Lack of association of HIV-1 biological or molecular properties with neurotropism for brain cells

Journal of Molecular Neuroscience 131 Volume 29, 2006

*Author to whom all correspondence and reprint requests should be addressed. E-mail: mmunoz@ cbm.uam.es.

Introduction

Brain HIV-1 infection might result in a syndromeof profound cognitive, behavioral, and motor impair-ment known as AIDS dementia complex (ADC) inadults and HIV-related encephalopathy (EP) in child-ren (Glass and Johnson, 1996; Power and Johnson,2001). Although the advent of highly active antiretro-viral therapy (HAART) has reduced the incidence ofADC by approx. 50% (Maschke et al., 2000), neuro-logical disorders continue to be a major cause of mor-bidity and mortality in AIDS. On the other hand, asHIV-1 infection is now becoming a chronic illness,infected patients live longer and are more prone tosuffer from neurological complications (McArthur

Journal of Molecular NeuroscienceCopyright © 2006 Humana Press Inc.All rights of any nature whatsoever are reserved.ISSN0895-8696/06/29:131–144/$30.00JMN (Online)ISSN 1559-1166DOI 10.1385/JMN/29:02:131

ORIGINAL ARTICLE

et al., 2003). Moreover, central nervous system (CNS)disorders are more frequent and severe in infectedchildren, suggesting that a developing CNS could bemore vulnerable to HIV-1 (Epstein et al., 1986). In thosechildren, neurological dysfunction involves develop-mental problems and failure to acquire motor or cog-nitive skills. HIV-1 enters the CNS early after onset ofHIV-1 infection, as suggested by the detection of virusand viral antigens in the cerebrospinal fluid (CSF) andbrain tissue of patients even without symptoms(Lyman et al., 1990; Simpson, 1999).

The role of strain variability in the pathogenesis ofHIV-1 dementia is unknown. The abundance of virusin the brain and the biological phenotype have beensuggested as important determinants in neurological

Lack of Association of HIV-1 Biological or MolecularProperties With Neurotropism for Brain Cells

Susana Álvarez, José Luis Jiménez, Ma Jesús Serramía, Milagros González,Carmen Cantó-Nogués, and Ma Angeles Muñoz-Fernández*

Molecular Immunobiology Laboratory, Gregorio Marañón University General Hospital, 28807Madrid, Spain

Received December 11, 2005; Accepted February 3, 2006

Abstract

Despite HAART, a significant number of HIV-1-infected patients develop neurological complications. How-ever, the presence of specific neurotropic HIV-1 strains, the extent of viral replication in the brain, and the type ofcells infected remain controversial issues. To address this controversy we have analyzed different V3 loop sequencesof viral isolates from four vertically HIV-1-infected children who developed HIV-1-related encephalopathy. More-over, we have determined that some biological and molecular properties of HIV-1 might contribute to AIDS neuro-logical dysfunctions. We detected very different HIV-1 isolates (X4 and R5) in the brain despite no great differencesin clinical, pathological, or immunological parameters. In vitro, no differences in replicative competence in glialor neuroblastoma cells were observed between virus isolated from the blood of children with or without clinicalneurological symptoms. The expression of both CXCR4 and CCR5 RNAs was observed in the brain independentlyof HIV-1 infection and viral strain predominant in this location. Our results failed to show a particular phenotypicproperty of the HIV-1 virus that might explain its neurovirulence and/or neurotropism.

DOI 10.1385/JMN/29:02:131

Index Entries: Neurotropism; brain cells; HIV-related encephalopathy; children.

JMN_29_2_JMN05_0068_Munoz 6/29/06 1:16 PM Page 131

132 Álvarez et al.

Journal of Molecular Neuroscience Volume 29, 2006

disease (Power et al., 2002). Despite frequent detec-tion of viral genome and proteins in the brains ofAIDS patients, their abundance is not unequivocallyrelated to neuropathology (Lyman et al., 1990; Simp-son, 1999). In addition, it has been described that themajority of brain isolates use CCR5 as coreceptor(Power et al., 2002), as well as the presence of HIV-1variants with increased CCR5 affinity in the brainof AIDS patients with ADC (Gorry et al., 2002). Incontrast, reports indicate that neurological disorderscorrelate with viral load in CSF but not with viralphenotype (Di Stefano et al., 1998).

Cells mainly infected by HIV-1 in the brain areblood-derived macrophages and resident microglia,whereas infected astrocytes and neurons have beenobserved rarely in vivo (Wiley et al., 1986; Saito etal., 1994; Bagasra et al., 1996; Balluz et al., 1996; Anet al., 1999). Nonetheless, HIV-1-infected neuronshave been described occasionally in the brains ofAIDS patients (Nuovo et al., 1994), and recently wehave clearly shown infected neurons in the brainsof vertically infected children with EP (Canto-Nogues et al., 2005). Moreover, primary humanneuroblast cells, as well as neuronal cell lines, aresusceptible to HIV-1 infection in vitro (Ensoli et al.,1995; Obregon et al., 1999; Alvarez Losada et al.,2002).

