Hepatitis C virus (HCV) continues to infect millions of people worldwide and remains a leading cause of serious liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). A majority of the patients (∼70%-80%) with acute infection fail to eliminate this virus and consequently develop chronic hepatitis C (CHC).[1-3] Hepatic cancer resulting from HCV infection is a rapidly rising reason for cancer-related deaths in the United States. Although there is no effective vaccine, the future of HCV antiviral therapy appears optimistic with the advent of direct-acting antiviral agents (DAAs).
HCV is an enveloped positive-strand, RNA virus belonging to the family Flaviviridae. Hepatocytes are the primary sites of replication. HCV induces rearrangement of intracellular membranes resulting in formation of membranous webs, which serve as scaffolds for the assembly of replication complexes. The viral genome consists of a single open reading frame (ORF), which is flanked by 5′ and 3′ nontranslated regions. This ORF encodes for a polyprotein that is cotranslationally and posttranslationally cleaved by host and viral proteases to yield at least 10 proteins. These include three structural (core, E1, and E2) and seven nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins. Infectious virus particles are assembled on the surface of cytoplasmic lipid droplets (LDs). Thus, the viral life cycle is a complex multistep process. It requires a large number of host cellular proteins in addition to viral. The main goal of antiviral therapy is to cure CHC by a sustained elimination of the virus, also called sustained virological response (SVR, undetectable serum HCV-RNA for 6 months posttreatment cessation).
Is there a direct or indirect role for HCV in HCC? HCV has a remarkable ability to cause chronic infection, which eventually leads to HCC. In the majority of CHC patients, inflammation results in fibrosis, followed by cirrhosis. It is well known that cirrhosis increases the risk for HCC. However, in a marginal case, HCC develops even in the absence of cirrhosis, signifying that HCV is directly oncogenic. Over the past few years, enormous substantiation for the ability of viral proteins to modulate important host gene functions (transcription, cell proliferation, and apoptosis) have also emerged. The expression of core protein in transgenic mice can induce HCC. Another multifunctional HCV protein, NS3, has protease, helicase, and NTPase activities.[1, 10] NS3 also promotes carcinogenesis by interacting with p53 in an NS3 sequence-dependent manner. HCV-Core protein expression both in vitro and in vivo has a direct effect on mitochondria and results in oxidative stress. Oxidative stress, ROS, repeated liver damage and repair can eventually lead to HCC. Even though there have been advances, we do not understand the precise mechanism by which HCV infection results in HCC. Knowledge of this specific mechanism would allow us to intervene and prevent HCC. The frequency of HCC has tripled over the past 2 decades, while the 5-year survival rate has remained below 12% in the U.S. In this issue of Hepatology, El-Shamy et al. have asked an important question regarding the role of viral factors (HCV 1b) in HCC development. While it is known that viral factors impact the outcome of HCV therapy, this study further proposes the possibility of a link between HCV 1b isolates and HCC. The authors report specific sequences of the structural (Core) and nonstructural (NS3 and NS5A) proteins that associate with the development of HCC.
In this retrospective study, the authors selected 49 patients infected with HCV genotype 1b who eventually developed HCC. In addition, 100 HCV-infected patients who did not develop HCC, even after 15 years of follow-up, served as a control group. All patients were confirmed for CHC by liver biopsy and there was no significant difference in viral load between the two groups. The study outcome led the authors to propose that patients infected with HCV isolates with core-Gln70 and NS3-Tyr1082/Gln1112 have a higher risk to develop HCC compared to those who lacked these residues. HCV core protein is the main structural component of the viral nucleocapsid and has also been proposed to be involved in a wide array of functions such as modulating viral and cellular gene expression, host signaling pathways, and lipid metabolism. Amino acid residues at position 70 and 91 in the core protein have been associated with the outcome of the standard of care (SOC) treatment, specifically in Japanese patients infected with HCV 1b. A few studies have also suggested a correlation between polymorphism at positions 70 and 91 of core protein (HCV 1b) and progression to HCC.[16, 17]
The present study clearly demonstrates a greater propensity of HCV 1b isolates with core-Q70 and NS3-Y1082/Q1112 residues to cause HCC. How does the expression of core-Gln70 and NS3-Tyr1082/Gln1112 proteins contribute to HCC? Viral proteins are multifunctional, therefore perturbed interactions with signaling molecules, resulting in out-of-order signaling pathways, can be anticipated. Interestingly, a recent study found that the substitution pattern in the HCV 1b-core region does influence very early viral dynamics during the treatment (SOC plus telaprevir). The amino acid residues in the NS3 protease at positions 1082 and 1112 reported in this study are near the catalytic triad (His-1083, Asp-1107, and Ser-1165) of NS3-protease. It has also been shown that the N-terminal protease domain of NS3 transforms cells in culture.[8, 11, 12] However, the mechanism by which these polymorphic viral factors could affect virus-host interactions, as a result initiate, and finally cause HCC, needs further investigation. These studies will also help to further understand the complex life cycle of HCV. Many interesting questions can be asked: for instance, could phosphorylation of Y residues have an impact on the NS3 (protease or helicase) activity? Could these modified or unmodified residues alter protein-protein or protein-nucleic acid interactions in hepatocytes? It would be interesting to investigate these questions further.
NS5A is a proline-rich, RNA binding zinc metalloprotein with three proposed structural domains (domain I, II, and III) which are separated by two low complexity sequences. Owing to the high degree of conformational flexibility, domain II (DII) and domain III (DIII) are intrinsically disordered. This high degree of flexibility in D II and III imparts NS5A with the ability to interact with an array of biological partners. Domain II contains the interferon sensitivity-determining region (ISDR) which overlaps with protein kinase R (PKR) binding site. Mutations in this central region of NS5A-ISDR are reported to associate with treatment response in HCV 1b patients. In the current study, Asn residue at position 2218 of the NS5a protein was detected more frequently in pre-HCC isolates than in the control isolates. It is worth noting that this Asn residue is located in the ISDR (D II) region of NS5A. The significance of this observation is not clear and more studies are required to fully understand and elucidate its role in HCC development, if any.
Another part of the study looked at the evolution of core, NS3, and NS5A-IRRDR sequences during the interval between CHC and HCC. No significant change in sequences occurred (core-Q-70, NS3-Y1082/Q1112 residues) in a progression from CHC to HCC. Interestingly, an IRRDR region in the post-HCC isolates showed a very high degree of sequence heterogeneity. NS5A-Domian III contains the IFN-RBV resistance-determining region (amino acids 2334-2379). The current study found that a high degree of heterogeneity in the IRRDR region was significantly associated with HCC. This difference between pre- and post-HCC sequence in IRRDR suggests that this region evolves rapidly during the course of HCV infection, conceivably due to strong selective pressure. This region is intrinsically disordered, known to interact with multiple host factors, and, most important, also regulates virus production and consequently pathogenesis.
In conclusion, the present study argues that HCV-1b isolates with core-Q-70, NS3-Y1082/Q1112 residues or NS5A-IRRDR≥6 are significantly associated with HCC. These clinical studies provide the basis for a broader investigation of viral populations in a hope to decipher the precise mechanism leading to HCC. More important, such studies can also help in the design of vaccines matched to dominant/circulating viruses. Rigorous research and development efforts have led to the discovery of several DAAs. High hopes are pinned on the forthcoming DAAs, which have the potential to boost the treatment potency and eliminate the morbidity and mortality associated with CHC.
Suresh D. Sharma, Ph.D.
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA