TLR7 dosage polymorphism shapes interferogenesis and HIV-1 acute viremia in women

Type I interferon (IFN-I) production by plasmacytoid dendritic cells (pDCs) occurs during acute HIV-1 infection in response to TLR7 stimulation, but the role of pDC-derived IFN-I in controlling or promoting HIV-1 infection is ambiguous. We report here a sex-biased interferogenic phenotype for a frequent single-nucleotide polymorphism of human TLR7, rs179008, displaying an impact on key parameters of acute HIV-1 infection. We show allele rs179008 T to determine lower TLR7 protein abundance in cells from women specifically, likely by diminishing TLR7 mRNA translation efficiency through codon usage. The hypomorphic TLR7 phenotype is mirrored by decreased TLR7-driven IFN-I production by female pDCs. Among women from the French ANRS PRIMO cohort of acute HIV-1 patients, carriage of allele rs179008 T associated with lower viremia, cell-associated HIV-1 DNA, and CXCL10 (IP-10) plasma concentrations. RNA viral load was decreased by 0.85 log 10 (95% CI, − 1.51 to − 0.18) among T/T homozygotes, who also exhibited a lower frequency of acute symptoms. TLR7 emerges as an important control locus for acute HIV-1 viremia, and the clinical phenotype for allele rs179008 T, carried by 30%–50% of European women, supports a beneficial effect of toning down TLR7-driven IFN-I production by pDCs during acute HIV-1 infection. Analysis of IFN- a production by blood mononuclear cells . PBMCs were freshly isolated from whole blood by Pancoll (PAN-Biotech) gradient separation, and resuspended in R10 medium, i.e., RPMI 1640 (Sigma-Aldrich) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The cells were seeded into 96-well plates (3 × 10 5 cells/well) and stimulated with either resiquimod (R-848; 1.5 µ g/ml or 0.3 µ g/ml) or imiquimod (R-837; 1.5 µ g/ml; both from InvivoGen). IFN- a was quantified by ELISA (PBL Biomedical Laboratories) in culture supernatants sampled 6, 12 and 24 hours after stimulation. Supernatants from 24-hour cell cultures without any TLR7 ligands were used as controls. ELISA values were normalized to the percentage of pDCs in the respective PBMC preparation, and expressed as IFN- a produced per 10 3 pDCs as described elsewhere (25).


Introduction
The intracellular Toll-like receptors, TLR3, TLR7, TLR8 and TLR9 recognize autologous and microbial nucleic acids and are critical components of innate antiviral immunity (1). RNA and DNA ligands engaging the endosomal TLRs of plasmacytoid dendritic cells (pDCs), respectively TLR7 and TLR9, elicit a vigorous production of the type I interferons, IFN-a and -b (IFN-I) (2). Cell-associated and, less efficiently, cell-free HIV-1 virions signal predominantly through TLR7 (3,4), and elevated plasma IFN-I marks the acute and late chronic phases of HIV-1 infection (5,6).
Studies of macaques infected with simian immunodeficiency virus (SIV) to model the pathogenesis of human AIDS identified pDCs as the critical source of IFN-I in vivo (7)(8)(9)(10). Blocking the IFN-I receptor early during acute SIV infection increased the viral burden and led to distinctly shorter times to death from AIDS (11). Conversely, continued treatment with IFN-I resulted in increased SIV replication and accelerated CD4 T cell loss, suggesting that the time pattern of IFN-I signaling is determinant in the outcome of SIV infection (11).
In line with the macaque model, depleting the pDCs of humanized mice prior to HIV-1 infection boosted viral replication, and abolished serum IFN-I elevation and the expression of interferon-stimulated genes (12). These investigations are supportive of a beneficial role for IFN-I during early-stage HIV-1 infection, opposing the spread of the virus and its expansion within lymph nodes. Nonetheless, innate immunity might play a paradoxical role at the mucosal epithelial barrier shortly after HIV-1 entry by facilitating viral dissemination (13,14). A double-edged effect of IFN-I is revealed also by the course of SIV infection in its natural host, such as African green monkeys, where the IFN-I response is toned down during the acute to chronic phase transition and the animals do not progress to AIDS despite patent viremia (15). The non-pathogenicity of SIV in African green monkeys is linked to lower expression of interferon-stimulated genes in the blood and tissues (lymph nodes, gut) of this host during chronic SIV infection, by comparison with macaques and humans infected respectively with SIVmac and HIV-1 (15,16). A deleterious role of pDC-derived IFN-I production has been illustrated also by the lymphocytic choriomeningitis virus (LCMV) murine model of persistent viral infection. Here, blockade of IFN-I signalling, induced even before infection, promoted viral clearance (17,18). Long-term viral control brought about by early IFNAR-blockade was associated with down-regulation of IL-10 and PD-L1, improved CD4 + T cell function and restoration of lymphoid architecture (17,18).