Different biological properties of HIV-1 isolates,such as syncytium-inducing (SI) capacity, cellular tro-pism, X4/R5 (T lymphocyte-adapted strain, HIV-1NL4.3/monocyte/macrophage-adapted strain,HIV-1Bal, respectively) coreceptor usage, and replica-tive rate (Koot et al., 1992) might affect their patho-genicity. HIV-1 primary infection is characterized byuse of CCR5 as a coreceptor for entry and non-SI(NSI) phenotype (Simmons et al., 1997). SI/X4 vari-ants that use CXCR4 as a coreceptor appear inadvanced stages of HIV-1 infection (Fauci, 1996) andare associated with poor prognosis (Galan et al., 2004),whereas dual tropic viruses (R5/X4) can use bothreceptors. HIV-1 disease progression is generallyassociated with a general broadening of virus tro-pism by expansion of coreceptor usage and the emer-gence of X4 or R5/X4 variants (Connor et al., 1997).

Several chemokine receptors that mediate HIV-1entry are expressed in the CNS (reviewed in Hes-selgesser and Horuk, 1999). Thus, it has been demonstrated that neurons and glial cells in thehippocampus and other regions of the brain expressCCR2, CCR3, and CXCR4 but not CCR5, whereasmicroglial cells express low levels of CD4 (Dick etal., 1997), CXCR4, CCR5, and CCR3 (Lavi et al., 1998;

Albright et al., 1999). It is assumed that HIV-1 in thebrain uses CCR5 as the principal coreceptor for infect-ing target cells (van der Meer et al., 2000), althoughR5, X4, and R5/X4 isolates all have been demon-strated in the CNS of HIV-1-infected individuals(Gorry et al., 2001).

To better understand whether some biological andmolecular properties of HIV-1 isolates contribute toneuropathology and neurotropism, we have char-acterized viruses isolated from brain tissue, blood,and plasma samples of four HIV-infected childrenwith neurological disorders. Moreover, we havetested how the different isolates varied in their abi-lity to infect in vitro glial or neuroblastoma (NB)cells. Finally, we have investigated in vivo the exis-tence of differential expression of CCR5 and CXCR4chemokine receptors in the brains of infected child-ren. Our results failed to show a particular pheno-typic property of the virus that might explain theneurovirulence and/or neurotropism.

Materials and MethodsCell Lines

The human NB cell line SK-N-MC and the glioblas-toma human cell line U-87 were grown in RPMI 1640(Biochrom KG Seromed, Berlin, Germany), supple-mented with 10% heat-inactivated fetal calf serum,1% penicillin/streptomycin, and 2 mM L-glutamine(ICN Pharmaceuticals, Costa Mesa, CA) at 37°C ina humidified atmosphere of 5% CO2. Humanperipheral blood mononuclear cells (PBMCs) wereisolated from blood by Ficoll-Hypaque density gra-dient centrifugation (Pharmacia, Uppsala, Sweden)(Muñoz-Fernández et al., 1996). The MT-2 (T-cellleukemia) cells were grown routinely under the sameconditions as the NB cells. The parental HOS-CD4line is a human osteogenic sarcoma cell line stablyexpressing high levels of CD4.

Assay for Coreceptor UsageHOS-CD4 cells transfected with genes encoding

either CCR5 or CXCR4 in addition to CD4 (cell linesHOS-CD4-CCR5 and HOS-CD4-CXCR4, respec-tively) were used as indicator lines for coreceptorusage (Deng et al., 1996). To determine coreceptorusage, HOS-CD4-CCR5 and HOS-CD4-CXCR4 cellswere seeded into 12-well plates and inoculated witha standard amount of titered virus after 24 h. HIV-1NL4.3 and HIV-1Bal were inoculated in parallel as X4-and R5-specific positive control viruses, respectively,and uninfected cells were used as negative control.


Lack of Association of HIV-1 133


To eliminate any artifacts resulting from infectiondue to low levels of endogenous coreceptor expres-sion, parental HOS-CD4 cells were inoculated withprimary and control virus. Cell supernatants wereharvested at day 7 after infection to monitor p24 viralcore antigen production using an antigen capture(INNOTEST HIV-1 antigen MAb, Innogenetics N.V., Zwijndrecht, Belgium).

