Conservation of the α 4 β 7 Lymphocyte Homing Receptor in HIV-Infected Patients with Distinct Transmission Routes and Disease Progression Profiles

Abstract

The α₄β₇ is a lymphocyte homing receptor to the gut-associated lymphoid tissue (GALT). HIV-1 gp120 binds to α₄β₇ integrin through a mimetic tripeptide in V2 and ensures successful infection of GALT. In the present report, we investigated the presence of polymorphisms in the α₄β₇ cytoplasmic and α₄ N-terminal binding domains and their potential association with susceptibility to HIV infection or disease progression. Subjects displaying distinct categories of disease progression or transmission routes (HIV-positive adults, vertically infected infants, and seronegative subjects) had their ITGA4 and ITGB7 gene segments corresponding to virus binding sites and C-terminal domains PCR amplified and sequenced. An absolute conservation of the studied regions was observed in all patients and controls. Albeit polymorphisms in α₄β₇ may exist in other regions not tracked in this study, α₄β₇ activation and binding domains do not seem to be polymorphic and are not correlated with distinct patterns of HIV transmission or disease progression.

Integrins comprise a family of heterodimeric α/β transmembrane cell adhesion receptors involved in cell–extracellular matrix and cell–cell interactions in the presence of divalent cations.¹ They are associated with numerous complex biological processes including cell migration and growth, tissue organization, inflammatory response, and target recognition by lymphocytes.² Currently, 18 α and eight β subunits have already been identified, which originate 24 distinct αβ combinations.¹ The integrin α₄β₇ is encoded respectively by ITGA4 (location 2q31.3) and ITGB7 (location 12q13.13) genes, and is expressed in activated effector/memory T cells that migrate to the gut-associated lymphoid tissues (GALT). This site constitutes the largest lymphoid tissue of the human body and is home to a high concentration of target cells for HIV replication,³ causing irrevocable impairment to the immune system.

It has been shown that the HIV-1 envelope is capable of recognizing integrin α₄β₇ by mimicking an epitope common to its natural ligands VCAM1, MadCAM1, and fibronectin.⁴ This interaction drives the virus to its primary site of replication and plays a critical role in the early phases of infection. Integrin natural ligands exhibit a conserved tripeptide required for α₄β₇ binding (LDV/I). The homologous binding motif in HIV gp120 is located in the V1/V2 variable loop. Integrin α₄β₇ plays a decisive role in signaling mechanisms that triggers LFA-1 activation to form immunological synapses. This mechanism increases viral dissemination during the acute phase of infection and also features a significant role in HIV pathogenesis.⁴

Essential regions for ligand binding have been located to the N-terminal portions of the α and β subunits.⁵ The N-terminal region of the α₄ protein encompasses the β-propeller domain, a helical structure formed by seven repeats of 60 amino acids. The main ligand-binding region in integrin α₄ is located on the third repeat of the helical structure and is encoded specifically by exons 5 and 6 of the ITGA4 gene.⁶

α₄β₇ is present at the lymphocyte surface in different conformational states (extended, partially bent, or completely bent) that regulate avidity of adhesion molecules and also of the gp120 molecule. Intracellular activation signals—known as inside-out signaling—targeting cytoplasmic domains from both subunits induce a transition between these affinity states.² Interaction between the α₄ and β₇ cytoplasmic domains (encoded respectively by exons 28 of the ITGA4 gene and 15–16 of the ITGB7 gene) coordinates the integrin activation state. Although cytoplasmic tails of α₄ and β₇ chains are relatively short (31 and 52 amino acids, respectively), cytoplasmic tail interactions constitute crucial regions for inside-out signaling.⁷

HIV-1 infection outcomes have been associated with genes involved in immune responses. Innate immunity genes such as those encoding toll-like receptors and defensins have been shown to play an important role in HIV-1 infection susceptibility and disease.⁸ Allelic variants in chemokine receptors such as CCR5, CCR2, and CXCR4 are well characterized as host restriction factors capable of suppressing HIV-1 replication.⁹ Little is known, however, about the HIV-1 and α₄β₇ interaction and its influence on infection susceptibility or progression to AIDS. Recently, our group showed that variants of ITGA4 exons 5 and 6 present in New World primates (NWP) displayed significant loss of avidity for HIV-1 gp120, which likely contributes to NWP resistance to lentiviral infections.¹⁰

In the present study we hypothesized whether polymorphisms could be found in the genomic regions corresponding to exons 5, 6, and 28 of ITGA4 and to exons 15 and 16 of ITGB7 in HIV-infected patients that could be linked to specific HIV transmission routes or to disease progression outcomes.

