Loci Polymorphisms of the APOBEC3G Gene in HIV Type 1-Infected Brazilians

Abstract

The human APOBEC3G (A3G) protein activity obstructs retrovirus infection by inducing mutations of guanosines to adenosines (G → A) in the viral DNA. These G → A mutations may disrupt the reading frames of the viral genes. It has been observed that A3G polymorphisms can affect the degree of G → A mutations and the disease progression. For example, one study showed that the nonsynonymous substitution H186R was linked to AIDS progression in African Americans. Other studies, however, found no association between A3G polymorphisms and progression to AIDS in Europeans or in Asians. The genetic structure of a host population likely affects the dynamics of HIV-1 infection. The AIDS infection in Brazil is unique because of the high incidence of isolates with an unusual motif (GWGR) at the V3 region of the env gene. Since the Brazilian population is a mix of Portuguese, native Amerindians, and Africans we aimed to explore the influence of A3G polymorphisms in HIV-1 infection in this heterogeneous host population. We analyzed seven loci polymorphisms of A3G in 400 HIV-1-infected individuals naive to drug therapy. Our findings indicated no significant influence of A3G polymorphisms on disease status. The exception was the SNP −571 (rs5757463) in which heterozygous individuals (C/G) and homozygous individuals (G/G) presented lower CD4⁺ T cell counts compared to homozygous (C/C) individuals (Mann–Whitney test p-value = 0.0076). Furthermore, the loci diversity of A3G in Brazilians was similar to that of Europeans. Consequently, if there is any host factor that could be used to explain the peculiar subtype B HIV-1 infection in Brazil it is not associated with the innate immunity of the A3G gene.

Introduction

T he AID/APOBEC proteins are found in mammals and have cytidine deaminase activities that modify RNA or DNA.^1
–3 The inhibitory effect of cellular proteins APOBECs (apolipoprotein B mRNA-editing catalytic polypeptide) during human immunodeficiency virus type 1 (HIV-1) infection, particularly APOBEC3G, has been described as a component of innate antiviral immunity.^4,5 In the absence of human immunodeficiency virus type 1 (HIV-1) virion infectivity factor (Vif ), APOBEC3G is incorporated into newly synthesized viral particles through an interaction with Gag protein molecules,⁶ and hypermutate the viral DNA by deamination of cytosine (C) to uracil (U) in nascent minus-strand HIV-1 DNA, resulting in guanine (G) to adenine (A) substitutions in the coding-strand DNA.^7

–10 Previous studies have defined polynucleotides motifs within single-stranded DNA that are preferentially targeted by APOBEC3G (GG → AG).¹¹ The frequency of the mutation increased over the genome in the 5′ → 3′ direction, with the lowest frequency in the 5′ LTR and the highest in the 5′ portion of nef.¹²

To overcome the effects of the A3G protein activity, the viral Vif protein counteracts this by assembling a viral-specific E3 ubiquitin ligase through its interaction with cellular Cullin5 (Cul5)-ElonginB-ElonginC proteins. Vif induces polyubiquitination of APOBEC3G by tagging them for the proteasomal degradation pathway.^12

–15 Studies suggested involvement of Vif residues 33 to 83 in APOBEC3G binding,¹⁶ and that mutation of the Vif PPLP motif reduces Vif binding to A3G without affecting its interaction with ElonginC and Cullin5.¹⁷ In the A3G protein, amino acids 105–156 are required for interaction with Vif, and the degradation process requires amino acids 157–245.¹⁰

Single nucleotide polymorphisms (SNPs) have been described occurring along human A3G gene, but the effect on HIV-1 disease progression is not completely clear. The A3G gene was screened in American⁴ and European¹⁸ cohorts to identify genetic variants that might affect the progression of AIDS. An et al. ⁴ showed a correlation between A3G SNPs and disease progression; mainly the codon-changing variant H186R was linked with accelerated disease progression in African-Americans. Conversely, Do et al. ¹⁸ found no association between the variant H186R and progression to AIDS. A similar result was found by Valcke et al.,¹⁹ reporting a low prevalence of the H186R variant in whites (3%) and no association with disease progression. Recently, a study evaluating individuals from northern India has not found this genetic variant in the population.²⁰ It has been suggested that polymorphisms in the A3G gene are remnants of evolutionary encounters between our primate ancestors and retroviruses.²¹

