A Review on The Nobel Prize in Medicine or Physiology 2020: The Discovery of Hepatitis C

Written by Sarp Turan, Zeynep Özörnek, Ece Sağıroğlu and Simge Ocak.

A. INTRODUCTION

Viral hepatitis is the general name for a ranging array of liver conditions which are resulted from viral infections of the liver hepatocytes.[1] Viral hepatitis is often caused by a variety of viral infections, however, the most frequent cases are associated with five hepatocyte-specific viruses which primarily demonstrate themselves as the causes of chronic liver diseases.[1,2] These sets of viral pathogens were hence named as the “human hepatitis viruses” and abbreviated as HAV, HBV, HCV, HDV, and HEV respectively.[1] The understanding of viral hepatitis was largely constructed during World War II. As large-scale transfusion therapies and crude vaccines from human serum got popular, during the time of war, the frequency of jaundice and liver diseases drastically increased among soldiers. This evidence allowed scientists to discover HAV and HBV as the common causes of these conditions and further reveal various ailments that these viruses can result in, such as, hepatocellular carcinoma. [1,2] However, even after the discovery of these viruses and the development of their respective scanning techniques, there seemed to be an estimation of a 30% risk in the 1960s to acquire hepatitis from a single transfusion. [1] Consequently, it was considered that there existed another viral pathogen, which was neither HAV nor HBV, that wasn’t distinguishable with the current scanning methods. Hence, this agent was initially named as the non-A, non-B (NANB) hepatitis virus. This issue would later be enlightened by the work of Michael Houghton, Harvey J. Alter, and Charles M. Rice which involved the application of a large-scale screening technique to identify the virus, which would later be called HCV. [2,3] Due to the importance of their work, Houghton, Alter, and Rice would go on to be awarded the 2020 Nobel Prize in Physiology and Medicine. In this post, the biology, pathogenesis, and the experimental procedure behind the discovery of HCV will be introduced with a considerable emphasis on the importance of the scientific methodology applied by Houghton et al.

B. BIOLOGY OF THE ORGANISM

HCV is an enveloped, positive sense RNA virus.[4] It is a member of the Flaviviridae family in the Hepacivirus genus, whose other well-known members are the yellow fever virus, West Nile virus, and dengue virus.[6] The particles of HCV are shaped spherically and have a varying size profile which displays itself between 40 and 80 nanometers.[7] Eighty-four different subtypes and 7 genotypes of HCV have been noted by today.[5]

The viral capsid is composed of the protein named HCV core.[8] The outer side of the core connects with the lipid layer of the viral envelope and the inner side of it ties-up with the F segments of HCV genome.[7] The capsid acquires its final conformation through these interactions between the HCV core proteins and the designated regions of the single stranded RNA. The nucleocapsid in its final form, acquires a spherical shape with an approximate diameter of 30 nanometers.[7]

The viral envelope contains a lipid membrane which is formed out of cholesterol, cholesteryl esters, phosphatidylcholine, and sphingomyelin.[7] HCV has a membrane that is very rich in incorporated cholesterol and this makes the virus assume a configuration that is alike very-low-density lipoproteins in humans. One can differentiate a HCV virus from other viruses and host cells, by the special lipid ratio that its membrane has.[7] Both cholesterol and sphingolipids are very important agents that enable the virus to get into the host cell and cause an infection.[7] The viral envelope also contains multiple E1-E2 glycoprotein complexes.[8,9] These two glycoproteins play an important role in membrane fusion and viral assembly.[9] The E1 glycoprotein facilitates the binding of the HCV virion to the host cell not through receptor attractions but with the interactions it has with the E2 glycoprotein.[9] The manipulation of E1 glycoprotein on the E2 complex allows for direct receptor binding and membrane fusion with the host-cell.[9] This allows for the release of the genomic material within the host cell, which later causes an infection.

C. HCV PATHOGENESIS

HCV infections are acquired from direct contact with bearers’ contaminated blood and due to the infection of hepatocytes, they show themselves primarily as liver pathologies.[10] These infections also account for 15–20% of acute hepatitis cases and 50–80% of chronic hepatitis which, due to viral persistence, carries the potential to develop liver inflammation, cirrhosis, and hepatocellular carcinoma (HCC).[10] There exist 8 different genotypes and 84 subtypes of HCV which results in a wide range of pathologies and corresponding treatments.[5] However, all types of HCV share a common protein, called, HCV Core which is regarded as the primary viral agent in the development of HCV-driven sicknesses.[15,10,13] Even though the gene responsible for the production of the core protein is one of the highly conserved ones among different HCV genotypes, small differences in the sequencing of the gene are what lead to a varying number of pathologies which are associated with HCV.[13] Pathogenicity of the core protein was examined in transgenic mouse models which expressed both core and a variety of other viral proteins.[11] It has been recorded that the mice models developed a variety of cases, namely, hepatic steatosis, lipid accumulation in hepatocytes, hepatocellular adenoma, hepatocellular carcinoma, and upregulation of enzymes that take part in lipogenesis.[14,13] The way this is accomplished, is that the HCV core protein inhibits the activity of microsomal triglyceride transfer protein (MTP) and upregulates the promoter of sterol regulatory element-binding protein lc (SREBP-lc). These interactions result in de novo lipogenesis and accumulation of un-secreted triglycerides within the hepatocytes which later cause steatosis and cirrhosis.[14] As for the development of hepatocellular carcinoma, mitosis of chronic HCV and hepatocyte necrosis both, favor nodular regeneration which leads to hepatocyte dysplasia and hepatocellular carcinoma. However, HCV, itself has not yet been proven to be oncogenic but still, the direct oncogenic effect it has on individuals cannot be overlooked.[14] HCV also displays a long-time dormancy within its host, which can sometimes be asymptomatic. Due to this, HCV possesses the ability to co-infect with other viruses such as HIV and cause severe infections.[12]