Viral StrainsPrimary HIV-1 strains HIV-1LRH (SI/X4), HIV-1CGP

(SI/X4), HIV-1CSB (NSI/R5), HIV-1ASS (NSI/R5), HIV-1ADR (SI/X4), and HIV-1ILG (NSI/R5) were isolatedfrom HIV-1-infected children (Muñoz-Fernández et al., 1996) in different clinical courses of infectionwith or without neurological symptoms. The T lymphocyte–adapted strain, HIV-1NL4.3 (X4), and themonocyte/macrophage-adapted strain, HIV-1Bal(R5), were also used as controls. Because we havedemonstrated that after HAART there is a changefrom X4 to R5 phenotype in viral isolates (Galan etal., 2004), the capacity of infection of two HIV-1 iso-lates from one child before and after HAART in NBand glial cells was also studied. These two isolatesare HIV-1ESR-1 (SI/X4) before treatment and HIV-1ESR-2(NSI/R5) after HAART. The phenotype of those HIV-1 strains was determined by sequencing, andclassified according to their SI capacity by coculti-vation of patient PBMCs and MT-2 and coreceptoruse of the HOS-CD4 system (Muñoz-Fernández et al., 1996).

Virus stocks were prepared by expanding viralisolates in PBMCs and were titrated using the end-point dilution method. The virus stocks gave 104–105

TCID50 when PBMCs were inoculated on micro-dilution plates and read 10 d after with Agp24 assay(INNOTEST HIV antigen MAb) according to themanufacturer’s instructions. The sensitivity of thisassay was at least 10 pg/mL.

HIV-1 Infection of NB and Glioblastoma CellsNB, as well as U-87, cells were exposed to differ-

ent HIV-1 isolates, described above, at 0.5 m.o.i for2 h at 37°C. Virus titers were evaluated in the lastwashing buffer (time 0). Cell supernatants were har-vested every 3 and 6 d postinfection to monitor p24viral core antigen production using INNOTEST HIV-1antigen MAb. The sensitivity of the assay was at least10 pg/mL. Viral infection was also detected by PCRwith a set of nested primers specific for a region ofthe pol gene (JA17–JA20), described previously(Muñoz-Fernández et al., 1996). In parallel, DNase

pretreatment (92 U/µL) of viral stocks and cell lysateswas performed for 1 h at 37°C. Only samples thatgave positive signals with primers specific for thehuman housekeeping GADPH gene were thereforesuitable and used for amplification. The PCR prod-uct was analyzed by electrophoresis on 1.5% agarosegels stained with ethidium bromide.

Expression of CXCR4 and CCR5 in NB and U-87Cell LinesThe expression of CCR5 and CXCR4 on NB cell

surfaces was evaluated by reverse transcriptase poly-merase chain reaction (RT-PCR) and direct immuno-fluorescence assay.

The mRNA from 105 cells was isolated usingoligo(dT)-coated magnetic beads and subsequentreverse transcription (PolyAtract Series 9600, mRNAisolation, and cDNA synthesis system [Promega,Madison, WI]), according to the manufacturer’sinstructions. The PCR was carried out in an auto-matic thermal cycler (Perkin-Elmer GeneAmp PCRsystem 9600).

SK-N-MC and U-87 cells were adhered to coverslides for 2 h at 37°C and incubated with anti-CXCR4-FITC or anti-CCR5-PE MAbs (Santa Cruz, Heidel-berg, Germany [1:500]) for 30 min at 20°C. Slideswere mounted with DAKO Ultramount.

DNA Sequence Analysis of V3 Loop Region of gp120 Viral ProteinDNA and RNA were isolated from PBMCs, for-

malin-fixed embedded brain specimens, and plasmasamples. DNA from PBMCs was extracted usingHigh Pure PCR Template Preparation Kit (RocheDiagnostics, Indianapolis, IN) and from paraffinsamples using NucleoSpin Tissue Kit (BD Bio-sciences, Belgium) according to the manufacturer’sinstructions. HIV-RNA was obtained by isolation ofplasma by ultracentrifugation, viral lysis, and latertreatment with isopropanol and ethanol.

The env (envelope) proviral genome was amplifiedby a nested PCR from total cellular DNA. Oligonu-cleotide primers A, 5′-TACAATGTACACATGGAATT-3′, and D, 5′-ATTACAGTAGAAAAATTCC-3′,were designed to amplify a 425-bp region, and thefollowing amplification was made with primers B, 5′-TGGCAGTCTAGCAGAAGAAG-3′, and C, 5′-TCTGGGTCCCCTCCTGAGGA-3′; 15 µL of extractedDNA was added to a PCR Master Mix (PromegaM7502), following conditions recommended by themanufacturer. The reaction mix was subjected to 35cycles of amplification. Each cycle consisted of three


134 Álvarez et al.


steps after denaturation (94°C, 2 min): denaturation(94°C, 45 s), annealing (primers A/D, 52°C, 45 s;primers B/C, 65°C, 45 sc), and extension (72°C, 1 min), with a final extension at 72°C for 7 min.