In this work we surveyed 400 HIV-1-infected Brazilians and analyzed the allele frequencies in seven previously described A3G loci to determine A3G influence on viral loads and the levels of CD4⁺ T lymphocytes. To gain further insight about the A3G nucleotide variation and HIV-1 infection, we took advantage of the heterogeneous characteristic of the Brazilian population that is formed mainly by European explorers, settlers, and immigrants mixed with native Amerindians and African slaves.²²

Materials and Methods

HIV-1-infected individuals

Proviral DNA was extracted from heparinized peripheral blood obtained from 400 seropositive drug-naive patients enrolled in the AIDS program of the Brazilian Ministry of Health. The CD4 counts (cells/mm³) ranged from 20 to 5362 and the virus loaded ranged from 80 to 7.8 × 10⁷. The study group represented almost equally the male (55.5%) and female (45.5%) populations and was composed of three ethnic groups: white (49.2%), black (9.2%), and mulatto (41.6%) individuals. HIV-1-infected individuals were sampled from São Paulo City between 1989 and 2006. São Paulo City has an estimated population of more than 11,000,000 individuals living in the urban area (http://www.ibge.gov.br/) and is the most ethnically diverse in Brazil. It is estimated that more than 600,000 individuals are infected with HIV-1 in Brazil, with 46% of them located in São Paulo State (http://www.aids.gov.br/). All HIV lineages present in the Brazilian HIV epidemics have also been detected in São Paulo City (http://www.hiv.lanl.gov/). All samples were obtained from HIV-1-infected patients who regularly received medical assistance in the ambulatory care unit of the Federal University of São Paulo. The study was approved by the Ethics Committee of the Federal University of São Paulo, and the biological samples were obtained in full accordance with signed informed consent forms.

A3G polymorphisms

The A3G gene spans 9 kb on 22q13.1–13.2 and has eight exons and seven introns. DNA sequencing was performed to detect seven SNPs (seen Table 1 for the NCBI identities of these loci): three in the putative 5′ regulatory region (−571, −199, and −90), F119F in exon 3, H186R in exon 4, and two within introns (197 and 193). The DNA was extracted from blood samples with a QIAGEN DNA extraction kit according to the manufacturer's instructions. The entire APOBEC3G gene, including 2 kb upstream of the transcription start site and 1.0 kb downstream of the 3′ untranslated region, was amplified as two products of approximately 6.5 kb (3′ half ) and 8 kb (5′ half ) in size.

Table 1.

Genotypic Frequencies Loci Polymorphisms of A3G of Brazilians

	Population (sample)
Locus (NCBI SNP ID)	Brazilians	Europeans ^a	SubSaharan Africans ^a
−571 (rs5757463)	N = 400	N = 120	N = 120
GG	4 (1.0)	0 (0)	2 (1.7)
CC	348 (87.0)	112 (93.3)	102 (85.0)
G/C	48 (12.0)	8 (6.7)	16 (13.3)
−199 (rs34550797)	N = 400	N = 42	N = 48
GG	396 (99.0)	42 (100.0)	44 (91.6)
AA	0 (0)	0 (0)	2 (4.2)
G/A	4 (1.0)	0 (0)	2 (4.2)
−90 (rs5750743)	N = 400	N = 44	N = 48
CC	39 (10.0)	4 (9.1)	12 (25.0)
GG	194 (48.4)	20 (45.5)	18 (37.5)
G/C	167 (41.6)	20 (45.5)	18 (37.5)
119 (rs5757465)	N = 400	N = 44	N = 44
CC	45 (11.3)	6 (13.6)	0
TT	184 (46.0)	20 (45.5)	44 (100)
T/C	171 (42.7)	18 (40.9)	0
186 (rs8177832)	N = 400	N = 120	N = 120
AA	316 (79.0)	112 (93.3)	34 (28.3)
GG	9 (2.0)	2 (4.3)	26 (21.7)
A/G	75 (18.7)	6 (5.0)	60 (50.0)
197 (rs3736685)	N = 400	N = 46	N = 48
TT	316 (79.0)	42 (91.3)	20 (41.7)
CC	9 (2.0)	2 (4.3)	4 (8.3)
T/C	75 (18.7)	2 (4.3)	24 (50.0)
199 (rs2294367)	N = 400	N = 44	N = 46
CC	125 (31.2)	12 (27.3)	42 (91.3)
GG	107 (26.7)	14 (31.8)	0 (0)
G/C	168 (42.0)	18 (40.9)	4 (8.7)

Data from NCBI SNP databank at www.ncbi.nlm.nih.gov/projects/SNP/.

