HEPATITIS C VIRUS
Hepatitis C virus (HCV), causing viral hepatitis, belongs to Hepacivirus genus of the Flaviviridae family. The error prone viral genome replication enables host immune evasion, which in turn facilitate viral persistence, leading to liver fibrosis, cirrhosis and hepatocellular carcinoma. HCV consists of a 9.6-kb single-stranded positive-sense RNA genome containing an open reading frame (ORF) flanked at both ends by highly structured and conserved untranslated regions (5’ and 3’UTRs). HCV translation is mediated by an internal ribosome entry site (IRES) located within the 341-nucleotide (nt) of 5′ UTR which further extends 30 to 40 nt downstream of the initiator AUG codon. On the other hand, HCV replication is mostly mediated by the 3′ UTR, which synthesizes a negative strand RNA. This negative-strand RNA then serves as a template for the generation of the plus-strand copies of viral RNA.
The error prone replication for HCV also leads to a challenge for development of specific antiviral therapies against all viral genotypes. The current therapy includes a combination of pegylated-IFNα (peg-IFNα) and ribavirin with either telaprevir or boceprevir. However, this triple therapy regimen has additional limitations due to selection of resistance variants.
The major focus of the study in our lab is to understand the basics of the various biological processes of the virus and implementation of the knowledge for the development of effective and suitable antivirals targeting those processes by use of different high end technology.
Translation of Hepatitis C Virus (HCV) is mediated by an internal ribosome entry site (IRES) located within the 5’ UTR. HCV IRES binds to the 40S ribosomal subunit stably even in absence of initiation factors. Several cellular trans-acting factors are known to bind to HCV IRES and influence the internal initiation. Our lab has characterized interaction of La (a cellular trans-acting factor), with HCV IRES, both in vivo and in vitro. Also, highlighted (in collaboration with Prof. N.Srinivasan, MBU, IISc) the structural insight of the RNA recognition motif of La protein critical for the interaction with HCV RNA. (Pudi et al. Journal of Biological Chemistry, 2003 and 2004). Human La autoantigen has been shown to influence internal initiation of translation of hepatitis C virus (HCV) RNA. Previously, we have demonstrated that, among the three RRMs of La protein, the RRM2 interacts with HCV internal ribosome entry site (IRES) around the GCAC motif near the initiator AUG present in the stem region of stem-loop IV (SL IV).
We have demonstrated that the mutations in the GCAC motif, which altered the binding to RRM2, had drastic effect on HCV IRES-mediated translation, both in vitro and in vivo. The results indicated that the primary sequence of the stem region of SL IV plays an important role in mediating internal initiation. Furthermore, we have shown that the mutations also altered the ability to bind to ribosomal protein S5 (p25), through which 40S ribosomal subunit is known to contact the HCV IRES RNA. Our observations strongly support the hypothesis that La protein binding near the initiator AUG facilitates the interactions with ribosomal protein S5 and 48 S ribosomal assembly and influences the formation of functional initiation complex on the HCV IRES RNA to mediate efficient internal initiation of translation (Pudi et al. Journal of Biological Chemistry, 2004 ).
Different stages of hepatitis C virus (HCV) life cycle are coordinated by a complex interplay of cis-acting elements, viral proteins and host factors. One of these stages is replication. HCV non-structural protein 3 (NS3) plays crucial role in viral polyprotein processing and RNA replication whereas human La protein (a cellular factor) is important for both HCV translation as well as replication. From our laboratory, we showed that interplay between NS3 and La regulates translation- replication switch in HCV. We found that both NS3 protease and La interact with stem-loop IVof HCV IRES at similar positions. Further, we observed that interaction of NS3 protease to HCV IRES inhibits its function. This inhibition of HCV translation makes HCV RNA free to undergo La mediated replication. Thus the competition between the host factor (La) and the viral protein (NS3-protease) for binding to HCV-IRES contribute to the molecular switch from translation to replication of the HCV-RNA (Ray and Das, Scientific Report, 2011).