PCR samples were purified with High Pure PCRproduct Purification kit (Roche Diagnostics GMBH,Germany) and sequenced with an ABI Prism 3100analyzer and Big Dye v3.1 chemistry (AppliedBiosystems). Several individual sequences weredirectly amplified from patients’ tissue to give theconsensus sequence.

All sequences were analyzed and aligned usingpackaged software (DNASTAR, Madison WI). Con-sensus sequences at both nucleotide and proteinlevels were generated for each individual proviralparticle and were compared to determine the V3divergence between samples for each patient., Wealso made a homology search by BLASTn(http://www.ncbi.nlm.nih.gov/BLAST/) throughGenBank data base, and we followed the qualitycontrol recommendation for HIV-1 sequencing(http://hiv-web.lanl.gov [Los Alamos NationalLaboratory]). All V3 sequences were studied toidentify ambiguous regions that are produced whena mixture of two sequences is detected. We anno-tated the net charge of each protein sequence at pH7.40 (http://bioweb.pasteur.fr/seqanal/interfaces/iep.html).

In Situ HybridizationFormalin-fixed, paraffin-embedded brain speci-

mens from HIV-infected children, as well as one addi-tional control child (HIV-1 seronegative), who diedfrom congenital cardiopathy, were taken from thefiles of the Department of Pathology in the GregorioMarañón Hospital in Madrid, Spain.

In situ hybridization (ISH) of sections from paraffin-embedded tissues was performed with minor mod-ifications, as described previously (Mueller et al.,1988). Sections of 5 µM from wax-embedded brainblocks were mounted onto capillary gap slides of 75 µM coated with an adhesive (Fisher HealthCare).Prior to hybridization, sections were dewaxed, rehy-drated, and permeabilized with proteinase K beforerefixing and dehydration in ethanol.

Tissues were then hybridized with the indicatedlabeled sense or antisense probes overnight at 50°Cin a moist chamber using asymmetric nested PCR,as described previously (Canto-Nogues et al., 2001).Hybridization with labeled probes was performedusing the MicroProbe Staining Station (Fisher Sci-entific, Pittsburgh, PA). The presence of the probes

were visualized using an alkaline phosphatase–conjugated sheep anti-digoxigenin antibody (RocheMolecular Biochemicals) and NBT/BCIP solution(GIBCO-BRL, Life Technologies) as chromogenicsubstrate following conditions recommended by themanufacturers.

ResultsCharacterization of HIV-1 Isolates

in Different Locations from HIV-InfectedChildrenHIV-1 isolates present in the brain, PBMCs, and

plasma from the four HIV-infected children withneurological disorders were characterized bysequencing to identify any changes in the V3 regionof the HIV-1 envelope glycoprotein gp120 thatcould explain the neurotropism/neurovirulence.Thus, the proviral genome was isolated of totalpurified cellular DNA, and envelope sequenceswere amplified by PCR. Next, the variable V3 regionof viruses from the different locations of the chil-dren was sequenced and compared (Table 1). Wehave found that NSI/R5 viruses present V3 domainamino acid sequences with low net positive charge,allowing the assignment of putative viral core-ceptor. Interestingly, despite no great differencesin clinical, pathological, or immunological para-meters and the course of HIV-1 infection, we foundthat the children harbored very different viral iso-lates. DNAsequencing of the viral V3 region of HIV-1 from PBMCs and paraffin-embedded brain tissuefrom autopsy revealed that this region was iden-tical between PBMC-associated proviral DNA andbrain (both being SI/X4 virus) in child no. 2 butshowed significant differences with virus presentin plasma (NSI/R5). The other three children pre-sented different V3 sequences in brain viral iso-lates than those from PBMCs or plasma, althoughthe degree of variation was significant. Thus, childno. 4 had an identical V3 region (most likely reflect-ing identical NSI/R5 virus) in PBMCs and circu-lating in plasma but very different from that presentin the brain (SI/X4). In contrast, child no. 3 had analmost identical virus in plasma and PBMCs(NSI/R5) but not too different from the brain.Finally, child no. 1 had different NSI/R5 virusesin the brain and plasma. On the other hand, look-ing at coreceptor usage, child no. 2 and child no. 4had X4 HIV-1 strains in the brain, whereas childno. 1 and child no. 3 had R5 HIV-1 strains in thislocalization.


Tabl

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Am

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et c

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135


136 Álvarez et al.


