HIV-1 Co-Receptor Usage

Immunological Investigations,40:597–613,2011Copyright ©Informa Healthcare USA,Inc.ISSN:0882-0139print /1532-4311onlineDOI:10.3109/08820139.2011.569673failure while taking a CCR5antagonist.Keywords HIV Co-receptor,V3loop,CXCR4,CCR5.Both authors Jiao and Wang contributed equally to this work.Address correspondence to Prof.Hao Wu,Center for Infectious Diseases,Beijing You’an Hospital,Capital Medical University ,Beijing 100069,Peoples Republic of China;E-mail:whdoc900@I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F o r p e r s o n a l u s e o n l y .598Y .Jiao et al.INTRODUCTION Highly active antiretroviral therapy (HAART)is highly efficient at reducing virus replication and increasing CD4+T-cell numbers,resulting in signif-icant decreases in acquired immune deficiency syndrome (AIDS)-associated morbidity and mortality .In response to HAART ,Human immunodeficiency virus type 1(HIV-1)RNA levels in plasma often decline to below the limit of detection by sensitive molecular assays in many patients;however,cells harboring replication-competent HIV-1persist even in patients who have been on suppressive HAART for many years,which is a major obstacle to virus eradication (Wong et al.,1997,Delobel et al.,2005).A relationship between coreceptor usage and HIV disease progression has been established,but the influence of antiretroviral therapy on coreceptor tropism remains controversial.Some studies reported a preferential suppression of X4viruses with HAART ,while other studies suggested a predominant R5-to-X4switch in cell reservoirs with effective therapy (Souliéet al.,2007;Equils et al.,2000;Philpott et al.,2001;Skrabal et al.,2003;Galán et al.,2004;Johnston et al.,2003;Saracino et al.,2009).Hence,the relationship between the use of antiretroviral therapy and the prevalence of X4and/or R5strains requires further clarification.HIV-1binds to host cells via the envelope glycoprotein.HIV-1envelope protein binds to its primary receptor CD4,and undergoes a conformational change and then binds to one of the chemokine receptor CCR5or CXCR4orboth via its V3loop (Xu et al.,2007).This tri-molecular interaction leads to the viral membrane fusion and viral entry into the host cell (Dragic el al.,1996).Based on the ability to use CCR5or CXCR4for entry into the host cell,HIV-1variants are classified as CCR5-tropic (R5),CXCR4-tropic (X4),or dual-mixed tropic (X4-R5)(Garrido et al.,2008).The HIV-1envelope is composed of relatively conserved (C1to C5)and variable regions (V1to V5).The V3region governs co-receptor usage (Dragic et al.,1996).Replacements of charged amino acids within the V3region are known to alter the co-receptor usage (Hoffman et al.,2002;Milch et al.,1993).During the early stages of disease,HIV-1isolates are preferentially monocytotropic with a non-syncytium-inducing (NSI)phenotype and use CCR5as a coreceptor.Progression of disease is associated with the emergence of syncytium-inducing (SI)viruses that are able to bind the CXCR4coreceptor.Persistence of CCR5usage is seen in association with delayed progression of disease,such as that observed in long-term non-progressors (LTNPs)(Schuitemaker et al.,1992;Zhu et al.,1993;Xiao et al.,1998;Connors et al.,1997).In addition to the relationship between coreceptor usage and HIV pathogenesis,the interest in HIV tropism has increased because of the introduction of CCR5antagonists in the anti-HIV arsenal (Poveda et al.,2006;Weber et al.,2006).Since these drugs show activity against R5viruses only ,I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F orpersonaluseonl y .Preferential Suppression of CXCR4Virus after ART 599viral tropism should be tested before prescribing these drugs in addition to the course of administration to exclude the presence of X4viruses (Garrido et al.,2008).Co-receptor tropism of individual viral strains can be delineated using reporter cells expressing different coreceptors (Skrabal et al.,2007),however recently alternate,bioinformatics strategies have been developed to predict viral co-receptor usage from envelope (env)gene sequence information (Jensen et al.,2003;Poveda et al.,2006).Due to its direct exposure to host immunological pressure,the envelope gene (env)of HIV-1is the most variable region in