These observations highlight the importance of the timing and duration of IFN-I signalling to the balance of its opposite antiviral and immunosuppressive effects during acute and chronic HIV-1 infection (19). We have exploited natural genetic variation to investigate the direct contribution of the TLR7-driven production of IFN-I by pDCs in the course of acute HIV-1 infection. Variation in the genes for TLR7 and the other nucleic acid-sensing intracellular TLRs is constrained by strong purifying selection against mutations suppressing or modifying the function of the encoded receptor (20). Single-nucleotide polymorphisms (SNPs) of the TLR7 gene, which is located on the X-chromosome, have nevertheless been associated with changes in parameters of HIV-1 infection (21,22). We chose to study a frequent SNP of the TLR7 coding sequence, rs179008 (NM_016562.3:c.32A>T), which was found to be associated with higher viral load at set point and accelerated decline of CD4 + T cell counts in male patients, and with a diminished cytokine response to a TLR7 agonist in the leukocytes of male allele-T carriers (22). This SNP was also, intriguingly, associated with higher prevalence of HIV-1 infection in women (22). The minor allele rs179008 T is especially frequent among European populations, where 30%-50% of women are homozygous or heterozygous carriers, and common worldwide except East Asia. Moreover, rs179008 is a missense SNP substituting a leucine for a glutamine (p.Gln11Leu) at protein level, which warrants enquiry into the impact of this variation on TLR7 biology. In this work, we analyzed the functional differences between the rs179008 alleles in the production of IFN-I by pDCs, and the mechanism of action of the SNP on TLR7 protein expression. In addition, we describe a pattern of association between this TLR7 polymorphism and key parameters of acute HIV-1 infection in women that supports the notion of a deleterious role for excess TLR7-driven IFN-I production during the early stages of infection.

TLR7-dependent IFN-a production is impaired in female carriers of rs179008 T.
There is a strong sex-bias in the TLR7-driven production of IFN-a by human pDCs, with higher frequencies of IFN-a-producing cells in women than in men (23-25). We therefore investigated the impact of rs179008 in either sex separately. We first examined PBMC production of IFN-a in response to TLR7 agonists in male (i.e., hemizygous) carriers of either rs179008 allele, and in A/T heterozygous women relative to A/A homozygotes. In this context, the pDC fraction of PBMCs is solely responsible for the early-onset production of IFN-I (25, 26). We accordingly normalized IFN-a measurements to pDC numbers to quantify the effect of rs179008 in time-course experiments where male or female PBMCs were stimulated ex vivo with either R-837 (Imiquimod; a TLR7 ligand) or R-848 (Resiquimod; a ligand of TLR7 and TLR8). Contrary to the earlier study (22), we observed no significant differences in IFN-a production between A/0 and T/0 males regardless of agonist dosage (Figure 1, A-C). In female PBMCs, by contrast, heterozygous carriage of allele T was associated with substantially lower production of IFN-a upon stimulation with either 1.5 µg/ml R-837 or 0.3 µg/ml R-848 (Figure 1, D and E). The response to 1.5 µg/ml R-848, possibly a near-saturation dose (26), showed a similar trend but differences by genotype were not statistically significant ( Figure 1F). In line with previous reports of female-biased responses (23), PBMCs from A/A women consistently produced much more IFN-a than comparable male cells of A/0 genotype in response to R-848 or R-837 regardless of dosage (Supplemental Figure 1). IFN-a production by pDCs was thus selectively blunted in female carriers of the rs179008 T allele, and the responses for the genotypes represented in these experiments ranked A/A > A/T > A/0 ≈ T/0.
We assessed also the effect of rs179008 on the frequency of IFN-a-or TNF-aproducing pDCs following stimulation with TLR7 ligands, using the flow cytometric strategy in Supplemental Figure 2. We stimulated PBMCs with GagRNA1166, a HIV-1-derived synthetic RNA ligand of TLR7 and TLR8 (24), in parallel to the imidazoquinoline compounds, R-837 and R-848. IFN-a-producing pDCs were boosted most efficiently by  Figure 3C). This argues against a general dysfunction of pDCs in allele T carriers, and suggests that the rs179008 genotype fine-tunes the TLR7driven production of IFN-a in female pDCs specifically.
