Two single nucleotide polymorphisms in IRF-1 associated with resistance to HIV-1 infection
The identification of IRF-1 as a candidate gene to explain, at least partly, natural resistance to infection by HIV-1 is supported by considerable data demonstrating that IRF-1 plays a key role in host immunity [19,20], as well as mediating HIV-1 early transcription and replication [21,22]. A previous comparative sequence analysis of the IRF-1 gene and its promoter region demonstrated that IRF-1 is highly polymorphic containing at least 53 individual single nucleotide polymorphisms (SNP) . Association analysis of all polymorphisms revealed that two SNP at position 619 (A > C; rs17848395) and 6516 (G > T; rs17848424) were differentially represented in HIV-1-resistant women compared with HIV-1-susceptible subjects (Table 2). The other polymorphisms showed no association with the HIV resistance phenotype. The 619A and 6516G alleles were significantly associated with HIV-1 resistance, whereas the alternative alleles (619C and 6516T) were associated with the HIV-1-susceptible phenotype (P = 0.00068 and 0.030, respectively). Genotype analysis confirmed this, demonstrating that genotypes containing 619A, 179 at IRF-1 microsatellite and 6516G were all significantly overrepresented in HIV-1-resistant subjects (Table 2). After correcting for multiple comparisons only the 619 SNP remained significant (data not shown). Kaplan–Meier and Cox regression survival analysis demonstrated that initially HIV-1-uninfected subjects with at least one copy of the 619 allele showed significantly longer HIV-1-free survival and decreased seroconversion compared with those lacking this allele (P = 0.002, HR 0.528, 95% CI 0.353∼0.790, log rank 10.00; Fig. 1b). Although not significant, a trend was observed for the 6516G allele (Fig. 1c).
Polymorphisms associated with resistance to HIV infection co-segregate, revealing the 619 single nucleotide polymorphism as the strongest independent determinant
Linkage disequilibrium (LD) is common in IRF-1  and among the three polymorphisms associated with resistance to HIV-1 infection. The three protective IRF-1 alleles (619A, 179 and 6516G) tend to co-segregate. Pairwise LD analysis of 619A: 179 microsatellite; 179 microsatellite: 6516G; or 619A: 6516G showed that these three polymorphisms were in significant LD (for all P < 0.0001). Log linear multivariate modelling and logistic regression analysis identified the 619A allele as the primary independent association with HIV-1 resistance (P = 0.0022), whereas the other polymorphisms were associated with the resistance phenotype probably as a result of LD with 619A (P = NS for both excluding LD effects). Furthermore, multivariate regression analysis determined that this effect was independent of the previously reported association with the HLA A2/6802 supertype (data not shown) [13,14].
Haplotypes containing more than one protective allele associated with resistance to HIV-1 infection
Although 619A was the primary allele associated with HIV-1 resistance, it is possible that combinations of protective alleles provide a stronger association with HIV-1 resistance and reduced seroconversion as suggested in Table 2 and Figure 1, respectively. We conducted haplotype analysis, where possible, in individuals homozygous for two or more of these alleles. We found that all combinations of protective alleles (619A + 179, 179 + 6516G, 619A + 6516G and 619A + 179 + 6516G) showed an even more significant association with protection from HIV-1 infection (OR 1.8–2.71 and P = 0.0114, 0.005, 0.0006, and 0.008, respectively; Table 2). Therefore, compared with individual alleles, additional alleles provide additive protection against HIV-1 infection. The numbers of subjects with discrete haplotypes were insufficient to conduct survival analysis.
When we assessed the combinatorial effects of having at least one copy of either the 619A, 179 or the 6516G allele, we found that 79.3% of HIV-1-resistant women carried at least one of these alleles compared with 55.6% of susceptible women (P = 0.0003, OR 2.97, 95% CI 1.61∼5.53). In survival analysis, we found that initially uninfected subjects having any one of these protective alleles had significantly longer HIV-1-free survival and reduced likelihood of seroconversion compared with individuals lacking these alleles (P = 0.0001, HR 0.458, 95% CI 0.305∼0.689, log rank 14.81; Fig. 1d). Interestingly, the strength of these associations as determined by hazard ratios suggested that the protective effect may be similar to individual alleles. Efforts to determine the combinatorial protective effects of one, two or all three of the protective alleles were unfeasible as a result of small sample sizes.
IRF-1 polymorphisms not associated with varied HIV-1 disease progression
To determine whether polymorphisms in IRF-1 play any role in HIV disease progression, we conducted Kaplan–Meier survival analysis to compare the duration between the first positive day in the cohort and time to CD4 cell decline to less than 200 cells/μl, clinically defined as progression to AIDS. The results are depicted in Figure 2. No significant differences were observed between subjects with or without the 619A or 6516G alleles (P = 0.8572 and 0.2405). A marginally significant difference was observed for the 179 microsatellite, with heterozygous subjects appearing to show faster progression (P = 0.0426). These data suggest that the protection offered by specific IRF-1 genotypes may only be active during the initiation of HIV-1 infection but not after infection is fully established.