Another study from lab deciphered the mechanism of regulation of HCV replication by interaction of cis-acting element GCAC within HCV IRES with human La protein. Using mutational analysis, we found that GCAC sequence plays role in replication independent of translation. We have shown that human La protein interacts with both 5’and 3’UTR which is crucial for linking 5’ and 3’ends of HCV genome. Results demonstrate the mechanism of regulation of HCV replication by interaction of cis-acting element GCAC within HCV IRES with human La protein, unravelling yet one more specific target to inhibit HCV replication (Kumar et al, Journal of Virology, 2013).
Highly restricted species specificity of HCV has prevented the development of small animal models to facilitate drug discovery and better understanding of host-virus interactions. Recently, in collaboration with Prof. Siddhartha Roy, IICB, Kolkata, we showed that a highly conserved unique beta-turn in human La protein may contribute to host tropism of HCV. We observed that chimeric mouse La bearing human beta turn sequence, but not mouse La alone, could promote increased HCV RNA replication in mouse cells. Our results provided molecular insight into one of the post entry factors that might be considered in generating HCV mouse model. (Kumar and Manna et al, Journal of Virology, 2014).
HCV-induced hepatitis is a serious medical problem of modern age with more than 170 million chronic carriers throughout the world. Chronic HCV infection can lead to liver cirrhosis and hepatocellular carcinoma (HCC). No vaccine is available yet. Different anti-HCV agents have been explored in recent past. Our research group demonstrated that a synthetic peptide LaR2C, derived from La-RRM (112-184) could interact with HCV RNA and interferes with the formation of 48S initiation complexes by acting as a dominant negative inhibitor of viral RNA translation (Pudi et al, Journal of Virology, 2005).
We have shown that competition between the host factor (La) and the viral protein (NS3-protease) for binding to HCV-IRES contribute to the molecular switch from translation to replication of the HCV-RNA. Recently we have targeted the translation-replication switch using a peptide derived from the RNA binding region of the NS3pro protein. A 30-mer peptide was designed from the predicted RNA-binding region of NS3 protease that inhibited IRES-mediated translation. Further, this peptide was truncated to 15-mer and this could also inhibit HCV translation as well as replication (in collaboration with Prof. N. Srinivasan, MBU, IISc). More importantly, in collaboration with Prof. Debi Sarkar, UDSC, we tested its activity in an in vivo mouse model using Sendai virus based virosome system (Ray and Roy et al, Molecular therapy, 2013).
Additionally, our group (in collaboration with Dr. Akhil Banerjea, NII) demonstrated an efficient use of DNAzymes as inhibitor of HCV-RNA translation and replication (Roy et al, Journal of General Virology, 2008). Our laboratory (in collaboration with Prof. Debi Sarkar, UDSC) successfully demonstrated efficient delivery of antiviral shRNAs into mouse liver using Sendai virus fusion protein-based virosome system, and inhibition of HCV-RNA translation in whole animal (Subramanian et al. Journal of General Virology, 2009).
Further, we designed different shorter RNAs to identify the minimum region of SLIII e+f required to inhibit HCV translation. Among them a short RNA, SLRef containing both SLIIIe and SLIIIf stem loops, sequestered La protein and ribosomal protein S5 to inhibit HCV translation and viral RNA synthesis.
More importantly, using Sendai virosome they have preferentially delivered this small RNA into mice liver and demonstrated inhibition inhibit HCV RNA (Bhat et al., RNA Biology, 2012). These observations constitute the “proof of the concept” that the ‘ribosome-viral RNA’ interaction can be specifically targeted to develop novel antiviral to combat HCV infection. Stabilizing the SLRef RNA by chemicalmodification and its targeted delivery to liver cells using efficient delivery system is currently in progress in the lab.