D. METHODOLOGY BEHIND THE DISCOVERY

As addressed before, the transfusion — acquired hepatitis C was named as the non — A non — B hepatitis (NANBH) at the time of its discovery.[2] That was due to serological methods, which were used in detecting HAV and HBV, being proven to be insufficient at recognizing the agent responsible for the development of NANBH.[16] The first experiments conducted on chimpanzees allowed the scientists to observe a NANBH related agent which caused the formation of characteristic cytoplasmic tubules of large sizes within the infected hepatocytes of the chimpanzees.[17,18] This agent was later named as the Tubule Forming Agent (TFA) and was discussed to be very sensitive towards organic solvents while being capable of getting filtered through 80 nanometer filters.[ 19] These evidences lead to the belief that NANBH would be related to the families of flaviviridae, togaviridae or better yet an unidentified family of viruses.[20] Harvey J. Alter was the leading scientist who conducted these experiments and gave the name NANBH to HCV. He later on published a series of articles focused on the pathogenicity of NANBH and whether it can result in chronic illnesses or not.[21] With this knowledge at hand, Michael Houghton and his colleagues decided to conduct a series of experiments in order to define the NANBH causing microbe. They have first tried to identify NANBH specific mRNAs from infected chimpanzee hepatocytes, however, this did not succeed as the used probe cDNAs were seen to be included in the pre-existing libraries indicating that no new genetic material was obtained through this method.[22] Following this, it was decided that a cross-hybridization between already known viral genomes could provide some insight into the situation. Since, it was already discussed by Harvey J. Alter that the virus might be a flavivirus, togavirus, hepadna virus or a picornavirus, NANBH RNA and DNA received from infected chimpanzee hepatocytes were hybridized into potent radioactive probes which assumed the aforementioned viral genomes as their templates.[22] Through these experiments, the familiarity of NANBH genome with the HAV and HBV genomes could be determined, along with the rarity of the NANBH agent within the system.[22] With the discovery of delta hepatitis (HDV) by Mario Rizzetto and his experiments on the pathogenesis of the virus, it was seen that HDV resulted in the same membranous tubules which formed in NANBH infections.[22,23] This led Houghton to think that HDV and NANBH could be relative species which led to him performing the mentioned hybridization protocol on them. However, the hybridization protocol for HDV and NANBH led to no clear hybridization that could be observed.[22] Using his previous unsuccessful experiments and their results, Houghton went on to generate a new experimental method with the help of his peers. Along with the help of George Kuo when discussing the immuno-screening technique, they have decided to apply a radioactively labelled anti-human Ig called I125 as it had the necessary sensitivity in order to detect the binding of human Ig to cDNA clones.[22] Following this, the serum of a chronic NANBH patient was screened using I125 and a cDNA library specifically created for this protocol.[22] The screening resulted in a number of positive clones, however, all but one of them were believed to be host-derived. In the following months, Houghton and his colleagues were able to show that the clone 5–1–1 was derived from the NANBH directly.[22] By conducting further experiments, they were able to determine the length of its genomic material and its relations with the members of the flaviviridae family.[22] By proving the pathogenicity of clone 5–1–1 they have displayed the viral etiology of this clone clearly and named it HCV.[22] With a better understanding of the virus, further studies were wished to be conducted. However, HCV proved to be very difficult to propagate in vitro.[24] In 2005, HCV was successfully propagated through experiments conducted by a Japanese — American collaboration. One out of the two American teams that went to Japan was directed by Charles M. Rice.[24] Through these extensive researches, HCV has been understood to great extent and has been used within the protocols of in vitro experiments. Consequently, Michael Houghton, Harvey Alter and Charles Rice, have won the Nobel Prize in Medicine and Physiology in 2020.

