 So, good morning everyone. I have been spending my summers as the part of the Khorana program in Professor Robert Stryker's lab. Our lab works in the field of virology and currently focuses on hepatitis C virus. And my project focused on the role of a non-structural protein NS5A of the hepatitis C virus in the viral life cycle. So, motivation for the study. Around 170 million people worldwide are infected with the hepatitis C virus. Around 30% of these cases lead to liver, cellvosis and cancer. There are many treatments available, interferon based. However, these treatments have two problems. One being that they cause a lot of side effects. And the second is that because the HCV genome is highly variable, there is a continuous evolution of resistant strains. And hence these treatments are not effective against all strains of the virus. Studying the molecular mechanisms underlying the HCV virus life cycle would help us edge close up to a potential drug target is what we believe. Now, coming to the HCV strains, the HCV genome is highly variable and heterogeneous. And if you look at the genome of the HCV as compared to the HIV genome, it is highly variable. This could be attributed to two reasons. Primarily, the HCV virus is an RNA virus which has an dependent RNA polymerase which may not be high in its fidelity. And also, the HCV genome therefore is divided into many different genotypes because there is a continuous evolution of different strains of the virus. We frankly focus on the genotype 1 simply because it is called up to the accounts for the majority of the cases of HCV that we see. Different genotypes vary in their response to interferon-based treatment. So it's essential to look at drug targets while keeping in mind the genotype of the virus. When you are studying virology is to understand the vital life cycle. So once the HCV encounters the host cell, it releases its RNA into the cell because it's a positive-stranded RNA virus. The RNA along with the host machinery then is translated into a series of proteins, into two sets of proteins. One, the structural proteins and other the non-structural proteins. While the non-structural proteins help in every part of the vital life cycle, right from replication to infection to mounting of host immune defense, the non-structural set of proteins on the other hand form the viral capsule as well as the envelope. Once the proteins are being translated, the RNA then goes in for RNA replication along with the help of the non-structural proteins. So an important aspect of viral life cycle is RNA replication that is synthesis of multiple RNA molecules. The next step is the virus infection that is the viral assembly and release that involves assembly of the translated proteins as well as the RNA to form infectious variants which once formed leave the cell and go on to infect adjacent host cells. The HCV Geno, the HCV is a positive-stranded RNA virus which is translated as a complete polyprotein which is core and host translationally cleaved into the following set of proteins. As I mentioned these are the structural proteins that is C, E1 and E2 and these are the non-structural proteins out of which the focus of our lab is on the NS5A protein. Now why do we wish to focus on the NS5A protein? The NS5A protein plays a crucial role in viral replication, assembly as well as release. Now it is important to initiate the assembly of the viral replication complex. Apart from that it counterheals, counterattacks the host in your defense. It also forms a homodimer and buys to the RNA. This is crucial for replication and another interesting field that is in research is that it associates with lipid droplets to facilitate efficient replication. The recent reports on the NS5A C-terminal domain state that the C-terminal domain is essential for virus assembly. That is the phosphorylation of certain critical serine 3-on-3 residues in the virus is crucial for assembly and release. Now driven by these reports we decided to focus on the domain 3 of NS5A. Now we decided to focus on the domain 3 also because the domain 3 that is the C-terminal domain of NS5A is relatively unstructured and is poorly conserved upon different viral genotypes. Hence it would be extremely worthwhile studying what is the function of the C-terminal domain of particular genotypes in the viral life cycle. Now our reasons of interest that we decided to focus on is primarily the C-terminal domain which ranges from the amino acid 3.56 to 4.48. We also decided to focus on an extended region from 3.12 to 4.48 because we believe that this region might have certain serine 3-on-3 residues which are important for phosphorylation during the viral life cycle. We also decided to focus on the genotype 1B 3.12 to 4.47 again because we think these residues might have important roles in the viral life cycle. Now we decided to primarily focus on genotype 1 because as I said it accounts for most of the HCV cases that we see and also because it is not so the present treatments are not extremely effective against genotype 1 hence it's worth investigating what roles would these proteins play in the viral life cycle of this genotype. A permanent problem in HCV in vitro studies is that we hadn't come up till recently with a system in which the virus replicates well in vitro. And just recently researchers came up with a genotype that is genotype 2A which is the only virus that replicates and infects human hepatoma cells efficiently in vitro. So any study or other strains for example if we are concentrating on the genotype 1 strain would involve us cloning the region of our interest from genotype 1 into a strain that replicates efficiently and infects efficiently in culture so that we can study potential targets for drug disease in vitro. Now this basically shows a homology of the regions of our interest which I had shown earlier with the genotype 2A that is the strain