SI/X4 or NSI/R5 HIV-1 strains isolated from PBMCsamples of several infected patients with or withoutneurological symptoms. All isolates could infect gliaand NB, as demonstrated by viral DNA detection inthose cells (Fig. 2A). Likewise, we assessed the levelof viral replication by monitoring p24 viral core anti-gen production in the cell supernatants at serial timepoints (Fig. 2B). Although the values obtained werelower than those usually observed in productive infec-tion of T cells, these results indicate that both cell lines,SK-N-MC and U-87, could be productively infectedby all HIV-1 isolates tested, as shown by increasingAgp24 levels in the culture supernatants over time.The two established isolates were able to infect bothcell lines; however, HIV-1Bal (NSI) infected glial cellsmuch more efficiently than HIV-1NL4.3 (SI) did. Amongthe primary isolates, some variations could beobserved but no significant differences between SIand NSI isolates were found in the various experi-ments performed. Therefore, primary isolates repli-cate less efficiently than established ones in general,but no differences were apparent in SI/X4 versusNSI/R5 isolates from children with or without neuro-logical symptoms.

To further confirm whether the Agp24 detected inthe supernatants of HIV-infected cell lines repre-sented infectious virus, the highly sensitive MT-2 cellline or GHOST-R5 or X4 (using R5 or X4 strains,respectively) was cocultured with supernatant frominfected cells, harvested 3 d after infection, and testedagain for productive infection in those permissivecells. The result of MT-2 infection with supernatantof HIV-1NL4.3-infected SK-N-MC and U-87 cells is

Fig. 1. Brain expression of chemokine receptors. Shown are CXCR4 and CCR5 in vivo expression in brain samples ofHIV-infected children and uninfected control by ISH.

Expression of CCR5 and CXCR4 Receptors in Brain TissueBecause increased chemokine receptor expression

has been observed in the brain of HIV-infectedpatients (Sanders et al., 1998), we analyzed theexpression of CCR5 and CXCR4 coreceptors in post-mortem brain tissue from the four HIV-infected child-ren with neurological disorders and one uninfectedchild who died from congenital cardiopathy. Brainsamples tested of the four HIV-infected children werepositive for both coreceptors by ISH analysis. Nosignificant variation in the expression of CXCR4 andCCR5 RNAs was observed between brain samples,indicating that expression is independent of the pres-ence of active HIV-1 infection in that region (datanot shown), as well as of the viral strain (R5 or X4)predominant in those locations (Fig. 1).

In vitro Infection of Neuronal Cells and Astrocytes is Independent of the BiologicalCharacteristics of HIV-1 IsolatesPreviously, we have shown that although

microglial cells are the main cellular types infected inthe CNS, some neurons also can be productivelyinfected in vivo (Canto-Nogues et al., 2005). Thus, wetested whether neurotropism can be ascribed to a moreefficient infection of astroglial or neuronal cells.Because primary cultures of human brain cells arehard to obtain, we addressed this issue by testing invitro infectivity of NB and glial cells as surrogate sys-tems. SK-N-MC cells representing neurons at animmature differentiation stage and U-87 as glial cellswere infected with different viral isolates. We used




shown in Fig. 3A. High levels of Agp24 were detectedin those cultures, confirming that NB cells produceinfectious virus. All of the supernatants from the otherisolates gave results similar to HIV-1NL4.3; thus, theyproduced Agp24 levels upon infection of corres-ponding GHOST-R5 or X4 (data not shown). More-over, when supernatants of infected glial or NB cellsinfected with SI/X4 strains were cocultured with MT-2 cells, we also observed the formation of syncytiumin all cases (only HIV-1NL4.3 is shown in Fig. 3B).

Previously, we have reported that some anti-retroviral treatments not only affect viral load butalso produce a change in the viral phenotype of iso-lates (Galan et al., 2004). Because this treatment hasbeen shown to correlate with a reduction of neuro-logical impairment, we addressed whether viralisolates from a single patient, before and after

HAART (presenting a different phenotype), had adifferent ability to replicate in NB and glial cells.For this, we infected SK-N-MC and U-87 cells withviral isolates from one child before and afterHAART (HIV-1ESR-1 [SI/X4] and HIV-1ESR-2 [NSI/R5],respectively). The sequence of V3 loop from viralgp120 involved in cellular tropism from both PBMCisolates from that child is shown in Fig. 4A. Previ-ous to HAART, HIV-1ESR-1 had a similar sequenceto HIV-1NL4.3, being SI, whereas after HAART, therewas a switch to NSI in HIV-1ESR-2, as its V3 sequenceclosely resembled that of HIV-1ADA (a prototypeR5/NSI virus). As shown in Fig. 4B, both strainsfrom the same patient before and after HAART pre-sent similar replicative capacity in SK-N-MC or U-87 cells, indicating that HAART did not significantlychange neurotropism in vitro.

