer, E. Connick, and R. Akkina. 2006.HIV-1 infection and CD4 T cell depletion in the humanized Rag2/�c/ (RAG-hu)mouse model. Retrovirology 3: 76.

38. Blackley, S., Z. Kou, H. Chen, M. Quinn, R. C. Rose, J. J. Schlesinger,M. Coppage, and X. Jin. 2007. Primary human splenic macrophages, but not T orB cells, are the principal target cells for dengue virus infection in vitro. J. Virol.81: 13325–13334.

39. Rios, M., M. J. Zhang, A. Grinev, K. Srinivasan, D. Daniel, O. Wood,I. K. Hewlett, and A. I. Dayton. 2006. Monocytes-macrophages are a potentialtarget in human infection with West Nile virus through blood transfusion. Trans-fusion 46: 659–667.

40. Laskus, T., M. Radkowski, J. Jablonska, K. Kibler, J. Wilkinson, D. Adair, andJ. Rakela. 2004. Human immunodeficiency virus facilitates infection/replicationof hepatitis C virus in native human macrophages. Blood 103: 3854–3859.

41. Mentor, N. A., and I. Kurane. 1997. Dengue virus infection of human T lym-phocytes. Acta Virol. 41: 175–176.

42. Denman, A. M., B. Rager-Zisman, T. C. Merigan, and D. A. Tyrell. 1974. Rep-lication or inactivation of different viruses by human lymphocyte preparations.Infect. Immun. 9: 373–376.

43. Garcia-Tapia, D., C. M. Loiacono, and S. B. Kleiboeker. 2006. Replication ofWest Nile virus in equine peripheral blood mononuclear cells. Vet. Immunol.Immunopathol. 110: 229–244.

44. Laskus, T., M. Radkowski, L. F. Wang, S. J. Jang, H. Vargas, and J. Rakela.1998. Hepatitis C virus quasispecies in patients infected with HIV-1: correlationwith extrahepatic replication. Virology 248: 164–171.

9The Journal of Immunology

by guest on April 10, 2019

http://ww

w.jim

munol.org/

Dow

nloaded from

45. Kondo, Y., V. M. Sung, K. Machida, M. Liu, and M. M. Lai. 2007. Hepatitis Cvirus infects T cells and affects interferon-� signaling in T cell lines. Virology361: 161–173.

46. Lin, Y. W., K. J. Wang, H. Y. Lei, Y. S. Lin, T. M. Yeh, H. S. Liu, C. C. Liu,and S. H. Chen. 2002. Virus replication and cytokine production in dengue virus-infected human B lymphocytes. J. Virol. 76: 12242–12249.

47. Schlesinger, J. J., and M. W. Brandriss. 1981. Antibody-mediated infection ofmacrophages and macrophage-like cell lines with 17D-yellow fever virus.J. Med. Virol. 8: 103–117.

48. Miceli, M. C., and J. R. Parnes. 1993. Role of CD4 and CD8 in T cell activationand differentiation. Adv. Immunol. 53: 59–122.

49. Dalgleish, A. G., P. C. L. Beverley, P. R. Clapham, D. H. Crawford,M. F. Greaves, and R. A. Weiss. 1984. The CD4 (T4) antigen is an essentialcomponent of the receptor for human retrovirus LAV. Nature 312: 763–767.

50. Bowers, K., C. Pitcher, and M. Marsh. 1997. CD4: a coreceptor in the immuneresponse and HIV infection. Int. J. Biochem. Cell Biol. 29: 871–875.

51. Douek, D. C. 2003. Disrupting T-cell homeostasis: how HIV-1 infection causesdisease. AIDS Rev. 5: 172–177.

52. Douek, D. C. 2007. HIV disease progression: immune activation, microbes, anda leaky gut. Top. HIV Med. 15: 114–117.

53. Stapleton, J. T., K. Chaloner, J. Zhang, D. Klinzman, I. E. Souza, J. Xiang,A. Landay, J. Fahey, R. Pollard, and R. Mitsuyasu. 2008. GB virus C viremia isassociated with reduced CD4 expansion following interleukin 2 therapy in HIV-infected people receiving HAART. AIDS 23: 605–610.

54. Hoxie, J. A., J. D. Alpers, J. L. Rackowski, K. Huebner, B. S. Haggarty,A. J. Cedarbaum, and J. C. Reed. 1986. Alterations in T4 (CD4) protein andmRNA synthesis in cells infected with HIV. Science 234: 1123–1127.

55. Lisco, A., J. C. Grivel, A. Biancotto, C. Vanpoulle, F. Origgi, M. S. Malnati,D. Schols, P. Lusso, and L. B. Margolis. 2007. Viral interactions in human lym-phoid tissue: human herpesvirus 7 suppresses the replication of CCR5-tropichuman immunodeficiency virus type 1 via CD4 modulation. J. Virol. 81:708–717.

56. Barry, M., S. F. Lee, L. Boshkov, and G. McFadden. 1995. Myxoma virus in-duces extensive CD4 downregulation and dissociation of p56lck in infected rabbitCD4� T lymphocytes. Virology 69: 5243–5251.

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