the viral genome.This variability influences the penetration of HIV-1into different cell types,according to the two main chemokine coreceptors binding variants (X4and R5)(Saracino A et al 2009).The third hypervariable region (V3)of the HIV-1envelope glycoprotein gp120is known to be a key determinant of HIV-1coreceptor usage (Low et al.,2008).The amino acids at positions 11and 25of V3and the overall V3charges have also been linked to HIV-1coreceptor usage (Mild et al.,2010,Hartley et al.,2005,De Jong et al.,1992,Fouchier et al.,1995).It is reported that the V3loop of X4strains carry more positively charged amino acids than R5strains,and coreceptor usage can be predicted by analyzing the putative V3loop amino acid sequences using different bioinformatics tools,such as support vector machine,PART ,11/25charge rule,Geno2pheno <http://coreceptor.bioinf.mpi-inf.mpg.de/>,and position-specific scoring matrix (PSSM)(Sing et al.,2007,Chen et al.,2009).Based on several special residues (especially amino acid profiles at sites 11and 25in the V3loop)or the net charges of the V3loop,these approaches employ a multiple linear regression (Briggs et al.,2000),a neural network strategy (Resch et al.,2001)a machine-learning method (Pillai et al.,2003),or a position-specific scoring matrix (PSSM)(Jensen et al.,2003;Jensen and Van’tWout,2003),to predict HIV-1coreceptor usage and phenotype.The PSSM approach appears to have better predictive power over other methods (Jensen,2003).In this study ,genomic DNA was extracted from the peripheral blood mononuclear cells (PBMC)of seven AIDS patients and ten monoclonal sequences of HIV-1env C2–V5region were sequenced for each patient before and after 24weeks of HAART treatment.Putative V3loop amino acids were compared using the Geno2pheno and PSSM methods to predict coreceptor (R5/X4)usage.Our results indicate that 24weeks after start of the ART regimen,the HIV virus is more likely to use CCR5coreceptor.Additionally ,we observed that the positive charges and the net charges of the V3amino acids were significantly lower than those found prior to ART treatment.The potential clinical impacts of changes in viral tropism during periods of HAART are enormous,since it is believed that the X4variants are inherently more pathogenic and directly responsible for a more rapid disease progression I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F o r p e r s o n a l u s e o n l y .600Y .Jiao et al.since their emergence is believed to be a consequence of progressive immune dysfunction.MATERIALS AND METHODS Patients Seven HIV-1infected patients from Beijing Youan Hospital were enrolled,and all patients started receiving HAART in 2009.Whole blood was collected in tubes containing EDTA anticoagulant from each patient before treatment (baseline)and 6months after treatment (24weeks).The project was approved by the Institutional Ethics Committee and the patients participated in the study following informed consent.Demographic and immunologic characteristics of the patients are reported in Table 1.Cloning and Sequencing of C2–V5Region of HIV-1env The C2–V5region of the HIV-1env gene of the study patients was analyzed.Briefly ,PBMC were obtained by centrifuging whole blood on a Ficoll-Hypaque density gradient.The genomic DNA was extracted from PBMC using QIAamp DNA Mini Kit (QIAGEN,Hilden,Germany)and HIV-1proviral DNA were amplified by a nested PCR (2rounds of 30cycles,first round:95◦Cfor 3min,94◦C for 30s,annealing at 50◦C for 30s,extension at 72◦C for 80s and final elongation at 72◦C for 5min;second round:95◦C for 3min,94◦C for 30s,annealing at 52◦C for 30s,extension at 72◦C for 50s and final elongation at 72◦C for 5min)using outer primers TF1(forward):5’-ATG GGA TCA AAG CCT AAA GCC ATG TGT-3’(HXB2nt 5933–5959)and TR1(reverse):5’-GCG CCC ATA GTG CTT CCT GCT GCT GC-3’(HXB2nt 7203–7181),and inner Table 1:Demographic and immunologic characteristics of the study patients.CD4cell count Plasma viral load ∗∗∗baseline 24weeks baseline 24weeksPatient Age(years)Gender ∗HAART ∗∗(cells/µl)(cells/µl)(copies/ml)(copies/ml)Pt142M D4T +3TC +NVP 29735631513257Pt225M D4T +3TC +NVP 34971522968458Pt363M D4T +3TC +NVP 8919027846TND Pt436M D4T +3TC +NVP 256320115741799Pt528M D4T +3TC +NVP 23831218622TND Pt629M AZT +3TC +NVP 3352159264TND Pt723M AZT +3TC +NVP 27835722291164∗M:male;F:female.