This study included a total of 86 samples obtained from seropositive patients followed at two HIV/AIDS clinics from southern and southeastern Brazil. HIV⁺ patients included 51 infected adults (IAs) from the Hospital de Clínicas de Porto Alegre, RS. Twenty-one were parenterally infected through intravenous drug use (IDU) or blood transfusion. Fourteen IA patients were categorized as long-term nonprogressors (LTNP) or did not progress to AIDS before 8 years of follow-up (probable LTNP) according to standardized definitions.¹¹ A second group of patients comprised 45 vertically infected children (VIC) enrolled from 2002 to 2004 at the Pediatric Unit of Federal University of Rio de Janeiro and followed regularly since birth. Of those, 25 were part of a previous prospective study; hence time of infection and profile of disease progression could be defined according to HIV viral load and CD4⁺ T cell counts.¹² Four infants remained asymptomatic and presented CD4 counts >25% and therefore were classified as LTNP. Ten infants, classified as rapid progressors, progressed to disease or had CD4 counts decreased to <15% within 18 months of follow-up. Eleven patients did not fit into any of the categories and were defined as typical progressors. The other 20 seropositive pediatric samples were part of a cross-sectional study and, therefore, were not categorized according to disease progression patterns. The use of these samples was approved by the Institutional Review Board of the Federal University of Rio de Janeiro.

Seronegative patients and controls were obtained from two distinct groups. The first comprised 10 exposed-uninfected children (EUC) born from HIV⁺ mothers not subject to antiretroviral treatment during pregnancy, and followed up at the HIV/AIDS Program of the Federal University of Rio de Janeiro. These children were younger brothers or sisters to the seropositive pediatric patients described above and thus were definitely exposed to HIV. The second group consisted of 100 adult control samples previously available at the Brazilian Cancer Institute (INCA). The use of all samples was approved by ethics committees of their institutes of origin.

Blood samples from HIV-positive or negative patients or controls had their peripheral blood mononuclear cells (PBMCs) isolated using Ficoll density gradient. PBMCs were used for genomic DNA extraction with the QIAamp DNA Mini Kit (QIAgen, Chatsworth, CA) according to the manufacturer's specifications. DNA fragments corresponding to genomic regions encoding exons 5 and 6 (functional α₄ binding site) and 28 (cytoplasmic region– Pfam ID: PF08441) of ITGA4 and exons 15 and 16 (cytoplasmic region–Pfam ID: PF08725) of ITGB7 were polymerase chain reaction (PCR) amplified. Amplification of both ITGB7 exons 15 and 16 covered the internal intron, enabling amplification and sequencing of both regions in one fragment. Primers were designed using the online tool Primer3 v.0.4.0 (http://frodo.wi.mit.edu/) according to the ITGA4 and ITGB7 human reference genes retrieved from GenBank (ITGA4 GenBank ID: 3676; ITGB7 GenBank ID: 3695). PCR reactions of α₄ and β₇ fragments used 100–200 ng of genomic DNA, 5 μl 10×PCR buffer, 0.4 μl of 25 mM dNTP mix, 25 pmol of each primer, 2 μl of 25 mM MgCl₂, and 0.4 μl of 5 IU/ ml Taq Platinum DNA polymerase (Life Technologies, Carlsbad, CA). PCR was performed in a C1000 thermal cycler (Bio-Rad). Cycling conditions were the same for all fragments, except for varying the annealing temperature according to the primers used (Table 1): 2 min of denaturation at 94°C, 40 cycles including 30 s at 94°C, 30 s of annealing at 46–53°C, 40 s of extension at 72°C, and a final extension of 72°C for 5 min. Products were run on an agarose gel and purified using the Illustra GFX PCR DNA and Gel Band Purification kit (GE HealthCare, São Paulo, Brazil).