The primers used for A3G gene amplification were modifications from previously described oligonucleotides.^4,5 For the amplification of each product, 1 μl of DNA was amplified in a volume of 50 μl containing 10 × Hi-Fi PCR buffer, 2 mM MgSO₄ (1.5 mM MgSO₄ for the 3′ half ), 0.8 mM deoxynucleoside triphosphate mix, 100 nM of each forward and reverse primer, and 1 U of PlatinumTaq Hi-Fi. The following conditions were used: 95°C for 1 min, followed by 35 cycles of 95°C for 30 s, 61°C for 30 s (62°C for the 5′ half ), and a 6-min extension step at 68°C. Polymerase chain reactions (PCRs) were performed on Veriti thermocyclers.

The PCR products were then purified using the ChargeSwitch PCR Clean-Up Kit (Invitrogen, Carlsbad, CA, EUA) according to the manufacturer's protocol. Sequencing was performed using the BigDye Terminator kit, version 3.1 (Applied Biosystems/Perkin Elmer, Foster City, CA). The samples were electrophoresed on an ABI 3130 genetic analyzer, and the sequencing data were analyzed using ABI software Sequencing Analysis Software. The allele frequencies of the pooled DNA samples were determined by the chromatograph relative peak height and were compared to the allele frequencies for the reference sequence (NT_011520).

Statistical analyses

Linkage disequilibrium (LD) between pairs of loci was calculated using the absolute value of Lewontin's D method,²³ which assumes absolute values ranging from 0 for independence between loci to 1 when the pairs are completely linked. p-values represent the null hypothesis of independence. Haplotypes were inferred by the expectation maximization algorithm and the data were tested for Hardy–Weinberg equilibrium adequacy by calculating expected genotype frequencies and comparing them to the observed values using Arlequin software version 3.1 (http://cmpg.unibe.ch/software/arlequin3). Either chi-square with Yates' correction or Fisher's exact test (http://www.graphpad.com/quickcalcs/contingency1.cfm) was used to compare the observed allele and genotype frequencies of the Brazilian population to those of European and Africans.

Results and Discussion

The genotypic frequencies of seven loci in A3G are listed in Table 1. For comparative purposes, A3G frequencies found in Brazilians were compared to distinct datasets of Europeans and Africans (sub-Saharan) obtained from the Single Nucleotide Polymorphism database (www.ncbi.nlm.nih.gov/projects/SNP/). At all A3G loci, the genotypic frequencies found that Brazilians were similar to Europeans, with the exception of loci −571 and −199, which have identical frequencies in all datasets (Table 1). In addition, the H186R substitution that has been linked to AIDS acceleration in African-Americans⁴ was at a low frequency (2%) in Brazilians. Until the early nineteenth century, Portuguese, native Amerindians, and Africans (brought to work as slaves in sugar cane plantations) comprised the Brazilian population. However, between 1820 and 1964, at least three large waves of immigration occurred in Brazil; the main groups were Portuguese, Spaniards, Italians, Japanese, and Germans. They mainly colonized the south and southeast regions of the country (http://www.ibge.gov.br). These ethnic groups have been mixing to a varying degree; however, the genetic composition of the Brazilian population is not heterogeneous. It has been shown that there is a strong genetic bias due to an admixture predominantly of European men with Amerindian and African women.^24,25 Our results with the genotypic frequencies of A3G are in conformity with previous studies based on other genes,^26,27 which also indicated that the frequencies of European colonization had a strong impact on the genetic composition of the Brazilian population.

A nonindependence of allele segregation was detected due to the strong linkage in all pairs of loci in the Brazilian A3G dataset (Table 2). The LD revealed a nonindependence of allele segregation because all pairs of loci analyzed were strongly linked in the Brazilian A3G gene. It is likely that the strong linkage detected in all alleles reflects the physical proximity of these loci in the gene region of A3G, which is limited to 9 kb.

Table 2.