∗∗D4T:Stavudine;3TC:Lamivudine;NVP:nevirapine;AZT:Zidovudine.∗∗∗TND:Target not detected.I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F orpersonaluseonl y .Preferential Suppression of CXCR4Virus after ART 601primers TF2(forward):5’-CTG TTA AAT GGC AGT CTA GC-3’(HXB2nt 6405–6424)and TR2(reverse):5’-ACT TCT CCA ATT GTC CCT CAT-3’(HXB2nt 7046–7026).A PCR product of 642bp was obtained,including the C2–V5region of the HIV-1env gene.Multiple HIV-1negative controls were included with each PCR run to detect any possible contamination.For each patient,proviral DNA at baseline (before HAART treatment)and six months after treatment were amplified.Purified products from PCR were separately ligated to the pMD-18T vectors (Takara,Dalian,China)according to manufacturer’s instructions.For each sample,10single colonies of transformed Escherichia coli JM109were picked and cultured.The plasmid DNA was purified using Rapid Extraction of a small amount of high purity plasmid kit (BioMed,Beijing,China)and digested with EcoRI restriction enzyme to confirm the presence of the insert.The plasmids containing inserts were then sequenced using M13-47primers synthesized by BioMed Technology Development Company (Beijing,China).Sequences were assembled and checked for errors using the Vector NTI software (version 9.0;Invitrogen,Carlsbad,USA).Coreceptor Usage Prediction and V3Loop Characteristics Analysis The sequences of the C2–V5region were aligned with a reference sequence data set (Tamura et al.,2007)of all major subtypes and circulating recombinant forms (CRF)(downloaded from Los Alamos Sequence Database)by the ClustalW multiple sequence alignment program from MEGA 4.The sequences of the V3region were translated into amino acid sequences using the BioEdit Sequence Alignment Editor 7.0software and coreceptor usage predictions based on V3amino acid sequences were performed by Geno2phen (http://coreceptor.bioinf.mpisb.mpg.de/cgi-bin/coreceptor.pl)with a false-positive rate of 0.1and the Position-Specific Scoring Matrix (PSSM)approach (/com puting/pssm/)with higher the score,the more closely the sequence resembles those of known X4viruses (Chen et al.,2009).For the purpose of this study ,we considered a sequence as X4-tropic if one of the two approaches predicted it to be X4-tropic.The net amino acid charges of the V3region were calculated by Innovagen peptide property calculator (www .innovagen.se/custom-peptide-synthesis/peptide-property-calculator.asp)(Chen et al.,2009).The V3loop central motifs of each sequence were analyzed with respect to each subtype.Statistical AnalysisComparisons were performed using independent or paired sample t -test,all reported P-values were two-sided and were considered to be significant I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F o r p e r s o n a l u s e o n l y .602Y .Jiao et al.only when P-values were <0.05.All data were analyzed using SPSS statistical software (version 13.0;SPSS,Chicago,USA).RESULTS Viro-Immunological Response to HAART Viral loads and CD4+T-cell counts of the 7enrolled patients are shown in Table 1.The mean viral load prior to starting the HAART regimen was 224,791±425,722copies/mL and viral load decreased 117±161copies/mL within 6months of HAART .In parallel a significant increase in the absolute number of CD4+T cells was also observed,with a CD4count of 263±87cells/µL pre-treatment as compared to 352±173cells/µL at 6months post HAART .With the exception of patient 6,all patients showed significant increase in absolute CD4counts.These data show that commencement of HAART results in significant improvement in immune response in all patients studied.Changes in the Predicted Coreceptor Usage During HAART Ten HIV-1env gene V3region sequences for each patient pre-and post-HAART were analyzed and translated into amino acid sequences.V3genotyp-ing was performed from plasma proviral DNA before starting HAART and fromproviral DNA 6months (24weeks)post suppressive HAART .Viral tropism was interpreted using geno2pheno (false positive rate =10%)and an optimized version of position specific scoring matrices (PSSM)with a greater sensitivity to detect X4variants.Coreceptor (CCR5/CXCR4)usage data are shown in Supplementary Table 1and statistical analyses are reported in Table 2.Table 2:Predicted coreceptor usage based on 10clones of V3loopsequence before and after HAART.Baseline 24weeksPatient X4∗R5∗∗X4∗R5∗∗Pt1100010Pt237010Pt310073Pt446010Pt5010010Pt628010Pt710046∗Number of V3sequences predicted as CXCR4-tropic in all the 10monoclonalsequences analyzedfor eachpatient.