Fig. 2. Infection of SK-N-MC and U-87 cell lines with various HIV-1 isolates. (A) Cell lines were infected and viralDNA was detected 12 d after infection in the cell cultures by a nested PCR of the pol region of HIV-1. Left: Lanes 1–8,SK-N-MC cells infected with HIV-1CSB, HIV-1ASS, HIV-1ILG, HIV-1LRH, HIV-1CGP, as well as the established strains HIV-1NL4.3 and HIV-1Bal, or mock infected with heat inactivated HIV-1NL4.3 (10 m.o.i.); lane 9, positive control of an infectedT-cell line. Right: Lanes 1–6, U-87 cells infected with HIV-1CSB, HIV-1ADR, HIV-1ASS, HIV-1ILG, HIV-1Bal, and heat-inacti-vated HIV-1NL4.3, respectively; lane 7, HIV-1NL4.3. (B) SK-N-MC or U-87 cells were infected with primary isolates HIV-1ADR, HIV-1CSB, HIV-1ASS, HIV-1ILG, HIV-1LRH, HIV-1CGP, as well as the established strains HIV-1NL4.3 or HIV-1Bal, or mockinfected with heat-inactivated HIV-1NL4.3 (10 m.o.i.). Agp24 was quantified in the supernatants of cultures at 0, 3, and 6 d after infection.


138 Álvarez et al.


Fig. 3. Infection with culture supernatants from infected NB and U-87 cells. MT-2 cells were incubated with culturesupernatants from SK-N-MC and U-87 cells infected with HIV-1NL4.3 isolate. (A) Levels of Agp24 were quantified in thesupernatant of MT-2 culture at 3 and 6 d after infection. (B) Syncytia formation 10 d after infection. Panel 1, Culturesupernatants from SK-N-MC; panel 2, supernatants from U-87; panel 3, with infectious HIV-1NL4.3 isolate.

Fig. 4. Infection of SK-N-MC and U-87 cell lines with HIV-1 isolates before and after HAART. (A) Sequencing data ofthe different HIV-1 isolates from HIV-infected children before and after HAART. Amino acid alignments are comparedwith gp120 from HIV-1ADA and HIV-1NL4.3. Dots indicate residues identical to the NL4.3 consensus; dashes indicate gaps.(B) SK-N-MC or U-87 cells were infected with HIV-1Bal, HIV-1NL4.3, HIV-1ESR-1, and HIV-1ESR-2 (0.5 m.o.i.) or with heatinactivated HIV-1NL4.3 (10 m.o.i.). Agp24 was quantified in the supernatants of cultures at 0, 3, and 6 d after infection.




On the other hand, the SK-N-MC cell line wasinfected with the established HIV-1NL4.3 and HIV-1Balstrains and was routinely grown for >6 mo. Everymonth, the virus was isolated and sequenced, andno variation in the genotype and phenotype of HIV-1NL4.3 or HIV-1Bal virus was found (data notshown), which might account for in vitro adaptationto grow in brain cells.

Expression of CXCR4 and CCR5 in NB and Glial CellsAs we found both X4 and R5 viral strains in the

brain, we analyzed the expression of HIV-1 core-ceptors CCR5 and CXCR4 on SK-N-MC and U-87cells by RT-PCR and immunofluorescence analysis.Thus, both coreceptors tested were detected in theNB cell line (Fig. 5A), as well as U-87 cells (data notshown), by both techniques. Immunofluorescencestudies further supported the idea that NB cellsexpress more CCR5 than CXCR4, although the dif-ferences were not statistically different (Fig. 5B).Those results seem to eliminate any role of differentlevels of chemokine receptor expression in glial andNB cells as responsible for putative variations in invitro susceptibility to infection.

Taken together, the above findings suggest thatproductive infection can take place in these glial andneuronal cells and that infection is not limited to oneparticular strain of HIV-1.

DiscussionIn a significant number of infected individuals,

HIV-1 infection is associated with a variety of neuro-logical alterations that are independent of any oppor-tunistic infection. Although the introduction ofHAART has prolonged and improved the lives ofinfected individuals, it is clear that therapy does notprovide complete protection against neurologicaldamage in HIV/AIDS (Bouwman et al., 1998;Maschke et al., 2000). In addition, the molecular andcellular bases of neurotropism/neurovirulence arenot fully understood yet. In this regard, severalhypotheses have been proposed. Among them are(1) the increasing replicative ability in macrophagecells that can enter within the brain, as the replica-tion level in macrophages has been related to theseverity of HIV-1 encephalitis in murine models(Nukuna et al., 2004); (2) increasing viral load in theCSF (Di Stefano et al., 1998); (3) increasing ability toinfect neuronal or progenitor cells (Ensoli et al., 1995;Obregon et al., 1999; Lawrence et al., 2004); (4)increased expression of chemokine receptors after

infection (Sanders et al., 1998); and (5) specific neuro-tropic strains (Power and Johnson, 2001; Smit et al.,2001) and/or adaptation to grow in brain cells(Martin et al., 2001).