Female pDCs expressing the rs179008 T allele produce less IFN-a. The immune cells of women express either one or both alleles of TLR7 in mosaic fashion, as the allele on the inactive X chromosome may evade repression (27). More than half of the cells express monoallelic transcripts, and we developed an assay to determine whether IFN-a production following TLR7 engagement depends, at the cellular level, on the expressed rs179008 allele.
PBMCs from A/T heterozygous women were stimulated with either R-848 or GagRNA1166; next, pDCs were labeled for lineage-specific markers, surface-immunostained for selfsecreted IFN-a, and sorted into IFN-a pos or IFN-a neg pDC pools (Figure 2A) that were analyzed for the proportions of transcripts from either allele (Figure 2, B-D). In a group of healthy women, IFN-a-secreting pDCs displayed enrichment in allele-A TLR7 transcripts relative to same-donor IFN-a neg cells, as measured by an allele-specific PCR assay (27) ( Figure 2D) and corroborated by deep-sequencing of the TLR7 amplimers ( Figure 2E). These experiments demonstrated a depressed interferogenic response to TLR7 engagement in female allele T-expressing pDCs, relative to the allele A-expressing cells from the same donor.

The rs179008 A>T substitution determines reduced protein expression in vitro.
In assessing the mechanism of the allelic bias in interferogenesis, neither in silico predictions nor PCR amplification of the relevant TLR7 cDNA region indicated differences in TLR7 pre-  Figure 3A), because the central h-region is critical to the interaction of a signal peptide with the signal recognition particle and with the ER pore complex (28, 29). Overall, this argued against a deleterious effect of the Gln to Leu substitution per se on signal peptide function.
To experimentally address the functional fitness of the TLR7 signal peptide, we engineered a pair of secretion reporter vectors encoding a secreted alkaline phosphatase (SEAP) with its autologous signal peptide replaced by amino acids 1-30 from TLR7; the vectors were identical except for the single-nucleotide change in codon 11 ( Figure 3A These vectors retained approximately 70% of measured SEAP activity in cell culture supernatants, and again showed lower activity for the Leu11 version ( Figure 3B). These results evidenced lower activity attributable to the A-to-T change specifically, but were in contradiction with the hierarchy of scores from the in silico signal peptide predictions.
We transfected HEK 293T cells with expression vectors for each TLR7 variant tagged with a C-terminal HA or FLAG epitope and analyzed recombinant TLR7 expression by Western blot. In line with the SEAP reporter experiments, we observed a 30% to 40% decrease in TLR7 synthesis for the Leu11 (rs179008 T) form relative to the Gln11 protein ( Figure 3, C and D). Next, we co-expressed FLAG-tagged TLR7 c.32A with either c.32A or rs179008 T HA-tagged TLR7 to analyze TLR7 intracellular dimers. Immunoprecipitation with anti-FLAG antibodies followed by Western blot analysis with anti-HA antibodies confirmed that the rs179008 A>T substitution was associated with decreased TLR7 protein synthesis and homodimer formation in vitro ( Figure 3G), codon-specific effects were dissected from signal peptide function in the reporter system. SEAP reporter activity was positively correlated with relative codon usage ( Figure 3H), with the most (CUG) and least frequent (CUA) codons at opposite ends of the range of activities. Codon 11 is thus a likely control point for TLR7 translation, and this effect could involve interference with translation initiation, the rate-limiting step in protein synthesis, owing to the proximity of the polymorphic codon to the 5ʹ end of the TLR7 coding sequence (36).

The T allele lowers TLR7 protein expression in female PBMCs specifically.
We recently characterized a commercially available monoclonal antibody to the C-terminus of human TLR7 that correctly detects natural and recombinant TLR7 in Western blotting, but not TLR7 modified with a C-terminal epitope tag (reference (27) and Supplemental Figure   6). We quantified by this method the full-length (140-kDa) and proteolytically mature (75-kDa) forms of TLR7 in a deconvolution experiment to characterize the principal TLR7producing cell populations among steady-state PBMCs (Figure 4, A and B). Most of the TLR7 signal came from CD14 + monocytes, followed by pDCs and then by B cells, with regard to both the full-length and mature forms of TLR7 ( Figure 4C). A small proportion of TLR7 (< 5%) came from the CD14 − CD19 − BDCA4 − cell fraction mainly comprising T lymphocytes and NK cells. When the densitometric data were expressed on a per-cell basis ( Figure 4D), pDCs exhibited the highest level of full-length TLR7, with a 100-fold enrichment over unsorted PBMCs, followed by CD14 + monocytes and B cells. A similar hierarchy was found for the mature form of TLR7.