∗∗Number of V3sequences predicted as CCR5-tropic in all the 10monoclonal sequences analyzed for each patient.I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F orpersonaluseonl y .Preferential Suppression of CXCR4Virus after ART 603Our data show that prior to commencement of HAART ,the predominant virus in the therapy naïve patients was the T tropic X4virus,however six months post HAART ,the proportion of X4viruses in majority of the patients decreased,and the proportion of R5viruses increased,this transition of viral tropism from X4to R5as a consequence of HAART was statically significant (p =0.0017),in all patients,with the exception of patient 5who was M-tropic to begin with and in whom the R5virus remain unchanged.V3Loop Charge After the sequences of HIV-1env gene V3regions were translated into amino acid sequences,the number of positively-charged amino acids (K,H and R)and net charges of the V3loop were analyzed (Supplementary Table 1).We found that both the positive charges and the net charges of the V3loop decreased significantly (p <0.05)after treatment.Both kinds of charges of V3loop in the X4viruses were significantly higher (p <0.05)than those in R5viruses (Table 3).Subtypes and Central Motifs of theV3Loop While using the Geno2pheno approach to predict coreceptor usage,the subtype of V3loop could be predicted at the same time (Supplementary Table S1).Of the 140sequences obtained in this study ,subtype B (42.14%),C (30.00%)and CRF01_AE (26.43%)were the three main predicted subtypes (Table 4).The V3loop could induce the host to produce tropism-restricted neutralizing antibodies and cytotoxic lymphocytes,and the central motifs of the V3loop are some of the most important neutralizing antibody determinants.The distribution of the types of V3loop central motifs in the seven patients were GPGQ (55.00%),GPGR (15.00%),GWGR (11.43%),andsome other rare types.As with each subtype,subtype B had a higher variability of V3loop central motifs,while subtype C and CRF01-AE were typically GPGQ-based (Table 4).Table 3:Predicted V3loop charges of HIV-1sequences before and after HAART.X4R5Baseline 24weeks Sig.∗viruses viruses Sig.Positive charges 6.80 6.440.0067.08 6.34<0.001Net charges+5.74+5.04<0.001+6.00+5.02<0.001∗Two-tailed P-value.I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F o r p e r s o n a l u s e o n l y .604Y .Jiao et al.Table 4:Subtypes and central motifs of the V3loop.V3subtype B C AE A/AG V3loop central motifs GPGR 21∗GPGQ 35GWGR 16GSGQ 1GPGK 10APGR 6GPGQ 42GSGQ 1VGGR 5GGGR 1RPKK 1GTGQ 1Total 5942372percentage 42.14%30.00%26.43% 1.43%∗Number of the certain central motifs of each subtype in the total 140sequences analyzed.DISCUSSION The approval of CCR5antagonists for the treatment of HIV-1infection has raised significant interest in viral tropism dynamics in infected patients.The use of the CCR5antagonist maraviroc as part of the HAART regimen is hampered by the difficulty of assessing viral tropism in patients with undetectable viremia (Seclen et al.,2010).In general,coreceptor usage must be known before these drugs are prescribed (Garrido et al.,2008).The influence of antiretroviral therapy on coreceptor tropism remains controversial(Saracino et al.,2009).However,it is important to recognize that a change in viral tropism is an important indicator of ongoing viral evolution.In the current study ,we compared the difference between the viral phenotypes in HIV-infected patients before and after 24weeks of HAART ,and our results indicate that there was a preferential suppression of X4virus with HAART .Transition from a R5to X4or dual/mixed (D/M)virus is observed in untreated individuals and is associated with declining CD4count and possibly attributed to diminished immune control or increased pathogenicity of X4viruses.The cell tropism of HIV-1is determined by the amino acid sequence of the