In this study we have investigated several possi-bilities in an attempt find a correlation between bio-logical and molecular properties of HIV-1 isolatesfrom children with or without HIV-related EP toexplain the reason for neurovirulence and/or neuro-tropism.

The presence of neuroinvasive strains and the abil-ity to replicate in the CNS remain controversial issuesin neuro-AIDS. Previous studies in adults havedemonstrated that HIV-1 strains derived frominfected HIV-1 with ADC differ from those derivedfrom nondemented patients (Power and Johnson,2001). However, most of the studies have not pro-vided any correlation between biological propertiesof viral isolates and the possibility of suffering withADC. A partial association of gp120 V3 sequencesand AIDS dementia has been observed (Keys et al.,1993; Donaldson et al., 1994; Korber et al., 1994;Chang et al., 1998), and some studies on the V3 region

Fig. 5. In vitro expression of chemokine receptors. (A)In vitro expression in SK-N-MC cells. Purified total RNAwas reverse-transcribed and amplified with CXCR4- andCCR5-specific primers. Lane 1, peripheral blood lympho-cytes; lane 2, macrophages; lane 3, SK-N-MC cells. The371- and 506-bp PCR products are shown. GADPH wasused as a positive control. (B) CXCR4 and CCR5 expres-sion in NB cells by immunofluorescence analysis:CXCR4/CCR5 in SK-N-MC (×40).


140 Álvarez et al.


have suggested that the molecular diversity betweenblood and brain isolates is high (Power et al., 1994).Furthermore, it has been claimed that brain HIV-1strains present several differences from those pre-sent in the circulation (Di Stefano et al., 1996). In thisregard, CNS has been proposed as a reservoir forHIV-1 viruses (Pierson et al., 2000) that might sufferan independent genetic evolution distinct from thatof lymphoid tissues (Chang et al., 1998; Gorry et al.,2001). Nevertheless, in our four children, we cannotconfirm those hypotheses, as we do not find anyassociation between specific brain virus sequencesand neuropathology. Thus, no evident differences inV3 loop sequences among virus isolates from PBMCs,plasma, or brain samples from the four children whodied from HIV-related EP were found that couldexplain neuronal tropism and/or damage. At leastin the V3 sequences, we detected many differentcombinations. We found NSI/R5 viral isolates inplasma/PBMCs, different from the one found in thebrain (SI/X4) in one child and different from one inanother (changed from NSI/R5 to SI/X4 in the brain).However, at the same time, one of the other two chil-dren harbors identical SI/R4 viruses in the brain andPBMCs (but surprisingly very different from the onecirculating in plasma); in the other child, closely simi-lar, although not identical, NSI/R5 viruses werefound in all samples tested. However, we cannotexclude that other viral components also involvedin neuropathology, such as Tat (Power et al., 1994)or the long terminal repeat (LTR), might be moredivergent and responsible for the pathology. In thisregard, several authors have shown that viruses iso-lated from the blood and brain of adults did not differstatistically in V3 sequences (Reddy et al., 1996;Dittmar et al., 1997), but they differ in LTR sequences(Ait-Khaled et al., 1995). In addition, the genetic evo-lution of HIV-1 virus within the brain might be dis-tinct from that observed in lymphoid tissues andplasma. Although it has been shown that differentviral strains exist in the brain and in lymphoid tis-sues of adults (Gorry et al., 2001), in this work wehave found one case with identical virus in PBMCsand brain but different from that circulating inplasma, which seems to contradict this view. Simi-lar to our results, Reddy et al. (1996) found no evi-dence of specific brain V3 sequences.

Studies on the isolation and characterization ofHIV-1 virus in the CNS are scarce, and they have beenperformed primarily in adults, showing primarilythat adult brain-derived viruses are R5 and, in fewerinstances, dual tropic, X4/R5, strains (Williams and

Hickey, 2002). Moreover, some studies have sug-gested a predominant expression of CCR5 corecep-tor in the brain, which could explain this preferentialfinding of R5 strains (van der Meer et al., 2000) in thislocation. In this study, however, we found a similarproportion of X4/SI and R5/NSI HIV-1 populationsin the brains of children with EP. In agreement withthis, previous results have described the presence ofR5, X4, and R5/X4 isolates from the brain tissue ofadult AIDS patients (Gorry et al., 2001) and foundthat both X4 and R5 viruses can contribute to patho-genesis (Yi et al., 2003, 2004).