Next, we quantified by Western blotting the impact of the T allele on TLR7 protein expression within PBMCs of different rs179008 genotypes (Supplemental Figure 6E).

PBMCs from women heterozygous (A/T) and homozygous (T/T) for the T allele expressed
significantly less TLR7 protein than A/A women ( Figure 4E). We observed no significant differences between A/0 and T/0 male donors ( Figure 4E). By contrast, we observed a significant two-fold excess of full-length TLR7 in A/A women over A/0 men (Fig 4E), and a trend in the same direction for the 75-kDa mature form (Fig 4E), in agreement with our earlier work (27) noting greater TLR7 protein levels in female than in male human PBMCs.

The hypomorphic effect of rs179008 T on TLR7 protein expression is maintained
in post-menopausal women. The functional responses of pDCs from women decrease after menopause as they are partly dependent on estrogen-receptor signaling (25, 37). Premenopausal plasma levels of estradiol, which range between 50 and 65 pg/ml in women of various ethnicities (38), decline below 20 pg/ml within one year after the final menstrual period in the majority of Caucasian women (38). Plotting the TLR7 protein quantitation data in Figure 4E against the age of donors ( Figure 5A), however, failed to show a dependency of the quantitative effect of rs179008 on age. Only a modest positive correlation [r = 0.397, p = 0.036] was observed here for mature TLR7 expression in A/A women ( Figure 5A). In men, TLR7 expression was non-correlated with age or with the rs179008 allele ( Figure 5A).
The median age of women at the final menstrual period is 51.5 years (38). In our data for likely post-menopausal women, above 53 years of age, A/A women displayed markedly higher levels of the full-length and mature forms of TLR7 than the carriers of allele T (p < 0.001; Figure 5B). These results show that the hypomorphic effect of rs179008 T on TLR7 protein expression is present on either side of menopause, which supports the hypothesis that this effect is not primarily dependent on estrogen signaling.

The T allele is associated with reduced clinical parameters of HIV-1 infection in women.
In studying the effect of rs179008 on HIV-1 infection, we explored the previously reported association (22) with the acquisition of HIV-1 by women in two separate casecontrol settings, restricted to Caucasian women to minimize genetic diversity regarding rs179008 allele frequency distribution and other potential genetic determinants. The ANRS EP53 X-LIBRIS study compared a cohort of female Caucasian patients infected by sexual Cell-associated HIV-1 DNA was also reduced in women harboring rs179008 T: 3.1 log10 DNA copies/10 6 PBMCs (IQR 2.6-3.6) for the combined A/T and T/T genotypes versus 3.4 log10 DNA copies/10 6 PBMCs (IQR 2.9-3.8) among A/A homozygotes (p = 0.038, Wilcoxon's test). The frequencies of circulating CD4 + and CD8 + T cells were not significantly different between carriers and non-carriers of allele rs179008 T (Table 1). We next looked for potentially greater contrasts by comparing the opposite A/A and T/T homozygous genotypes (Table 1). We controlled for the confounding of RNA viral loads dependent on time since infection by using a fractional polynomial regression (Supplemental  Table 1).
We measured additional plasmatic parameters of HIV-1 infection and the immune response, namely IFN-a and CXCL10 (IP-10) among the 10 T/T PRIMO patients plus randomly-drawn subsets of the A/A and A/T groups (n = 41 for each genotype; n = 92 total). IP-10 was used as a surrogate marker enabling more robust detection and quantitation than IFN-I; in the PRIMO sub-cohort, detectable IFN-a was in fact associated with significantly higher IP-10 values (Supplemental Figure 9). Splitting the sub-cohort based on the symptomatic or asymptomatic presentation of primary HIV-1 infection resulted in two groups displaying clear, significant differences with each other in respect to RNA viral load and cell-associated HIV-1 DNA, IP-10 levels, and detectable IFN-a. All these parameters were lower in the asymptomatic fraction (Supplemental Table 2), which, as noted above, was enriched among the rs179008 T/T relative to the A/A patients. This sub-cohort showed the previously noted association of rs179008 T carriage with lower values for RNA viral load and cell-associated HIV-1 DNA, confirming that the randomly-drawn subsets were representative of the source group. We also observed a trend for lower IP-10 values ( Figure   6A and Supplemental Table 3). These three parameters were in positive correlation with one another ( Figure 6B). Thus, the comparisons of the rs179008 T carrier (A/T, T/T) and noncarrier (A/A) groups, revealed significant differences for RNA viral load combined with cell-associated HIV-1 DNA, for IP-10 combined with cell-associated HIV-1 DNA ( Figure   6B), and for the combination of the three parameters (p = 0.023, Hotelling's T 2 test). Lastly, mean RNA viral load among T/T patients was here 0.97 log10 units lower than in rs179008 A/A patients in the data adjusted for time since infection, a 9-fold difference (p = 0.025; Student's t-test).