Table 1.

Primers Used for DNA Sequencing of ITGA4 and ITGB7 Gene Fragment PCR Products

Name	Primer sequence	Gene	Position ^a	T_m (°C)	Product length (bp)	Location
ITGA4-F sense	5′-GTTTAATATTTCATTTTA-3′	ITGA4	21843–21863	46°C	69	Exon 5
ITGA4-RI antisense	5′-GGTACTATAAAAATTGACAAAC-3′	ITGA4	22027–22049
ITGA4-FI sense	5′-CAGGATTTAATTGTGATGGG-3′	ITGA4	23241–23260	46°C	130	Exon 6
ITGA4-R antisense	5′-CAGACATGATGCAGATGTTGCAC-3′	ITGA4	23374–23394
Exon28-F sense	5′-GGGATGCAGTTTAAGAAATATTGG-3′	ITGA4	78429–78452	50°C	320	Exon 28 (cytoplasmic domain)
Exon28-R antisense	5′-CATGAGTAAAAGAAGTCCAAAC-3′	ITGA4	78748–78727
B7_C-terminal-F sense	5′-ACAGAGGGAGCAGACCACAC-3′	ITGB7	15192–15211	51°C	552	Exons 15–16 (cytoplasmic domain)
B7_C-terminal-R antisense	5′-TCTTACAGACCCACCCTTCC-3′	ITGB7	15743–15724

Positions are relative to the reference of ITGA4 and ITGB7 gene sequences (ITGA4 GenBank ID: 3676; ITGB7 GenBank ID: 3695).

Primers used in amplifications were used for DNA sequencing of the corresponding PCR products. Sequencing reactions were performed using the Big Dye v.3.1 kit (Life Technologies, Carlsbad, CA), according to the specifications indicated by the manufacturer. DNA sequencing was carried out in an automated ABI3130XL Genetic Analyzer (Life Technologies).

A few samples corresponding to exons 5 and 6 of the ITGA4 gene from the VIC and IA groups were randomly chosen and subject to cloning for assessing the presence of possible distinct alleles. The pMos blunt-ended cloning kit (GE Healthcare) was used according to the manufacturer's specifications. Approximately 10 clones were screened for each fragment and confirmation of insert was performed by colony PCR. Colony PCR products were purified and sequenced as described above.

Sequences were first visualized using Chromas (Technelysium Pty Ltd., Australia; www.technelysium.com.au), manually edited with the software SeqMan v.7.0 (DNASTAR Inc, Madison, WI) and aligned using Bioedit v.7.0 with reference sequences of ITGA4 and ITGB7 retrieved from GenBank (IDs NC_000002.11 and NC_000012.11, respectively). Exons 5 and 6 of ITGA4 comprised bases 556–754 of the α₄ mRNA (Gene Bank ID NM_000885.4) and residues 186–251 of its precursor protein (GenBank ID NP_000876.3). Exon 28 of ITGA4 starts at base 3004 of the α₄ mRNA and encodes 31 amino acid residues. Exons 15 and 16 of ITGB7 comprise bases 2,309–2,548 of the β₇ mRNA (GenBank ID NM_000889.1) and residues 720–798 of its precursor protein (GenBank ID NP_000880.1). Fragments corresponding to the screened functional regions had their deduced amino acid sequence compared to the α₄ and β₇ reference amino acid sequences.

All nucleotide sequences generated in this study have been submitted to the GenBank database and were assigned accession numbers KF671586 to KF671756.

