Genome-to-genome analysis highlights the effect of the human innate and adaptive immune systems on the hepatitis C virus

Abstract

Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control.

Figure 1. Human to hepatitis C virus genome-wide association study in 542 patients.

The lower arc shows the human autosomes from chromosome 1 to 22, and the upper arc shows the HCV proteome from core (C) to NS5B. The red line represents the most significant association, P<2x10^-11. The four blue lines represent suggestive associations, P<4x10^-9. The thin grey lines represent associations with P<10^-5. The outer mini-panels represent, on the upper arc, the viral diversity as measured by Shannon entropy and, on the lower arc, the density of human SNPs in bins of 1Mb, with higher values away from the centre for both upper and lower arcs.

Figure 2. Hepatitis C virus genome-wide association studies.

Association between HCV amino acids and (a) HLA alleles (Fisher’s exact test) (b) IFNL4 genotypes (Fisher’s exact test) and (c) pre-treatment viral load (log₁₀PTVL, linear regression). Sites in experimentally validated epitopes in HCV genotype 3 are indicated by a plus sign. Viral sites 1444, 2414 and 2570 are coloured as red. Dashed lines represent a 5% false discovery rate. (d) HCV polyprotein.

Figure 3. Association between IFNL4 genotypes and substitution rates in genotype 3a infected patients.

Stratification of substitution rates by IFNL4 CC and non-CC genotypes. (a) Rate of synonymous substitutions (dS, P=0.68), (b) Rate of non-synonymous substitutions (dN, P=1.6x10^-8) and (c) dN/dS ratio (P=1.28x10^-10). Each blue dot represents the mean dS, dN or dN/dS ratio per patient. The mean and 95% confidence intervals are shown as black dots and bars. (d) The joint distribution of dS and dN/dS in individuals with the IFNL4 non-CC genotypes (red dots) and with the CC genotype (blue dots).

Figure 4. Association between viral load and human and virus genetic variants in genotype 3a infected patients.

(a) Association between human SNPs and log₁₀PTVL, (b) Distribution (blue dots), estimated mean and 95% confidence interval (shown as black dots and bars) of log₁₀PTVL stratified by amino acids present at viral position 2414 (P=9.21x10^-7), (c) by IFNL4 (CC, CT and TT) genotypes in patients whose virus carries a serine at position 2414 (P=9.37x10^-9) or (d) in patients whose virus does not carry a serine amino acid at position 2414 (P=0.9).

Figure 5. Overview of the observations relating to the interplay between innate immune response and the viral genome in hepatitis C virus control.

(a) Infected individuals with IFNL4 CC genotype (37% of the HCV genotype 3a infected patients) show high rates of spontaneous and treatment-induced clearance of HCV. IFNL4 is not expressed, which in turn induces a weaker and possibly differential interferon-stimulated genes (ISG) expression. The host environment is associated with weaker purifying selection and allows viral mutations associated with a better replicative fitness to accumulate, leading to higher viral load. In this group of patients, 67% are infected by a virus with a serine at position 2414 and 33% with a different amino-acid. (b) Infected individuals with IFNL4 non-CC genotypes (63% of the HCV genotype 3a infected patients), have lower rates of spontaneous and treatment-induced clearance of HCV. IFNL4 is expressed and induces ISGs that collectively establish an antiviral state hostile to viral replication. This hostile environment induces a high selective pressure and fewer viral mutations can accumulate. Although the serine at position 2414 (compared to non serine) is associated with lower viral load, it is highly prevalent in this group of patients (85% are infected by a virus with a serine at position 2414 and 15% with a different amino-acid).