Genetic association with HIV-1 viral load in the wider context of TLR7
polymorphism. rs179008 is one of a group of frequent TLR7 SNPs within a 40-kb linkage disequilibrium (LD) block on the X chromosome ( Figure 7A). Pairwise LD is moderately high (r 2 = 0.63) between rs179008 and the frequent intronic tag SNP, rs179012, previously associated with HIV-1 set-point viral load (21), but weak (r 2 = 0.04) between rs179008 and a frequent functional SNP in the 3ʹ UTR of TLR7, rs3853839 (Figure 7, A and B), in genetic association with the risk of developing systemic lupus erythematosus (40). We carried out an estimation analysis to compare the effect of each SNP on HIV-1 viral load among the PRIMO female patients (n = 220) ( Figure 7C). For rs179008, we again observed a clear difference in mean viral loads between the opposite A/A and T/T homozygous genotypes ( Figure 7C, I), as expected from our earlier comparisons (Table 1). By contrast, we found the difference between opposite homozygous genotypes to be very weak or absent for rs179012 (G/G versus A/A; Figure 7C, II) and rs3853839 (C/C versus G/G; Figure 7C, III).
To control for confounding due to LD between rs179008 and rs179012, we considered rs179012-rs179008 diplotypes where the genotype for one of the SNPs was the same in the two groups of patients compared; in this way, the effect of the second SNP could be assessed while removing the effect of the first ( Figure 7C, IV-V). This fully corroborated the direction and magnitude of the effect for rs179008 with independence from rs179012. Taken together, our biochemical and genetic association studies support the conclusion that rs179008 is a functional polymorphism of TLR7 in its own right.

Discussion
We observed here that the rs179008 SNP of TLR7 exerts in female patients a significant effect on key parameters in the clinical course of HIV-1 acute infection. Although recent findings strongly suggest a detrimental effect of pDCs and IFN-I during the chronic phase of HIV-1 infection, the early-stage role of IFN-I produced by pDCs as a result of TLR7 activation is still not fully understood (41). We have addressed this issue through the functional characterization of a frequent TLR7 SNP, rs179008, which we found to exert a quantitative effect on TLR7 protein synthesis and pDC IFN-I production in women specifically, and by analyzing the association of TLR7 polymorphisms with clinical parameters of HIV-1 infection. Our results demonstrate a protein-level and interferogenic cellular phenotype of rs179008. Carriage of the rs179008 T allele lowered ex vivo TLR7driven IFN-I production by pDCs from heterozygous A/T women relative to A/A homozygotes. Mechanistically, we show this allele to determine lower TLR7 protein expression in female leukocytes, which mirrors the reduced frequency of IFN-a-producing female pDCs. Lastly, we observed a beneficial association between the lowerinterferogenesis allele, rs179008 T, and lower levels of informative correlates of acute HIV-1 infection severity in women.
These results may appear counter-intuitive in light of previous studies supporting a beneficial function of pDCs and IFN-I-signalling through the control of viral replication during acute infection in macaque and humanized mouse models (11,12). The effects of innate immunity on HIV-1 infection, however, are complex and not consistently positive.