Hardy–Weinberg Disequilibrium of A3G Loci

Locus	Chi-square	p-value
(−571)	2.4549	0.1172
(−199)	0.0101	0.9199
(−90)	0.122	0.7268
(119)	0.3041	0.5813
(186)	3.0601	0.0802
(197)	3.0601	0.0802
(199)	10.023	0.0015

Likewise, most of the A3G loci in Brazilians followed the Hardy–Weinberg (HW) equilibrium, except locus 199, which significantly deviated from the equilibrium (Table 3). The HW model states that allele frequencies remain constant over time (generation after generation) in idealized large populations where there is random mating, no selective mutation, no effect of genetic drift on the population, and no migration. Then, we would expect a departure from the HW model with the contribution of non-European ethnic groups to the genetic pool of the Brazilian population. However, our results showed that all loci were in equilibrium, except locus 199, which significantly deviated from equilibrium. It is likely that departures from the HW model may happen due to a small population, but it has no effect on association analysis.²⁸

Table 3.

Linkage Disequilibrium of A3G Loci ^a

Locus pair	Lewontin's D	p-value
(−571 × −199)	1	0.0343
(−571 × −90)	0.9228	0.0286
(−571 × 119)	1	0.0244
(−571 × 186)	1	0.0278
(−571 × 197)	1	0.0278
(−571 × 199)	0.9252	0.0204
(−199 × −90)	1	0.0309
(−199 × 119)	1	0.0251
(−199 × 186)	0.1513	0.0142
(−199 × 197)	0.1513	0.0142
(−199 × 199)	1	0.0221
(−90 × 119)	1	0.0216
(−90 × 186)	0.8596	0.0218
(−90 × 197)	0.8596	0.0218
(−90 × 199)	0.9145	0.0184
(119 × 186)	1	0.0239
(119 × 197)	1	0.0239
(119 × 199)	0.934	0.0215
(186 × 197)	1	0.0312
(186 × 199)	0.9099	0.0199
(197 × 199)	0.9099	0.0199

The measure of allele disequilibrium between pairs of locus was performed according to the Lewontin's D method.

Statistical analyses revealed that most loci of A3G were not correlated with CD4⁺ T cell counts or with the viral level in Brazilians. Specifically, no association was detected with a 5% significance level between A3G SNPs and CD4⁺ T counts. However, we found one exception with SNP −571 (rs5757463), which was correlated with CD4⁺ T cell counts (Mann–Whitney test p-value = 0.0076). This locus is in an untranslated region of the A3G gene. We found that heterozygous individuals (C/G) and homozygous individuals (G/G) tend to have lower CD4⁺ T cell counts compared to homozygous (C/C) individuals. It is not clear how this polymorphism influences A3G activity because it is located in an untranslated region of the gene, unless it is located in a DNA region that regulates the transcription of the A3G protein. Though A3G expression has been observed to change during HIV infection,^29,30 mRNA levels of A3G may not affect CD4⁺ T cell counts or viral load.³⁰

Conclusions

HIV-1 epidemics in Brazil are complex with distinct lineages and recombinant forms circulating in the same regions.^31

–37 In addition, subtype B infection in Brazil is characterized by the occurrence of more than 30% of viruses with the uncommon GWGR motif in the V3 loop of the env gene. These GWGR isolates have been associated with slower disease progression and restricted cellular receptor usage.^32,36 This diversity of distinct motifs at the V3 loop is not explained by characteristics related to the genetic composition of HIV-1 because all B isolates in Brazil share a common ancestor. The genetic makeup of an individual may equally influence the susceptibility to HIV-1 infection and disease progression. Thus, the genetic background of human hosts could help to explain the uniqueness of subtype B infection in Brazil. However, our findings indicated no significant influence of A3G polymorphisms on disease status. Furthermore, the loci diversity of A3G in Brazilians was similar to that of Europeans. Consequently, if there is any host factor that could be used to explain the peculiar subtype B HIV-1 infection in Brazil it is not associated with the innate immunity of the A3G gene.

In conclusion, although A3G alleles were fixed in the human population due to recurrent ancient viral infections, the antiviral property of this gene can still be observed in modern-day viruses, such as in HIV. A3G polymorphisms are likely to affect AIDS in many instances; however, our study showed that most A3G SNPs had little impact on disease parameters such as CD4⁺ T cell levels and viral load.

Footnotes

Acknowledgments

We would like to express our sincere gratitude to all patients who contributed to this study. The authors also extend their gratitude to the AIDS program of the Brazilian Ministry of Health for providing access to patient information and blood samples. This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Foundation for the Support of Research in the State of São Paulo; Grant 06/50109-5). The first two authors contributed equally to the study.

Author Disclosure Statement

No competing financial interests exist.

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