The analysis of ITGA4 exon 5 was performed in 14 samples from the VIC group (seven typical progressors, six rapid progressors, and one LTNP). All exon 5 sequences were revealed to be identical to the ITGA4 reference. Changes in exon 6 of ITGA4 have been highlighted as the most influential in binding efficiency of MAdCAM, VCAM, and, more significantly, the hypervariable V2 region of HIV gp120.¹⁰ Twenty IAs and 19 VICs (three slow, six rapid, and 10 typical progressors) were screened for ITGA4 exon 6, but again all deduced amino acid sequences were shown to be identical to the reference sequence. Cloning of fragments corresponding to ITGA4 exons 5 and 6 from pediatric and adult seropositive patients was also carried out. Fourteen pediatric samples were assessed for exon 5 (six VIC: three rapid progressors, two typical progressors, and one LTNP) and 19 for exon 6 (16 VIC: five rapid progressors, eight typical progressors, and three LTNP). Eleven IA samples were also subject to cloning. None of the screened clones showed nucleotide substitutions when compared to the reference exon 5 or 6 sequences (data not shown).

The cytoplasmic domain of α₄ subunit, encoded by exon 28, was sequenced and analyzed for 22 IA samples (14 LTNP or probable LTNP and 12 VIC of undefined disease progression profile). Exon 28 was extremely conserved, including the GFFKR motif of inside-out signaling regulation, not showing changes when compared to the reference sequence (data not shown). The DNA fragment corresponding to the first 90 base pairs of 3' UTR of ITGA4 was also analyzed in all samples amplified for exon 28. This fragment comprised target sites for microRNAs miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e (www.microrna.org/microrna/home.do). No polymorphisms or changes were observed.

Thirty-three IA patients had ITGB7 exons 15 and 16 screened, including 14 individuals classified as LTNP/probable LTNP and 13 infected by the parenteral route. For β₇ C-terminal sequences, 40 control samples from voluntary donors were included. As expected, changes in the intronic region between ITGB7 exons 15 and 16 were common in both infected patients and control samples (data not shown), but regions encoding the β₇ subunit cytoplasmic domain showed no changes when compared to the human reference sequence.

Integrins are surface receptors that play a key role in cell-to-cell and cell-to-extracellular matrix interactions in diverse animal species. In gut immune responses, lymphocytes are expected to migrate to lymphoid tissues according to their expressed surface markers, and β₇ integrins are responsible for their efficient trafficking and retention in sites such as Peyer's patches, mesenteric lymph nodes, and intestinal lamina propria.

Genomic analysis of sponge and fish genomes showed maintenance of the integrin structure during the course of evolution.¹³ For means of comparison, ITGA4 and ITGB7 sequences from several different vertebrate specimens were retrieved from GenBank and compared at the α₄ virus binding extracellular domains (exons 5 and 6) and at the cytoplasmic domains of both α₄ and β₇ subunits. The GenBank accession numbers for all database sequences used herein are listed in Table 2. A few changes have been observed in the extracellular domains of α₄ from other mammals compared to humans (Supplementary Fig. S1; Supplementary Data are available online at www.liebertpub.com/aid), in accordance to previous work by our group.¹⁰ Sequences of α₄β₇ cytoplasmic tails from varied primates, including the chimpanzee (Pan troglodytes), the Sumatran orangutan (Pongo abelli), the gibbon (Leucogenys nomascus), the olive baboon (Papio anubis), the bonobo (Pan paniscus), and the rhesus macaque (Macaca mulatta), as well as from other mammals such as horse (Equus caballus), cow (Bos taurus), rat (Rattus novergicus), and sheep (Ovis aries), were also evaluated. There was a great conservation of amino acids within the salt bridge domain between the two subunits (amino acids 969–999 and 728–779 of the α₄ and β₇ mature peptides, respectively; Supplementary Fig. S2). In the α₄ cytoplasmic tail, the GFFKR motif was widely preserved among mammals. The same was observed for the corresponding sequence in the β₇ subunit, in which no amino acids change was observed. Phosphoserine at position 988 (human mature peptide) was conserved even for nonmammalian sequences such as frog (Xenopus laevis) and chicken (Gallus gallus). In the β₇ cytoplasmic domain, the phosphotyrosine was present in all species. This indicates that the integrin may have a determinant role in the formation of animal tissues, since it is closely linked to the formation of adhesions and tissue structures, even though it is not directly involved in cell migration in all examined species.¹³

Table 2.

