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RNA Dependent RNA Polymerase from Hepatitis C Virus

Background Information –

The Hepatitis C virus (HCV) causes the disease Hepatitis C which is an infection in the liver. An HCV infection can either result in an acute infection or a chronic infection. Acute Hepatitis infections are most commonly asymptomatic and usually do not lead to a life-threatening condition. About 30% of acute infections are cured by the host without treatment in as little as six months. The infection, in the short term, causes yellow discoloration of the skin and eyes, referred to as jaundice. Multiple other symptoms are caused by Hepatitis C infection such as weight loss/loss of appetite, fatigue, spiderlike blood vessels, and various other symptoms. The remaining 70% of HCV infections result in a chronic infection. Long term symptoms or conditions caused by Hepatitis C commonly can lead to liver cirrhosis and liver cancer. In 2019, there were approximately 290,000 deaths due to a Hepatitis C infection. The majority of these deaths were largely due to the long-term adverse effects like, cirrhosis and liver cancer. Currently in the world, about 58 million people are chronically infected with the Hepatitis C virus and there are about 1.5 million new infections annually. In the United States, data estimates that 2.4 million people are infected with Hepatitis C. Due to the low percentage of diagnoses and the frequency of acute infection, it is believed that the number of Hepatitis C infections can range anywhere from 2.5 million to 4.7 million infected individuals. Currently, antiviral medicine can cure approximately 95% of HCV infected people, but lack of access to treatment and diagnosis is a major contributing factor to the prevalence of the disease. The antiviral medicine found most successful is termed Direct Acting Antivirals (DAAs). DAAs currently used in treatment include sofosbuvir and dasabuvir, while beclabuvir is currently in phase III clinical trials. The DAAs include nucleoside analogs that are similar to the natural substrate, but instead are phosphorylated before binding and lead to chain termination. Other antiviral medicines include non-nucleoside inhibitors (NNIs) that bind to allosteric sites and inhibit enzymatic activity. HCV is composed of a single-stranded, positive piece RNA. Due to HCV being a positive stranded RNA, it can function as mRNA and be immediately translated into viral proteins.

RNA Dependent RNA Polymerase (RdRp) Function –

Nearly all of RNA viruses depend on specific activities performed by RNA dependent RNA polymerases. These activities importantly include the copying of the RNA sequence and facilitating the process of transcription. Therefore, if a vaccine was created to target the RNA dependent RNA polymerase and inhibit the function of this protein, then the viral genome could not be replicated. If replication halts, the infection cannot spread to other cells.

Major Structural Features –

Specific to Hepatitis C virus family, the NS5B protein has a 21-24 residue hydrophobic membrane anchor at its C-termini that allows involvement with the replication complex that is located in membrane bound vesicles derived from the endoplasmic reticulum. RNA dependent RNA polymerases have 3 domains divided into the . The palm region is the most heavily conserved region across viral RNA dependent RNA polymerases and is the location of the catalytic site. The fingers, palm, and thumb region of the HCV contain 531 residues and is termed NS5B (non-structural protein 5B). The region, including all three domains, are composed of 21 alpha helices and 18 beta barrels. The fingers region of the HCV virus can be divided into two distinct sections: the palm-proximal region and the distal region. The palm-proximal region is folded as a bundle of alpha helices and the distal region is composed of a singular beta barrel. These regions are termed the fingertips of the HCV RdRp. The fingers domain interacts with the major groove of the template RNA and aids in recognition and binding. The fingers domain of HCV are composed of a set of 4 beta strands that can establish contact with the thumb region closing the active site. The thumb region of HCV is structurally larger in comparison to other RdRps. It is composed mainly of alpha helices, 7 of them, and two beta hairpins. It contains amino acids 370-531. The core of the thumb is composed of two repeating helix motifs. The thumb region of HCV also contains a “priming loop” that extends from the thumb and provides stability during initiation. The palm region of the HCV virus is composed of basic amino acids on the surface and conserved aspartic acid residues. The conservation of these residues is essential through RdRp because of the ability to coordinate Mg2+ metal ions. Conserved residues of the NS5B protein, excluding the surface of the palm region include the RNA and NTP binding groove, the back surface of the thumb region, the NTP tunnel, and an acidic area on the top front of the fingers region.

Motifs –

There are seven conserved structural motifs, A to G, throughout all RdRps. A to E are motifs present in the palm domain. Motif E is hypothesized to play a major role in the polymerase cycle. Motif G and F are part of the fingers domain. Due to HCV being a positive RNA strand, it contains an additional motif termed H. The H motif is present in the thumb domain. Motif C, a heavily conserved motif, is formed by a loop and two flanking beta strands. The loop region is essential for binding the Mg2+ ions. The conserved residues coordinate the metal ion. Motif F is comprised of a loop and a beta strand. This motif interacts with the phosphate group of an incoming NTP. In HCV it is predicted to promote RNA synthesis. Motif G is a loop that is a part of the template strand entrance tunnel in HCV. Not including motifs H, F, and G, the remaining motifs are functionally conserved in NS5B protein in regards to a traditional RdRp.

Catalytic Site –

The fingers domain help with guiding the RNA into the active site. More specifically, the RNA interacts directly with residues 14, 93, 95, 97, 98, 139, 141, and 160. A diagram better shows these interactions. The active site is "encircled" due to interactions between the thumb and fingers domain. In many polymerases, this structure undergoes a tightening conformation change when the substrate is bound. The HCV RdRp undergoes very minimal conformation change between bound and unbound active sites. The RdRp performs efficiently in native conformation. Internal cavities adjacent to the thumb domain are found between structural motifs C and E. This cavity is heavily preserved and could be a place for antiviral chemotherapy.

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