Published online Feb 14, 2018. doi: 10.3748/wjg.v24.i6.680
Peer-review started: September 28, 2017
First decision: October 18, 2017
Revised: December 25, 2017
Accepted: January 18, 2018
Article in press: January 18, 2018
Published online: February 14, 2018
The preS1 region of the hepatitis B virus (HBV) large envelope protein interacts with its cellular receptor, sodium-taurocholate cotransporting polypeptide (NTCP), to enable HBV infectivity. Identification of this interaction has led to the development of drugs that can block HBV entry by targeting NTCP, such as synthetic myristoylated lipopeptides derived from the domain of the preS1 N-terminal end which interacts with NTCP (hereafter, NTCP-interacting domain), that can dock to this receptor, blocking the HBV entry mechanism. Several clinical trials are currently testing this type of HBV entry inhibitor; for example, Myrcludex-B. Furthermore, HBV cellular entry may also be impaired by the single nucleotide polymorphism (SNP) rs2296651 in the SLC10A1 gene. This SNP causes the NTCP S267F variant, which can affect interactions between the receptor and viral particles. Study of these viral and host features may be relevant to understand the interaction of NTCP with the preS1 NTCP-interacting domain and, reasonably, to provide an indication of the potential effectiveness of treatments with synthetic myristoylated lipopeptides derived from this domain.
Sequence variability in the NTCP-interacting domain might change its affinity to attach to NTCP, and this would alter the dynamics of competition with synthetic myristoylated lipopeptide inhibitors of HBV entry, potentially weakening the effectiveness of these treatments. The presence of the rs2296651 SNP causing the S267F NTCP variant could also impair the effectiveness of HBV treatment, as synthetic myristoylated lipopeptides have the same amino acid (aa) sequence as the NTCP-interacting domain. These factors could be analyzed in specific patient populations to determine the potential effect they may have on treatment with the new therapies designed to block NTCP and avert HBV binding to hepatocytes. This was done in the chronic hepatitis B (CHB) patient population of our area (Barcelona, Spain), mainly Caucasians of European Mediterranean origin, using robust methods that can be applied in other patient populations.
The main objectives of the study were to determine the variability/conservation of NTCP-interacting domain and the prevalence of the rs2296651 SNP (S267F NTCP variant) in chronically infected HBV patients. Using a high-throughput analytical protocol based on next-generation sequencing (NGS) and an in-house developed PCR using fluorescence resonance energy transfer (FRET) probes, these objectives were realized in an exploratory sample of HBV patients from our setting. Analysis of these viral and host features could be relevant to understand the interactions between HBV and its receptor, and to determine their applicability as prognostic markers of response to treatment strategies based on NTCP blocking.
We performed two main analyses in serum samples from 246 individuals in 3 groups: patients with CHB, patients with resolved HBV infection, and HBV-uninfected individuals. First, the variability/conservation of aa sequences in the NTCP-interacting domain was analyzed and compared to that of a highly conserved preS1 C-terminal domain associated with virion morphogenesis. Comparison between the NTCP-interacting domain and the highly conserved virion morphogenesis domain gave an idea of the magnitude of sequence conservation of the former. To perform this analysis, we developed a high-throughput protocol based on NGS. The raw sequencing data obtained underwent a bioinformatics filtering and analysis procedure based on an in-house-developed pipeline with all computations done in R environment and language. The conservation/variation of these sequences was analyzed by calculating the information content of each position, which was represented graphically using sequence logos, as explained in detail in the Materials and Methods section of the main article.
Second, to estimate the prevalence of the rs2296651 polymorphism in our patient population, this SNP was determined in all samples from the patients included. To accomplish this aim we designed a new in-house real-time PCR method based on FRET probes.
High-throughput NGS analysis yielded viral sequences for most HBV genotypes (A to F and H). In general, the NTCP-interacting domain showed a high degree of conservation, which depended on viral genotype, particularly the sequence between aa 9 to 21. In comparison to the virion morphogenesis domain, the NTCP-interacting domain showed a smaller percentage of HBV genotype-unrelated changes, but greater variability between different HBV genotypes, according to consensus sequences from the GenBank patterns of genotypes A to H. Interestingly, proline residues showed a high degree of conservation in both domains, and serine residues were also particularly conserved in the virion morphogenesis domain, where changes above 1% of the quasispecies were always to potentially phosphorylatable aa. Finally, we demonstrated a low to null prevalence of the rs2296651 SNP in HBV patients from our area. The high degree of conservation of the NTCP-interacting and virion morphogenesis domains should to be confirmed in larger patient series, and the role of proline and potentially phosphorylatable residues and their implications on HBV activity should be clarified. The potential effect of additional NTCP polymorphisms on interactions between HBV and this receptor should also be investigated.
This study describes high-throughput NGS analysis of the preS1 NTCP-interacting domain, which showed overall high conservation that depended on HBV genotype, particularly between aa 9 to 21. These findings concur with previous in vitro results demonstrating that these aa are essential for HBV infectivity. In the comparison with the virion morphogenesis domain, we focused on the proline and serine residues in the two domains: proline showed a high degree of conservation and changes in > 1% of the quasispecies in serine residues were always to potentially phosphorylatable aa in the virion morphogenesis domain. Based on the physical-chemical properties of these aa, we hypothesized that proline residues could stabilize the structure of these two preS1 domains, and the tendency to keep phosphorylatable aa in specific positions of the virion morphogenesis domain suggested that they may be phosphorylated. In addition, we developed an in-house real-time PCR method that allowed us to estimate the prevalence of the rs2296651 SNP in our HBV patients, which turned out to be low to null. Bearing in mind that rs2296651 is reported to be prevalent in Asian populations, where HBV infection is endemic, we hypothesized that the low presence of this SNP in our area may be associated with the lower incidence of HBV infection. Taken together, the findings from this exploratory study suggest that inhibition of HBV entry by NTCP block therapies would be suitable treatment in our CHB patient population.
The present study illustrates the value of NGS to investigate the variability/conservation of the preS1 region of the LHBs. The reasons for the high degree of sequence conservation of the NTCP-interacting and virion morphogenesis domains should be investigated by preS1 structural simulations and site-directed mutagenesis experiments, in particular with modification of the proline and serine phosphorylatable residues. However, it should be borne in mind that the sequence conservation results found in both domains should be confirmed in larger patient samples. In addition, the new in-house real-time PCR method based on FRET probes used here provided fast, reliable detection of the rs2296651 SNP in serum samples of all patients, and revealed a low to null prevalence of this SNP in our patient population. Nonetheless, it cannot be excluded that Caucasian individuals might have additional functionally relevant NTCP SNPs, which would be expected to modify bile acid homeostasis and HBV cellular entry. Therefore, further studies should be performed to determine the prevalence of NTCP SNPs in different populations, and characterize their implications in NTCP function.