The recruitment and activation of pDCs and monocytes in the vaginal mucosa of female macaques early after local SIV inoculation helps attract CD4 + T cells, which can then be infected to boost viral production and dissemination (13,42). Conversely, prolonged exposure to IFN-I is associated with unfavourable outcomes in chronic HIV and SIV infection (19). A deleterious effect is thus conceivable for TLR7-driven activation of pDCs and other innate immune cells even during acute-phase HIV-1 infection, by recruiting virus target cells at mucosal portals of entry (14), or initiating immunosuppressive pathways mediated by pDC-derived IFN-I (17,18). Such early mechanisms might be toned down in women carrying the rs179008 T allele of TLR7, blunting the initial spread of the virus. Our We show also that this nonsynonymous polymorphism of the signal peptide controls protein dosage at the translational level through the mRNA sequence itself, rather than by altering signal peptide function. The connection of genotype to phenotype via translation is compatible with the expected loss of the protein's allelic character when the signal peptide is co-translationally cleaved off on entering the ER. Overall codon usage in TLR7 is known to be markedly suboptimal (43), and translation of the mRNA relatively inefficient (43,44), which makes translation a likely rate-limiting step in TLR7 expression. Local translation efficiency depends on the abundance of aminoacyl-tRNAs with compatible anticodons, and this is positively correlated with the relative usage of synonymous codons (45). Given the 5ʹ to 3ʹ mRNA scanning direction of ribosomes (34), the Leu11 CUA (rs179008 T) codon could play a translation rate-limiting role as the first rare codon in the path of a translationcompetent ribosome (Supplemental Figure 5). In addition, the ribosome at codon 11 may sterically hinder translation initiation, the rate-limiting step in protein synthesis, because the span between codons 1 and 11 is similar to the 20-to 30-nucleotide footprint of a ribosome (46) and less than the 32-to 39-nucleotide pitch of ribosomes in tightly packed polysomes (47). This would be in line with evidence that the effect of non-optimal codons on translation efficiency is greatest near the 5ʹ end of the coding sequence (36). Further experimental work addressing translation directly will be necessary to confirm this mechanism. To our knowledge, the notion of a sex bias in regulatory mechanisms of translation has yet to be explored in the context of human biology, and it is uncertain whether the sex dependency we have observed in the TLR7 quantitative protein phenotype for rs179008 (Figure 4) could be mediated by translational regulation. The non-dependency of TLR7 quantitation on women age ( Figure 5) strongly suggests that estrogens are not involved in the sex-biased phenotype, because this is observable beyond menopause despite the expected decline in estrogen levels (38).
In agreement with earlier reports (23-25), we observed a sex bias in the TLR7-driven production of IFN-a by human pDCs, with higher frequencies of IFN-a-producing cells in women than in men. This was most noticeable when comparing women and men of similar A/A and A/0 rs179008 genotypes. The sex bias in the TLR7-mediated response of pDCs arises from mechanisms implicating estrogen signaling (25, 37,48) and the X-chromosome complement (37). The X-linked TLR7 gene escapes X chromosome inactivation in female immune cells, including pDCs, and was associated with higher TLR7 protein expression in leukocytes from rs179008 A/A women compared with A/0 men (27). In line with the concept of TLR7 dosage-dependent effects determined at chromosome level, we found here the carriage of allele rs179008 T in females to be associated both with a decrease in TLR7 protein relative to A/A homozygous pDCs, and with lower per-cell IFN-I production. Future studies of the functional response of pDCs from women should therefore consider the genotype for this polymorphism of TLR7.
Oh and coll. (22) identified rs179008 as a determinant of HIV-1 infection prevalence in women. Our observations fail to support an effect of the SNP on HIV-1 acquisition by the sexual route in women. This discrepancy might be explained by the small size of the healthy control group in that study (22), where the frequency of the T allele was clearly lower than in relevant reference populations, leading to over-estimation of effect size (49). In that study, the Leu11 (rs179008 T) allele of TLR7 was associated with more severe HIV disease progression in men (22). We show that this allele is not associated with lower constitutive Our observations lead us to propose that increased dosage of TLR7 protein is conducive to a higher viral burden during acute HIV-1 infection in women, and correlates with a predictor of the speed of disease progression. The reduced TLR7 protein values and pDC interferogenesis associated with allele rs179008 T were accompanied by significantly lower viremia and cell-associated HIV-1 DNA measurements in women from the ANRS-PRIMO cohort. We investigated also the association between rs179008 T and further clinical parameters of HIV-1 infection, namely plasma IP-10 (CXCL10) and IFN-a levels, among a representative subset of these female patients. Plasma IP-10 is known to display a robust positive correlation with viral load (51)(52)(53), and is responsible for the recruitment of CXCR3 + target T cells, fueling viral dissemination and the establishment of a viral reservoir (54).