V3region of env and its net charges.The amino acid residues at specific positions affect the biological phenotype of the virus,particularly the amino acids at sites 11and 25.It has been reported that X4strains are rich in basic amino acids in these locations,carrying more positive charges than the R5strains (Jensen,2003;Polzer et al.,2004).Consistent with previous reports,the current study found that following HAART in the X4strain,the number of positively-charged residues and the net charges of the V3amino acids that were significantly lower than those at baseline,were significantly higher as compared to the R5strain.The most important determinants of V3loop are the central motifs,which play an important role in influencing the induction of neutralizing I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F orpersonaluseonl y .Preferential Suppression of CXCR4Virus after ART 605antibodies (Vogel et al.,1994;Cabello et al.,1995).It is reported that strains of different subtypes have different central V3motifs with GPGR mainly found in subtype B isolates,while B,C,and CRF01-AE subtypes mainly containing the GPGQ motif (Liang et al.,2006).In this study we found that the main central V3motifs of subtype B isolates were GPGR and GWGR,while those of subtype C and CRF01-AE were mainly GPGQ.The motifs found in this study were consistent with previously published motifs in the subtypes.Furthermore,our results also agree with other reports that suggest that X4strains are preferentially suppressed during the first year of therapy and that treatment may lead to a change in the predominant phenotype of the viral population (Wong et al.,1997;Philpott et al.,2001;Skrabal et al.,2003).The change in the charge of the V3loop and restriction of HIV coreceptor tropism to R5-tropic may lead to a decrease in T-cell and thymocyte destruction,and may allow the reconstitution of early progenitor cells that will ultimately be released into the peripheral blood.These findings may help explain the increase in naive T cells commonly observed in pediatric and adult patients treated with HAART ,despite modest reductions in viral replication (Anastos et al.,2004;Weiser et al.,2008).The potential clinical impacts and the pathogenic consequences of changes in viral tropism in HAART naïve patients and in non-compliant patients during periods of treatment interruption are difficult to predict.A switch in viral tropism may result from the emergence of minority quasi-species that are already present in the proviral DNA surfacing as a result of the absence of antiviral selective pressure during HAART .Additional studies are needed to evaluate the potential impacts of HIV tropism switches on prognostic outcomes and the use of co-receptor antagonists in both HAART naïve patients as well as patients undergoing HAART treatment.ACKNOWLEDGMENTSThis study was supported in part by the National 11th Five-Year Major Projects of China (2008ZX10001-006,2008ZX10001-001),and the National Natural Science Foundation of China (30872226).Declaration of interest:This statement indicates no financial conflicts of interest and contribution of all authors.The authors alone are responsible for the content and writing of the paper.YJ drafted the manuscript and statistical analyses.PW participated at the Cloning and sequencing of C2–V5region of HIV-1env .HWZ assisted with manuscript and data anlysis.TZ and YZ assisted with patients’care and data acquisition.HZZ conceived the study and participated in the data analysis.HW supervised and coordinated the study .I m m u n o l I n v e s t D o w n l o a d e d f r o m i n f o r m a h e a l t h c a r e .c o m b y C h i n e s e P L A o n 03/07/12F o r p e r s o n a l u s e o n l y .606Y .Jiao et al.REFERENCES Anastos,K.,Barrón,Y.,Cohen,M.,Greenblatt,R.,Minkoff,H.,Levine,A.,Young,M.,Gange,S.(2004).The prognostic importance of changes in CD4+cell count and HIV-1RNA level in women after initiating highly active antiretroviral therapy.Ann.Intern.Med.140:256–264.Briggs, D.,Tuttle, D.,Sleasman,J.,Goodenow ,M.(2000).Envelope V3amino acid sequence predicts HIV-1phenotype (co-receptor usage and tropism for macrophages).AIDS 14:2937–2939.Connor,R.I.,Sheridan,K.E.,Ceradini 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