IRF-1 genotypes that associate with resistance to HIV-1 correlate with reduced basal IRF-1 expression
With the exception of early embryonic cells, IRF-1 is expressed at low basal levels in all cell types . Western blot analysis showed no electrophoretic pattern difference between PBMC from subjects with different IRF-1 genotypes. Quantitative analysis indicated that PBMC from subjects homozygous for the 619A allele (619AA) had significantly less IRF-1 protein levels compared with those from 619A/C heterozygotes or 619CC homozygotes (P = 0.0011 and 0.0010, respectively, Fig. 3a). Similarly, 6516G homozygotes showed reduced basal IRF-1 protein compared with heterozygous (6516GT) or 6516TT homozygotes (P = 0.0028 and 0.0015, respectively, Fig. 3b). A similar trend was observed comparing subjects with different IRF-1 microsatellite genotypes, although a significant difference was observed only between individuals homozygous for the 179 allele and individuals lacking the 179 allele entirely (P = 0.009; Fig. 3c).
IRF-1 genotypes that associate with resistance to HIV-1 also correlate with reduced responsiveness to exogenous IFN-γ stimulation
Using IFN-γ, a potent and commonly used IRF-1 stimulus, we determined whether polymorphisms in IRF-1 associated with altered IRF-1 responsiveness to exogenous stimulation. PBMC from individuals homozygous for alleles associated with HIV-1 resistance at 619 and 6516 (619A and 6516G) showed a significantly lower fold increase in IRF-1 expression compared with individuals heterozygous or homozygous for the alternative alleles (P = 0.0017 and 0.0019 for 619AA versus 619AC or 619CC, respectively; P = 0.0019 and 0.0008 for 6516GG versus 6516GT or 6516TT, respectively). No differences were observed between individuals heterozygous and homozygous for the non-associated alleles (Fig. 3d and e), nor among the different IRF-1 microsatellite markers (Fig. 3f).
In the present study, we identified a single microsatellite allele (IRF-1 179) within the IL-4 gene cluster associating with the HIV-1 resistance phenotype and protecting against seroconversion in prospective survival analysis. Comparative sequencing of the IRF-1 gene in this population revealed two additional SNP (619A > C and 6516G > T) that correlated with the HIV-1-resistant phenotype and a reduced likelihood of seroconversion. Logistic regression suggested that 619A was the primary association with HIV-1 resistance. Haplotype analysis suggested, however, that the protective effect of these alleles was additive. Regardless of the dominance of one particular allele it is clear that one or more of these polymorphisms is associating strongly with resistance to infection by HIV-1. Further analysis is underway to try to understand the addition, or independence of these observations, including efforts to assign IRF-1 haplotypes better. Perhaps most interestingly, however, these polymorphisms did not associate with altered disease progression, suggesting that protection may be limited to the initial stages of HIV-1 infection but not after infection has been established.
These polymorphisms are all located within introns in the IRF-1 gene, and how these non-coding changes affected IRF-1 expression was not clear. Measuring IRF-1 protein levels directly, we demonstrated PBMC from subjects with protective IRF-1 genotypes had lower IRF-1 protein levels both at baseline and in response to exogenous IFN-γ stimulation. We note that differences in protein expression were most significant only between the extreme genotypes for all three polymorphisms (619AA versus 619CC, 6516GG versus 6516TT, two ‘179’ versus no ‘179’), whereas the genetic data suggest a dominant genetic effect. We believe that as IRF-1 regulation is extremely complex, we may only be capable of observing the biological effects between the most extreme genotypes. The mechanism of how these intronic polymorphisms alter IRF-1 expression is unknown. Polymorphism in introns can affect gene expression by influencing transcription, messenger RNA splicing, translation, mRNA stability, or promoter activity [24–30]. Functional mechanistic studies are underway to establish how these polymorphisms affect IRF-1 protein levels.
Why does reduced IRF-1 associate with resistance to infection by HIV-1? IRF-1 is a transcriptional activator of interferon-inducible genes via binding to interferon-stimulated response elements in their promoter regions . IRF-1 plays a critical role in the regulation of host cellular immune responses, and it has been well demonstrated in IRF-1 knockout mice that these mice demonstrate defects in CD8 T-cell development, a CD4 T-cell shift from Th1 to Th2 cytokine expression, decreases in the numbers and function of natural killer cells, and decreased MHC and polymeric immunoglobulin receptor expression [19,20]. Studies on HIV-1-resistant women from this cohort have demonstrated that these women exhibit a Th1-biased response with HIV-1-specific cellular, but not humoral, responses, as well as a compartmentalized mucosal antibody response [6,8,9]. IRF-1 probably plays a critical role in the generation of these immune responses. That the protective IRF-1 genotypes associate with lower, but not higher IRF-1 protein expression is somewhat counterintuitive, as one would assume that this would result in lower interferon responses and perhaps a blunted cellular response. This does not occur, however, as HIV-1-resistant women have cell-mediated immune responses to HIV-1. This suggests that the generation of presumably protective immune responses against HIV-1 is not solely dependent on IRF-1 levels or that only a low threshold level of IRF-1 is required to maintain its normal physiological functions.