Antivirals targeting HCV enzymes: HerbalsIn view of safe and effective drug development for anti HCV therapy, we identified, Phyllanthus amarus (Euphorbiaceae), a natural herbal plant, which inhibits HCV viral enzymes and replication most significantly (Ravikumar et al, 2011, Journal of virus Research). Our current efforts include logical stepwise elucidation process to purify and identify bioactive components from Phyllanthus amarus and tracking their inhibitory effect at different stages of Hepatitis C virus life cycle, such as viral entry, attachment, replication, translation and release mechanisms. Apart from this we have also standardized protocols to study directly viral enzymes inhibition mechanisms.
Further, we have evaluated the anti-HCV activity, toxicity and the bioavailability of the active components in mice and rats. For a proof of concept, the most promising compounds are being tested in HCV-mouse models.
Our lab (as part of an Indo-Australian project) has also initiated work on HCV vaccine inducing both humoral and cell mediated immunity. We are trying to develop a vaccination regimen to generate antibody-based and cell mediated immunity against hepatitis C virus against multiple HCV genotypes. This will be achieved by simultaneous vaccination with virus-like particles (VLPs) containing the core and envelope proteins for neutralizing antibody generation and DNA vaccine construct encoding selective non-structural proteins for inducing cell mediated immunity. The results of vaccination with the individual components of the vaccine will then be measured in large animal model which represent a suitable model to test the vaccines for eventual use in humans. These studies are expected to yield an effective HCV vaccine able to generate effective immunity against multiple genotypes in humans.
The current focus of our lab also involves the study of miRNA and proteome profiles in the serum of HCV infected patients. We have carried out a 2-DE based proteomic analysis and identified differentially regulated proteins in serum of Indian patients infected with HCV genotypes 1 and 3. We have identified eight such proteins and validated one of the proteins, retinol binding protein 4 (RBP4), which showed significant upregulation in HCV infected serum samples compared to healthy controls.
We have also used JFH1 infectious cell culture system to confirm our observation and are interested in characterizing the role of RBP4 in HCV infection in detail. (Gouthamchandra et al, Journal of General Virology, 2014 ).
In parallel, we have performed miRNA microarray studies with RNA from HCV infected serum samples. miR-320c and miR-483-5p were validated to be upregulated from the screen in the serum of infected patients as well as in HCV-JFH1 infection.
Delineation of the pathways affected by the upregulation of these miRNAs would provide novel insights into the mechanism of pathogenesis. (Shwetha et al, Scientific Reports, 2013)
LIVER TARGETED DELIVERY
Nano-carriers are materials with sizes ranging from a few nanometers to about 500-700nm. They are widely being tested for their applicability to delivery drugs and genes. Commonly used nanocarriers are miscelles, liposomes, polymers, carbon nanotubes, dendrimers, metal nanoparticles and other substances suitably modified for biocompatibility. Virus based delivery vectors, especially adenoviral therapy had been the main focus for gene delivery and gene therapy for many decades. However, the massive immune response and the tragic “gene therapy death” in 1990 has prompted the need to find safer, efficient and target specific vectors. Working towards this aspect, we collaborate with synthetic chemists (Prof. N. Jayaraman, Department of Organic Chemistry, IISc) and material engineers(Prof. Ashok Raichur, Department of Materials Engineering, IISc) inthe quest for developing novel, non-toxic and target specific vectors to combat HCV infection. We have shown that PETIM dendrimers, a novel class of monodispersed, non-toxic, biocompatible molecules which can complex and compact nucleic acids via electrostatic interactions and deliver them into mammalian cells. (Lakshminarayanan et al, Bioconjugate Chemistry, 2013). We are currently working on designing the PETIM dendrimers to suit specific purposes of delivery of a wide range of nucleic acids and drugs molecules. Silica based nanoparticles can be loaded with the gene or drug of interest and the sustained delivery of the cargo presents a major advantage over many known synthetic delivery vectors. We are also exploring the use of single walled carbon nanotubes and peptides as gene delivery vectors.
MECHANOBIOLOGY OF LIVER CELLS
We intend to look at the effects at single cell level and at population level.
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