Identification of Siglec-1 null individuals infected with HIV-1

Results

Genetic description of SIGLEC1 variants

We used data from the Exome Aggregation Consortium (ExAc.broadinstitute.org) to identify naturally occurring knockout mutations in SIGLEC1. In that sample of ∼63,000 individuals we observed 70 protein truncating variants, that is, stop-gain, frameshift or splice site (Fig. 1). With the exception of rs150358287, a stop-gain variant resulting in an early stop codon at amino acid position 88 (Glu88Ter), truncating variants are of very low frequency (<1%). The Glu88Ter variant occurs in the second exon of SIGLEC1 (C to A transversion at position 3706494 on chromosome 20, GRCh 38 build reference sequence) and is predicted to truncate both major transcripts ofSIGLEC1. The stop-gain allele is found at highest frequency in individuals of European and South Asian ancestry (∼1.3%) and is rare or absent in African and East Asian populations (<0.5%).

Figure 1: Location and frequency of SIGLEC1 protein truncating variants.

Protein domains are represented in different colours. Data from the Exome Aggregation Consortium (exac.broadinstitute.org) identifies 70 protein truncating variants in SIGLEC1including 33 stop gain (red), 24 frameshift (yellow) and 12 splice disrupting (blue) variants. Grey boxes indicate amino acid blocks encoded by each exon. With the exception of Glu88Ter, all protein truncating variants occur at <1% frequency. Glu88Ter is located in the V-set domain of Siglec-1, the region that recognizes sialyllactose in HIV-1 membrane gangliosides.

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To assess the frequency distribution of this polymorphism in an HIV-1-infected population, we combined genotype data from exome sequencing (n=392), exome chip (n=2,212) and direct genotyping (n=1,129) in participants of the Swiss HIV Cohort Study (SHCS). In 3,733 individuals whose clinical characteristics are detailed in Table 1 (95% reported European ancestry), we observed 85 Glu88Ter heterozygotes and 2 homozygotes for the stop-gain variant (allele frequency=1.2%). Thus, we identified individuals in whom to assess the consequences of Siglec-1 haploinsufficiency and knockout in vivo and ex vivo.

Table 1: Clinical characteristics of the SHCS cohort.

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Functional analysis of SIGLEC1 null variant

To confirm that Siglec-1 Glu88Ter homozygous individuals truly lack receptor expression, we performed functional assays with cryopreserved cells collected from individuals with all three possible genotypes. We induced Siglec-1 expression in isolated monocytes using IFNα and determined the absolute number of Siglec-1 antibody binding sites per monocyte (Fig. 2a). Compared to individuals homozygous for the common allele, heterozygous individuals expressed approximately half of the amount of protein observed, while null individuals showed only background expression levels (Fig. 2a). Next, we analyzed the ability of monocytes to capture fluorescent HIV-1 virus-like particles (VLPs) displaying specific gangliosides that are efficiently recognized by Siglec-1 (refs 3, 4). IFNα-activated monocytes from individuals with the common allele showed the highest viral capture capacity followed by heterozygous and then by null individuals (Fig. 2b), which captured only residual levels of VLPs. To investigate whether this binding was specific for Siglec-1, cells were pre-treated with a monoclonal antibody (mAb) against Siglec-1. Treatment led to a significant reduction of VLP uptake in monocytes from common allele and heterozygous individuals (Fig. 2b), while it had no inhibitory effect on SIGLEC1 null individuals. The VLP uptake of monocytes from distinct SIGLEC1 genotypes strongly correlated with the mean number of Siglec-1 antibody binding sites per cell (Fig. 2c). To assess the general HIV-1 transfer capacity of Siglec-1 compared to other possible receptors on IFNα-activated monocytes from homozygous individuals, we pulsed cells with equal amounts of infectious HIV-1NL4-3 in the presence or absence of blocking mAbs and co-cultured them with a CD4+ reporter cell line (Fig. 2d). Monocytes from individuals with the common allele had higher capacity to trans-infect than did SIGLEC1 null cells (Fig. 2d). Trans-infection was inhibited with a mAb against Siglec-1, which had no blocking effect on SIGLEC1null monocytes (Fig. 2d). Overall, these results indicated that SIGLEC1null individuals lack functional Siglec-1 expression and HIV-1 trans-infection capacity, ruling out genetic compensation mechanisms or a possible stop codon read-through that could alleviate the null status21.

Figure 2: Siglec-1 expression and trans-infection across distinctSIGLEC1 genotypes.

Monocytes were isolated and cultured 24 h in the presence of 1,000 U ml−1 of IFNα to induce Siglec-1 expression. (a) Quantification of Siglec-1 expression levels assessed by flow cytometry. Empty box represents a repeat analysis of one Siglec-1 null homozygote. (b) Capture of fluorescent HIV-1 VLPs by monocytes from distinct genotypes previously exposed to isotype or α-Siglec-1 mAbs. Geometric mean fluorescence intensity of monocytes not exposed to VLPs is also depicted to show the background levels of the assay (empty bars). (c) Correlation between Siglec-1 expression levels and viral capture values of isotype-treated monocytes. (d) HIV-1 transmission to a reporter CD4+ cell line from monocytes of opposing homozygous individuals pre-incubated with isotype or α-Siglec-1 mAbs. HIV-1 infection of reporter cells was determined by induced luciferase activity. Data show mean relative light units and SEM of cells from two homozygous individuals with the common allele and one Siglec-1 null homozygote.