GenBank Accession Numbers of Retrieved ITGA4 and ITGB7 Genes from Species Analyzed in the Present Study

Species name	Common name	ITGA4 GenBank accession	ITGB7 GenBank accession
Pan troglodytes	Chimpanzee	XM_525977.3	XM_001141622.2
Pongo abelli	Sumatran orangutan	XM_002812645.1	XM_002823316.2
Nomascus leucogenys	Gibbon	XM_003253800.2	XM_003252071.2
Papio anubis	Olive baboon	XM_003907685.1	XM_003906448.1
Pan paniscus	Bonobo	XM_003823539.1	XM_003831030.1
Macaca mulatta	Rhesus macaque	XM_001100929.2	XM_001087864.2
Equus caballus	Horse	XM_001917601.2	XM_001494867.1
Bos taurus	Cow	NM_174748.1	NM_001105365.1
Rattus novergicus	Rat	NM_001107737.1	NM_013171.1
Ovis aries	Sheep	XM_004004537.1	XM_004007388.1
Xenopus laevis	Frog	NM_001087975.1	—
Gallus gallus	Chicken	XM_421974.3	—

Recently, point mutations in the binding regions encoded by ITGA4 exons 5 and 6 were found to dramatically decrease the affinity of the HIV envelope for the α₄β₇ integrin.¹⁰ Functional assays of α₄ variants present in New World primates showed loss of binding to natural ligands such as VCAM-1 and MAdCAM-1.¹⁰ However, in light of our results we can infer that such changes are very unlikely to be encountered in humans.

A recent study showed that α₄β₇ integrin blocking induced by administration of anti-α₄β₇ monoclonal antibody in SIV infection during the acute phase directly decreased levels of plasma viremia.¹⁴ Such evidence highlights the importance of integrin binding domains in lentiviral pathogenesis and how mutations at these sites would be meaningful to the course of lentiviral disease progression. Even though SNPs in binding sites and cytoplasmic domains of ITGA4 and ITGB7 are reported in the NCBI dbSNP, the true frequency of these variants is not known and their statuses have not been validated. To date, no studies have been published examining the genotypic differences in ITGA4 and ITGB7 between HIV-seropositive patients and controls, and our results contribute by providing such information.

In addition to integrin extracellular sites responsible for adhesion molecule recognition, α₄β₇ function and affinity state also require specific elements present in both α₄ and β₇ cytoplasmic domains. The cytoplasmic tail of the β₇ subunit has an important role in the maintenance of high affinity or avidity to adhesive sites.¹⁵ Regulation of α₄β₇ conformation also depends on regulatory domains lying at the α₄ subunit. The membrane-proximal GFFKR motif in α₄ is a highly conserved site found in most α subunits. Conservation of carboxy-terminal cytoplasmic domains in α and β chains of integrin α₄β₇, just as in the extracellular regions, is therefore of pivotal importance to the role of integrins in the immune system.

In this study, we provided further understanding of the integrin conservation of binding sites of natural ligands such as VCAM-1 and MAdCAM-1, as well as of the cytoplasmic activation domains of both the α₄ and β₇ subunits. In our analysis, those selected regions were verified in both pediatric and adult infected patients, as well as in HIV-negative controls, and no amino acid changes were observed. These data are strongly congruent with the high conservation of the α₄β₇ integrin at domains crucial for integrin recognition of binding molecules and regulation of activation and affinity states. Hence, the absence of polymorphisms in the studied domains prevents any possible correlation with HIV patterns of disease progression or even viral acquisition as previously suggested.¹⁰

The present study did not assess variations in the virus ligand to the α₄β₇ integrin itself, that is, the LDI/V tripeptide motif found in the V2 of HIV-1 gp120. Yet a strong conservation of this tripeptide is apparent; entropic variations in this sequence have been recently traced to the differential dissemination potential of distinct HIV-1 genetic forms in China.¹⁶ Therefore, further investigation of the biology of this virus–host interaction is still warranted.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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