E. PERSPECTIVE OF THE SCHOLARS

Such discoveries, where a new understanding of a certain situation is established, have their impact on the academic cumulation. In order to emphasize the importance of such discoveries on the pre-existing knowledge and their applications, we have contacted Professor Onur Öztaş, who teaches a course in microbiology within Koç University, Turkey this semester. When asked about the methods by which a new virus is discovered, Öztaş states “We can get information about the life cycle of a virus by examining its genome and the genes that produce the viral proteins. So, the most important step in virus discovery is the process of determining the sequence of the genome by sequencing methods. The type of genome also should be determined since viruses can have DNA or RNA genomes and the genome could be single-stranded and double-stranded. By the development of sequencing methods, this step has become easier. Now, we can determine the viral composition of a sample, called viriome, by next generation sequencing. One of the effective methods for virus discovery is microscopy. We can observe the viruses under electron microscopes and identify their morphology. In addition, we can identify the structure of the virions by structural biology techniques. By recombinant DNA technology, we can produce and isolate viral proteins in other organisms and have information about the structure and functions of these proteins. The discovery of a new virus involves the contributions from several research groups that perform different methods to illuminate the mysteries in the lifestyle of this virus.”. Professor Öztaş also addresses the dynamicity of the branch of virology by emphasizing that these essential methods may have limitations when discovering new viruses and require certain improvements in efficiency, reliability and speed. We then ask Professor Öztaş about what scientists do in order to explore the functions of a newly discovered virus, under varying conditions and question whether computer simulations may be used or not, to which they respond by stating that the discovery of a new virus would not be sufficient for such a research. They further argue that the virus would first have to be isolated in order to understand the effects of the environment on the virus life- cycle. Professor Öztaş also forms a correlation between virology and computer science by stating “Computer simulations could be useful. With the advance of computational biology, we can predict the structural changes in molecules in different conditions, so we can use these methods for virions. The other contribution of computer science to virology would be creating a detailed database of virus types including their genomes and life cycles. Using this database, we might predict the life cycle of newly discovered viruses by only using its genome sequence in the future. “.

After these, we direct HCV specific questions to Professor Öztaş. When asked about how HCV differs from pre-existing HAV and HBV, Professor Öztaş explains “Hepatitis viruses get their names from the outcome of their infection. They all infect liver cells and cause inflammation of the liver. But these viruses are so different from each other based on their virion structure, genome type and life cycle. “and further adds by commenting “Hepatitis A, B and C belong to different virus families. While Hepatitis C is in the flavivirus family, Hepatitis A and Hepatitis B belong to hepatovirus and hepadnavirus families, respectively. The genome of Hepatitis C is single-stranded RNA, while Hepatitis B has a partially double-stranded DNA. When Hepatitis C is an enveloped virus, Hepatitis A does not have an envelope. We can give many examples for the differences in the life cycles of Hepatitis A, B and C. “. Whilst discussing the importance of the discovery of HCV and its further applications, Professor Öztaş adds that there exist many different viruses in nature and that we only know a small fraction of them, especially the pathogenic ones. He later adds that the discovery of new viruses allows scientists to observe diverse survival strategies applied by the viruses. “So, every time we discover a new type of virus, we are getting closer to understanding the viral world.” states Professor Öztaş and further comments on the discovery of Hepatitis C by stating “Hepatitis C discovery strengthened our knowledge on RNA viruses and provided us to develop more efficient treatments against disease-causing viruses and do it faster. And, Hepatitis C can be the first virus that is discovered by using only molecular biology methods. Still, there is no vaccine for Hepatitis C. The Hepatitis C case has shown that although we cannot develop vaccines against viruses, we can develop efficient antiviral drugs to stop or lessen their ability of infection.” On the topic of coinfection, we question the mechanism that underlies within the system and Professor Öztaş replies by giving a very relevant example, that being coronaviridae. They stress that viruses can apply different means to infect a cell and that each type of the coronavirus binds to a different receptor to accommodate themselves on the host cell surface. “So, this leads to the possibility of the coinfection of a cell by different virus types.” adds Öztaş and continues by discussing “The best example for coinfection is the simultaneous infection of amoeba cells by virions and megaviruses. And, different viral subtypes can also co-infect a cell. The diversity in the variants can increase the success of infection. Coinfection of a cell by different pathogens requires the survival of the host cell during infection. The Hepatitis C virions are released from the host cell by exocytosis, not by cell lysis, so it causes chronic infection, which is a more suitable lifestyle for coinfection with other types of pathogens.”. When arguing the number of subtypes that HCV has and the factors that gather them in unison, Professor Öztaş comments that the sub-types of the HCV have very similar life cycles and characteristics. They further evaluate this by stating that the genomic sequences of HCV subtypes vary by 15% on average and that this may result in a variety of lifestyles, infection and immune escape mechanisms among subtypes. At the end of the discussion Professor Öztaş clarifies the importance of the lipid membrane composition that HCV has by stressing that HCV leaves the host cell through exo-cytosis and not cell lysis. They further comment that during this process, the nucleocapsid is surrounded by an envelope which may or may not include outer surface molecules of the host cell. They conclude the discussion by stating “These surface molecules can prevent the detection of these viruses from immune cells and enable immune escape and safe travel through bloodstream. Hepatitis C enters through the host cell through clathrin-mediated endocytosis and the composition of its envelope is important for the attachment and for the beginning of endocytosis.”

ACKNOWLEDGEMENT

The authors of this text would like to offer their thanks, to Assistant Professor Onur Öztaş for his comments and discussions on the topic.

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