In vitro studies examining the presence of CCR5and CXCR4 receptors on human brain cell culturesdemonstrated not only microglial but also abundantneuronal expression (Sanders et al., 1998). Bajetto etal. (1999) have shown that CXCR4 is expressed incultured cortical type-I rat astrocytes, cortical neu-rons, and cerebellar granule cells. We present sev-eral lines of evidence showing that brain isolates canuse several chemokine receptors and can presentseveral viral phenotypes, SI/X4 and NSI/R5, sug-gesting that neurological dysfunction is indepen-dent of that viral property.

Our results show that there is no apparent rela-tionship between predominant HIV-1 variants anddamage to CNS, as two children had X4 and the othertwo had R5 strains. This suggests, in agreement withprevious studies (Gorry et al., 2001), that CXCR4usage by brain isolates could be more prevalent thanthought previously. The viral envelope proteins areimportant determinants of neurotoxicity (Power etal., 1998), and those with specificity for CXCR4 havebeen associated with greater neuronal apoptosis invitro (Zheng et al., 1999). Even though HIV-1 entersthe brain at early stages of infection, neurologicalsymptoms take place at a relatively late stage, whenX4 strains generally predominate. Thus, the indirectdamage of neurons could be greater in brains har-boring X4 virus. However, our findings indirectlysuggest that not only these viral isolates but also theR5 strains could induce damage of CNS and cellu-lar apoptosis in children.

It is generally accepted that microglial cells arepredominantly infected in vivo (Power and John-son, 2001; Wesselingh and Thompson, 2001), but itis still not clear whether neurons and astrocytes, inaddition to microglia, can be infected. In this respect,we have shown recently that cortical neurons are theones predominantly infected in vivo (Canto-Nogueset al., 2005) in AIDS-infected children with EP. Ourresults clearly show in vivo infection of neurons, at




least in children, in agreement with some data inadults (Budka, 1989; Takahashi et al., 1996), althoughthe physiological relevance of this issue remains tobe determined.

Here, we found that all isolates tested, showingSI/X4 or NSI/R5, can infect cells of glial or neuronalorigin in vitro. Thus, there were also no distinct dif-ferences in the ability of viral isolates from childrenwith or without neurological disease to infect andreplicate in NB or astroglioma cells in vitro. This isin agreement with previous reports indicating HIV-1 infection of NB and glial cells to a similar extent(Ensoli et al., 1995; Obregon et al., 1999). These resultscontrast with those of Trujillo et al. (1996), whoshowed that infection of SK-N-MC cells by HIV-1 ofdifferent tropism (NSI/R5 vs. SI/X4) varied con-siderably. Similar to our results, Dittmar et al. (1997)found that despite diversity in the V3 sequences frombrain-derived viral isolates compared with thosefrom lymphoid tissues, all recombinant HIV-1 clonesshowed identical cell tropism and replicative kine-tics in CD4 cells. Along similar lines, Sabri et al. (1999)found that HIV-1 isolates independent of their bio-logical phenotype and chemokine receptor usagewere able to infect fetal astrocytes. Anyway, it isunlikely that differential expression of chemokinereceptors can affect in vitro replication in our system,as both cellular types used in this study expressedsimilar levels of viral coreceptors.

Although HAART decreases neurological symp-toms (Maschke et al., 2000) in HIV-1-infected indi-viduals, its impact on neuroinvasiveness andneurovirulence remains to be established. Our dataindicate that HAART has an important impact onthe viral phenotype, as the virus strain predomi-nantly isolated in the HIV-1ESR patient was SI priorto HAART but changed to NSI after therapy, as wehave reported previously with several other chil-dren (Galan et al., 2004). Despite this, no differencein in vitro ability to infect NB or glial cells wasobserved between both strains.

In summary, our results, albeit with a limitednumber of children, owing to the scarcity of the sam-ples available, establish that a single phenotypicproperty of the HIV-1 virus does not exist that mightexplain its neurovirulence and/or neurotropism. Allviral isolates tested presented in vitro equal capa-city of infection of neural cells and astrocytes. Thissuggests that more than one factor might accountfor HIV-1 neurotoxicity: the abundance of the virusinto the brain, the viral strain, the number ofmacrophages infected, the ability to infect neuronal

cells, etc. Future analyses with a larger number ofpatients are necessary to confirm any of thesehypotheses.

AcknowledgmentsThis study was supported by Plan Nacional de

Salud (grant SAF 2003-09209), Fundación para la Inves-tigación y la Prevención del SIDA en España, FIPSE(grant 36365/02, 12456/03), Comunidad de Madrid,Red Temática Cooperativa de Investigación en SIDA(grant RIS G03/173) of FIS, and Red Temática Coop-erativa de Investigación en Genética Clínica y Mole-cular (grant RIG C03/07) of FIS. J. L. J. is supportedby a grant from FIS (CP03/00140).

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Lack of association of HIV-1 biological or molecular properties with neurotropism for brain cells