How then, may lowered IRF-1 expression result in resistance to infection by HIV-1? Reconciliation may lie with the potential role of IRF-1 in modulating HIV-1 replication. IRF-1 binds to an interferon-stimulated response element-like sequence in the HIV-1 5′–long terminal repeat and initiates HIV-1 transcription independently of Tat [21,31]. IRF-1 may act as the primary initiator of HIV-1 transcription before Tat and later synergize with Tat in amplifying HIV-1 replication . It is reasonable to speculate that lowered baseline IRF-1 expression and reduced IRF-1 responsiveness may limit HIV-1 transcription and reduce viral replication at the initial stages of infection. That is, reduced IRF-1 expression and responsiveness in women with protective IRF-1 genotypes may partly protect them from infection through an inability to support the initial phases of HIV replication. This ‘natural’ protection may not have been apparent in previous studies showing that cells from HIV-resistant women can support HIV replication , as these studies and others like them used polyclonal activators such as phytohaemagglutinin to stimulate target cells, which may overwhelm any deleterious effects of IRF-1 in cells from these women.
This reduced initial replication could lead to a widened period allowing host immune responses to develop. This is supported by several non-human primate studies in which suboptimal viral inoculation can elicit protective mucosal and systemic responses . Early control, or a reduction of HIV-1 replication before uncontrolled viral replication has been postulated as a key phase in blocking HIV-1 infection . That protective IRF-1 genotypes are associated with reduced susceptibility but not prolonged HIV disease progression add further weight to this hypothesis. Further studies on the functional consequences of the IRF-1 polymorphisms in IRF-1 mRNA splicing and IRF-1-regulated immune responsiveness will provide a more comprehensive understanding of the mechanisms underlying this association. A more direct proof of IRF-1 protection against infection may come from identifying HIV-1 infection in unstimulated cells from subjects with altered IRF-1 genotypes. These studies are underway.
This study adds to the number of factors both genetic and immune mediated that associate with altered susceptibility to HIV-1. As a result of the complex nature of HIV susceptibility and immunity to HIV, it is likely that resistance is polygenic in nature, and that there is more than a single factor involved. Whenever possible, we conducted multivariate analysis to determine that the association between IRF-1 polymorphisms and the HIV-1-resistant phenotype was independent of previously identified HLA supertypes . The interaction of these apparently independent genetic factors needs to be assessed in larger studies, and in other exposed uninfected cohorts. It is likely that all these different factors may provide alternative and perhaps complementary mechanisms protecting these women from infection. A previously identified polymorphism in the IL-4 promoter (−589C > T), which had been suggested to affect viral phenotype switch and HIV-1 disease progression [34,35], showed no correlation with the resistance phenotype (data not shown). We recently identified an association with the HIV-resistant phenotype and the expression of regulated upon activation: normal T-cell expressed/secreted in the genital tract. Although the numbers in the previous study were small (n = 10) further determination of both regulated upon activation: normal T-cell expressed/secreted levels in all HIV-resistant women and the association with IRF-1 genotypes are ongoing.
In conclusion, we have identified an association between polymorphisms in the IRF-1 gene and resistance to infection by HIV-1 in a Kenyan female sex worker cohort. Lowered IRF-1 expression may be the mechanism underlying this association. This study demonstrates that polymorphisms in an immunomodulatory and transcriptional regulatory gene were associated with altered susceptibility to HIV-1. This is the first report suggesting that altered host expression of a viral transcriptional regulator might contribute to the development of resistance to HIV-1. Clearly, these results are preliminary, and need to be confirmed in other study populations of exposed uninfected individuals, or more importantly, in a prospective, blinded manner to see if these polymorphisms affect HIV susceptibility directly.
The authors would like to thank Drs X. Mao, M. Luo, X. Yao, S. Ramdahin and L. Slaney, for technical support, as well as J. Waruk, L. McKinnon and S. Iqbal for critical review of the manuscript.
Sponsorship: This work was supported by grants from the National Institute of Health (RO1 A156980) and Canadian Institute of Health Research (CIHR, HOP43135). F.A.P. holds a tier I CIHR Canada Research Chair in resistance and susceptibility to infections. H.J. is a recipient of an International Center of Infectious Diseases (ICID)/CIHR national doctoral training award and Manitoba Health Research Council doctoral scholarship.
The first two authors contributed equally to this work.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
Gene polymorphism; IL-4 gene cluster; interferon regulatory factor 1; resistance to infection by HIV-1