HECT Domain and RCC1-Like Domain-Containing Protein 5 ( HERC-5 ) Gene Polymorphisms in HIV-1-Infected Individuals: A Study from India

Introduction

Human immunodeficiency virus-1 (HIV-1) primarily infects the T cells, integrates into the host genome, and uses its cellular machinery to produce progeny virus particles that are then released from the infected cells. ¹ Targeting the host cellular proteins that are vital for viral replication may help in controlling the viral replication. ² Hence, a better understanding of different host proteins that restrict or promote HIV replication is crucial toward the development of novel therapeutic targets. Various host factors have been implicated in restricting HIV growth by interfering at different stages of the HIV replication cycle, such as APOBEC3, TRIM5α, BST-2/Tetherin, SAMHD1, MOV10, ISG15, HERC5, as well as cellular miRNAs. ^{3 –6} HECT domain and RCC1-like domain-containing protein 5 (HERC-5) is one of the novel host restriction factors that has been recently shown to inhibit HIV-1 Gag particle assembly by a mechanism correlating with the post-translational modification of Pr55Gag-(P6) with interferon stimulating gene (ISG)-15 and, hence, restricting the release of mature virion in vitro. ¹ HERC-5 protein contains a C-terminal HECT domain and an N-terminal regulator of chromosome condensation 1 (RCC1)-like domain and is encoded by the HERC5 gene located on chromosome-4. In vitro, HERC-5 has been shown to inhibit HIV-1 particle production in the U2OS cell line. ¹ Higher HERC-5 expression was noted in HAART-treated patients with undetectable viral copies as well as in elite controllers as compared with HIV-1 seronegative individuals, ⁷ indicating its probable contribution toward restricting viral growth. Woods et al. have recently shown that the HERC5-C994A mutant failed to inhibit HIV-1 particle production under in vitro conditions ¹ ; hence, it is likely that HIV disease progressors express a nonfunctional HERC-5 variant, impairing the restriction of viral budding. A polymorphism in HERC5, single-nucleotide polymorphism (SNP)-rs34457268 (Exon-18), as predicted in human beings from the dbSNP database, leads to production of a truncated HERC-5 protein lacking the Cys994 active site, ^1,8 which subsequently facilitates the viral budding and release. Previously, polymorphisms in other host factors such as TRIM-5, ⁹ BST-2, ⁴ and SAMHD-1 ⁵ have been shown to influence HIV susceptibility and, hence, disease progression. However, the association of the polymorphism in the HERC-5 gene with HIV disease progression is hitherto not reported. We, therefore, studied the polymorphism of the HERC-5 gene in HIV-infected individuals with different clinical outcomes and explored its association with disease outcomes.

Forty-one (17M/24F) progressors (antiretroviral therapy [ART] naive HIV-1-infected individuals with CD4 < 500 cells/mm³), 39 (11M/28F) HIV-1 long-term nonprogressors (LTNPs are asymptomatic with a known HIV seropositivity, stable CD4 count above 500 cells/mm³ for the entire follow-up period of seven or more years and are ART naive), and 74 (54M/20F) HIV seronegative healthy individuals (HC) were enrolled in the study. The median age of LTNPs, progressors, and HC was 37 (range 28–58), 35 (range 17–49), and 28 (range 18–52) years, respectively. At the time of enrollment in this study, the median CD4 count was 320 and 699 cells/mm³ in progressors and LTNPs, respectively. Cryopreserved peripheral blood mononuclear cells (PBMCs) from 20 ml whole blood from all the study participants were used in this study. The study was approved by the Institutional Ethics Committee, and prior written informed consent was obtained from all the participants.

Overall, 1 × 10⁶ PBMCs were used for DNA extraction by using QIAamp DNA kit (QIAGEN, Inc., Valencia, CA) as per the manufacturer's instructions and stored at −20°C for further genotyping assay. Different sets of primers were designed by using Primer-3 and Oligo Analyzer software to determine the known SNP (rs34457268) as well as unknown SNP in the functional and regulatory regions UTR5′ and UTR3′ of HERC-5. The sequences of polymerase chain reaction (PCR) primers and the amplification condition for HERC5 SNP region (rs34457268), functional and regulatory regions are given in Table 1. For the amplification of UTR5′+Exon-1 region, 3% dimethyl sulfoxide was used to get an optimum amplified product due to its high GC content. Amplified DNA was checked on 1% agarose gel for expected bands of template size as mentioned in Table 1. PCR amplicons were purified by using High Prep PCR clean up kit (MagBio Genomics, Inc.) followed by sequencing by using Big Dye terminator sequencing kit (Applied Biosystems, Foster City, CA) as per the manufacturer's instructions. Sequencing was performed on an automated sequence analyser of 3730xl DNA analyzer (Applied Biosystems). About 10% of samples were randomly picked for repeat assays, and the final concordance rate was 100%. Sequenced data were acquired and analyzed by using SeqScape^® Software v2.6 data tracking, management, and storage system. The direct DNA sequencing technology was used to validate the genotypes of the three polymorphisms, and the results were consistent with reference genotypes. The UCSC database was used as a reference gene database to analyze any polymorphic changes if present in the HERC-5 gene at various regions.