Elevated blood IP-10 during primary HIV-1 infection predicts rapid disease onset more robustly than peak viremia or the CD4 + cell nadir (53,55). Although pDCs can produce IP-10 through direct TLR7 stimulation, this cytokine can be indirectly induced in cells such as monocytes and mDCs by pDC-derived IFN-I (56, 57). We found here, as expected, that patients with detectable plasma IFN-a exhibited higher levels of IP-10, and that both IFN-a and plasma IP-10 were associated with symptomatic HIV-1 infection. Importantly, rs179008 T was significantly associated with lower levels of a triad of positively correlated parameters: RNA viral load, the cell-associated HIV-1 DNA reservoir, and plasma IP-10. In addition, the higher frequency of asymptomatic clinical presentations at diagnosis for the T/T homozygous genotype implies a trend for a delayed onset of the symptoms of acute infection.
Heritable quantitative variation of TLR7 dosage and function provides here valuable insights into the role of this receptor as a determinant of acute viral load. Our data point to TLR7 as a key effector molecule, with a dominant deleterious action of higher TLR7 dosage during acute HIV-1 infection in women. This, in turn, argues for a beneficial effect of toning down a too strong type I interferon response early on, even before the acute-to-chronic phase transition. Corroboration of the genetic association with disease parameters in those populations where the rs179008 T allele is frequent will be important. Genomic DNA extraction and SNP genotyping. Genomic DNA was extracted from cryopreserved or freshly isolated PBMCs using a NucleoSpin Tissue kit (Macherey-Nagel) and genotyped for TLR7 SNPs by a KASP allele-specific PCR assay (LGC Genomics) run on a LightCycler 480 II instrument (Roche). and anti-Lin-FITC antibody conjugates (BD Biosciences). Cells were then fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 0.5% saponin, and stained for intracellular cytokine production using anti-IFN-a-phycoerythrin (Miltenyi Biotec) and anti-TNF-a-Alexa700 (BD Biosciences) antibody conjugates. Flow cytometry analysis was performed on a BD Biosciences LSRII instrument, and pDCs defined as the Lin − CD123 + BDCA-4 + cell fraction (Supplemental Figure 2). The frequency of cytokine-producing pDCs was determined by subsequent data processing with the FlowJo software (FlowJo, LLC).

Intra-individual analysis of the impact of rs179008 on IFN-a production by pDCs.
Freshly isolated PBMCs (10 × 10 6 cells/ml) from healthy rs179008 A/T heterozygous women were stimulated for 5 hours with 1 µg/ml R-848 or with GagRNA 1166 complexed with DOTAP. Cells were then surface-labeled for IFN-a production using an IFN-a secretion assay from Miltenyi Biotec, according to the manufacturer's instructions. Briefly, stimulated cells were coated with IFN-a capture reagent (anti-IFN-a monoclonal antibody conjugated to a cell surface-specific monoclonal antibody) and then incubated for 20 minutes at 37°C to allow IFN-a secretion. Cells were then surface-stained with IFN-a detection antibody (anti-IFN-a monoclonal antibody conjugated to phycoerythrin), and for BDCA-4, CD123 and Lin as above. pDCs were then sorted on a FACSAria II instrument (BD Biosciences) into Lin − CD123 + BDCA-4 + IFN-a + and Lin − CD123 + BDCA-4 + IFN-a − populations. RNA from both purified populations was extracted using the Absolutely RNA Nanoprep kit (Agilent Technologies) and retrotranscribed with the Maxima H Minus First Strand cDNA Synthesis kit (Thermo Fisher Scientific). The cDNA was subjected to target amplification using TLR7 PCR primers flanking the SNP (oligonucleotides 1F and 2R below) followed by KASP typing as above to determine the rs179008 allele of origin of TLR7 transcripts. The percentage of allele A expression in each cell population was given by the standard curve for the KASP PCR assay ( Figure 2C) as a four-parameter logistic function of the log-ratio of the A and T fluorescence intensities. The curve was generated using genomic TLR7 PCR amplimers from male (hemizygous) A/0 and T/0 subjects in different mix ratios.
Analysis of pre-mRNA splicing. RNA from PBMCs was isolated and reversetranscribed as above. The exon 2-exon 3 splice junction in the TLR7 cDNA was amplified by PCR with forward primer 1F: 5ʹ-CCATGCCATCAAGAAAGTTG-3ʹ straddling the exon 1-exon 2 junction, and reverse primer 2R: 5ʹ-TCTGTGCAGTCCACGATCA-3ʹ hybridizing 155 base pairs downstream from the canonical TLR7 exon 2-exon 3 junction. PCR products were analyzed by agarose gel electrophoresis.