that replicates well in culture. If you look at the homology between our regions of interest and the strains and the strain that replicates well in culture you would see a very low homology. So basically this is to drive the point that for one the C-terminal domain is quite variable among different genotypes and second it is worth studying this domain in the context of genotype 1A and 1B. So the objectives of my study one do these primary constructs modulate viral replication in vitro and second do these modulate viral assembly and release in vitro. If we do find an altered capability in replication or viral production in these kind of strains that we can implicate the role of possibly the role of certain serine and cloning residues that we think are important in the phosphorylation events at the C-terminal domain in the viral life cycle. Now coming to the approach we do two separate experiments for replication and infection. So first focusing on the replication the replication would involve cloning our regions of interest into this following construct. If you look at this construct it lacks the structural proteins that is the core and the envelope proteins and therefore this construct can replicate efficiently in culture but it would not be able to assemble and leave this in and hence virus infection is not possible. So this construct would help us monitor replication separately. This construct also has a luciferase region luciferase gene and this would help this acts as a reporter gene and it would help us modulate the replication levels in different time points. We then go ahead with the in vitro transcription of these plasmids since it's an RNA virus and then you re-electroporate the in vitro cells we use human hepatoma cells with the synthesized RNA and then you would monitor the replication levels or titers through luciferase readings every 24 hours. Focusing on the virus assembly and release it more or less follows the same protocol but now we do have the structural proteins and hence the virus would replicate as well as assemble and leave the cell. We are going to an in vitro transcription of these plasmids and electroporate the cells with the synthesized RNA but the difference being instead of monitoring the replication levels in this since we do not want to monitor replication levels we collect the supernated of this batch of cells at 24 hour time points because we assumed that the virus would have assembled and left and release the cells into the supernated. We then take the supernated and subject it to another batch of cells and then measure the luciferase reading 48 hours post infection and this would help us give a tighter value on what the infection levels were of the virus. Coming to the results so this is planning the replication data these are the luciferase readings plotted against the time so these are initially the controls that is the GND. The GND is a mutation in the NS5B that is the RNA dependent RNA polymerase of the virus and this mutation would not allow the virus to replicate and this is the wild type profile. Now if you look at the replication data for the other three strains what we found is we did not find a very significant difference between the different virus strains in the replication data therefore we concluded that we would now go on to infection and check for release. Now coming to the viral life cycle virus assembly and release so now I am focusing on the infection part. Now the cells of the assembly and release so we attained data for 48 hours till now and what we essentially found is in 48 hours there is a huge increase there is a significant increase in the NS5A1A that is from the 356 to the full 48 strain. So data for 48 hours should have a significant increase in viral virus assembly and release for genotype 1A kind of extremes that is this one and this one as compared to viral family this one. So the summary for one the replication data does not show significant differences in the luciferase readings among the different climatic strains virus production does show a significant increase in 48 hours for the genotype 1A strains. Future experiments will involve repeating the infectious rule experiments once more to check whether the data obtained is actually right and second information and confirmation of potential phosphorylation sites in the C-terminal domain of NS5A which would actually explain the increased virus productions in the genotype 1A kind of extremes which we found. Acknowledgements, I would sincerely like to thank my co-guide Chisrar for giving a lot of time with me for this project. I would also like to thank my guide Professor Robert Stryker for hosting me in his lab. It was an amazing experience Dipankar, Julie, Ismail, Jeremy thank you for making my lab experience so enjoyable. I would also like to thank the Kuranda Program, the Department of Biotechnology Government of India and the Alice S. Tia for the funding. Thank you. I'll tell you why your mutant polymerase cell grows. So there might be a few reasons behind it. One might be experimental but it's also possible that the GND since it's an RNA-dependent RNA polymerase that is involved and I had said the fidelity of the enzyme is low. There might be mutations which were leading to that but in the other set of experiments that we did, we did not find this so it generally doesn't happen but it might have happened due to the fidelity of the enzyme which might have lateral mutation. May be further analysis on that. How did you choose these mutants? Did you choose to make the mutation? Why did you make that particular mutation? This mutation because it's a change in the RNA-dependent RNA polymerase that is the active site of the enzyme which does not allow the virus to replicate. This is one amino acid change from GDD to GND in the active site so it does not allow the virus to replicate. That would serve as an ideal control.