The χ² goodness-of-fit test was used for any deviation from the Hardy–Weinberg equilibrium in healthy controls. We used the χ² statistic (Fisher's exact test for cell size <05) to compare the genotype frequency of the Intron-18 region polymorphism between the study groups. Odds ratios (ORs) and 95% confidence interval (95% CI) were calculated by unconditional binary logistic regression. All statistical analyses were performed by using the SPSS software version 17.0 (SPSS, Chicago, IL), and tests of statistical significance were two sided and taken as significant when p-value was ≤.05.

We screened a total of 154 individuals (41 progressors, 39 LTNPs, and 74 healthy individuals) for known SNP in Exon-18 (rs34457268) ^1,8 of the HERC5 gene. No change in the nucleotide sequence was observed for rs34457268 in the Exon-18 region. Instead, we noted a synonymous single-nucleotide change from T to C at another reference site of Exon-18, that is, rs6857425. Sequences identified with synonymous SNP can be accessed through GenBank and dbSNP ID, respectively (accession numbers KX356043 and ss2019497319). The HERC5 Exon-18 sequence of a total of 152 samples has been submitted to GenBank (Submission ID: MF198465-MF198616). The synonymous mutation at this site was present in all the study participants irrespective of their HIV status. Further sequence analysis revealed that there was no change in the other functional and regulatory sites of the HERC-5 gene: UTR-5′, Exon-1, Exon-23, and UTR-3′ in any of the study groups. Synonymous/silent mutations have gained significant interest over the past few years. Genome-wide association study (GWAS) and other studies have also highlighted that synonymous SNPs also contribute to human disease risk and other complex traits. ^{10 –14} Synonymous mutations may influence the biological effects by either affecting the cis regulatory splice sites or altering the transcription factor binding sites. In addition, these can also lead to an altered mRNA structure, resulting in low protein expression. ¹⁴ Chen et al. have further shown similar likelihood of disease association with synonymous SNPs as that of nonsynonymous SNPs. ¹² We found synonymous SNP at the Exon-18 region of the HERC-5 gene. Since the HERC-5 region contributes toward the postentry modification of various host and viral proteins, ^1,3 mutation in Exon-18, though synonymous, possibly indicates its role in restricting the release of viral proteins ¹⁴ ; however, it needs a more extensive investigation.

We also detected two variants of Intron-18 of the HERC-5 gene at chr4:88494400 (rs74876955) and chr4:88494489 (rs number not allotted to dbSNP). (NCBI dbSNP database accession numbers: ss2137497742 and ss2137497743, respectively). The genotype and allele frequencies of HERC-5 Intron-18 in HIV-infected individuals and healthy controls are shown in Tables 2 and 3. The genotype distributions of HERC5 Intron-18 polymorphisms in controls were in the Hardy–Weinberg equilibrium (p = .756). The frequency distribution of rs74876955 for C allele and CC genotype was predominant in all the study groups. No significant difference was noted in any genotype between LTNP and HIV-1 progressors (OR: 3.33, 95% CI: 0.33–33.49, p = .306), HIV-1 progressor and healthy controls (OR: 2.89, 95% CI: 0.327–25.69, p = .339), and LTNP and healthy control (OR: 0.870, 95% CI: 0.197–3.84, p = .854) (Table 2).

Similarly, the frequency distribution of another SNP at chr4:88494489 (rs- number not assigned in dbSNP) of G allele and GG genotype was predominant in all the study groups (Table 3), with no significant difference between LTNP and HIV-1 progressors (OR: 0.685, 95% CI: 0.10–4.33, p = .687), HIV-1 progressor and healthy controls (OR: 0.918, 95% CI: 0.208–4.05, p = .910), and LTNP and healthy control (OR: 1.34, 95% CI: 0.24–7.24, p = .734) (Table 3).

Overall, these findings indicate that the SNPs in Intron 18 at chr4:88494400 (rs74876955) and chr4:88494489 are not associated with HIV infection or disease progression. Our findings for the HERC5 polymorphism are similar to those observed with the APOBEC3G gene polymorphism, where Li et al. ⁶ have identified three intronic SNPs and a synonymous codon SNP at rs5757465 of the APOBEC3G gene that were not associated with HIV infection.

To the best of our knowledge, this is the first study to report HERC-5 gene polymorphisms among HIV-1-infected patients. We found that a known SNP (rs34457268) at Exon-18 of the HERC-5 gene is neither associated with HIV-1 susceptibility nor associated with disease progression in our study population. Instead, we report a synonymous SNP (rs6857425) in Exon-18 that was present in all the study groups, indicating that this SNP is rather conserved among Indians although the sample size is small in this exploratory study. Further, SNPs detected at the intronic region may have some role in the regulation of HERC-5, which needs further investigation with a larger group of individuals. Additional studies are, however, warranted to determine the functional consequences of novel variants in other exon, intron, and/or functional region(s) of HERC-5 to HIV susceptibility and/or disease progression.