Ultra-deep sequencing.
A 273-nucleotide-long TLR7 cDNA fragment encompassing the position of the rs179008 SNP was generated by PCR as above. The PCR products were amplified on capture beads in water-in-oil emulsion micro-reactors, and pyrosequencing performed on a Roche 454 GS Junior system. The sequence reads were quantified with the GS amplicon variant analyser (Roche).
Chemiluminescent detection was carried out with Amersham ECL Select or ECL Prime reagents (GE Healthcare) as necessary, and densitometric analysis performed with the Image Lab 5.0 software (Bio-Rad).
TLR7 protein quantification in healthy subjects of the different rs179008 genotypes was performed on cryopreserved PBMCs. PBMCs were thawed and cultured at 37°C for 2 hours before counting, cell lysate preparation and western blotting as above. The densitometric signals of the TLR7 145-kDa and 75-kDa forms were normalized to b-actin and then to an internal standard PBMC lysate that was loaded in each gel for inter-gel data normalization as described in Supplemental Figure 6.

TLR7-SEAP reporter vectors.
The signal peptide reporter vectors encoded a secreted alkaline phosphatase (SEAP) where the autologous signal peptide was replaced by an allelic form of the human TLR7 signal peptide (Fig. 3A). The TLR7 and SEAP moieties connected at a proline residue analogous to Pro31 in TLR7 and to Pro20 in SEAP (GenBank U89937.1).
Preparatory analyses with the SignalP 4.1 software (60) predicted in each case a signal peptide with a near-identical profile to that of the parental TLR7 form (not shown). The SEAP gene under the control of the human EF-1a promoter was released from pYSEAP (61) (Addgene plasmid #37326) as a 3.5-kb SpeI-SalI fragment and inserted into a modified pBC KS+ vector (Stratagene) to generate pYSEAP-JM562. Next, a 360-bp fragment of pYSEAP-JM562 overlapping the 5ʹ end of the SEAP coding sequence was excised with EcoRI and NdeI, and in-frame replaced by one of a series of synthetic TLR7-SEAP DNA fragments. The hybrid inserts were generated by successive PCR steps with the Phusion high-fidelity DNA polymerase (Thermo Scientific), and included 69 proximal nucleotides of the TLR7 5ʹ-untranslated region to preserve a predicted RNA stem-loop structure around the start codon (not shown). Initial TLR7 cDNA amplifications were seeded with reversetranscribed pDC or PBMC RNA from male donors carrying the relevant rs179008 allele.
Where necessary, single-base changes in TLR7 codon 1 or 11 (Fig. 3G) were introduced in the design of PCR primers: to force translation initiation at Met5, the major ATG initiation codon was made a TTG triplet; in the Leu11 synonymous versions, the natural CTA leucine codon was replaced by a CTC, CTT or CTG leucine codon; vectors for leucine codons TTA and TTG were not generated. In all plasmid constructs, any regions derived from synthetic DNA fragments were subjected to complete DNA sequencing to rule out unwanted sequence variation. The sequence of a prototypic TLR7-SEAP reporter vector has been deposited in the GenBank database under accession number KX035095.      Figure 2A for the Lin − CD123 + BDCA4 + gating strategy. Bar graphs show data as means ± standard error (SD). The number of donors by genotype is indicated in the plot legend. Groups were compared by a two-way ANOVA test followed by Sidak's multiple comparisons test. N.D., not detectable, i.e., below the 30 pg/ml detection threshold of the assay. (G-I) Frequency of IFN-a-producing pDCs. Freshly isolated male and female PBMCs were stimulated with 0.3 µg/ml R-848 (G), HIV-1-derived GagRNA1166 (H), or 1 µg/ml R-837 (I). The frequency of IFNa-producing pDCs was determined by flow cytometry as above. Each dot represents one donor; horizontal bars indicate mean values ± SD. Groups were compared by the Kruskal-Wallis test corrected for multiple comparisons by controlling the FDR (Benjamini-Hochberg method).   Table 1. Association between rs179008 genotypes and parameters of primary HIV-1 infection in women 1 Categorical data expressed as case counts, and percentages with 95% CI. Fisher's exact test. 2 Data for continuous variables represented by the median and IQR. Wilcoxon's test, twosided.