Building the foundation to combat HCV

     Hepatocellular carcinoma, or liver cancer, accounts for tens of thousands of deaths a year.  Many factors are implicated as causative agents, however most cases are associated with one of two viruses: hepatitis B and hepatitis C (1).  Unfortunately, these viruses are common amongst the human population.  It is estimated that about 175 million people in the world are infected with hepatitis C virus (HCV) and that 75% of these individuals are unable to clear the virus from their body. Consequently, most individuals are chronically infected with HCV providing the chance for long-term detrimental health effects like cirrhosis or liver cancer to develop.  Thus, research has been interested in developing both treatments and vaccines for HCV.

               
     HCV belongs to the Flaviviridae family of viruses, characteristic for their positive-sense, single-stranded RNA genome.  The viral genome encodes a polyprotein that is processed by both host and viral proteases.  In other words, HCV completes it's life cycle and produces new virions by encoding a large protein that is cleaved into smaller parts with differing biological functions.  Namely, the processed polyprotein leads to three structural proteins essential for virion assembly and seven non-structural proteins essential for the HCV life cycle (3). Drugs have been developed to specifically inhibit the non-structural proteins and function by shutting down the enzymatic functions of such proteins.  However, drugs targeting the non-structural 5A (NS5A) protein, a regulator of viral replication and assembly, do not function in this way given that NS5A does not bear apparent enzymatic functions (4).      

     It has been well-established that NS5A's functions are regulated in part by its phosphorylation  states.  Thus, drugs inhibiting a phosphorylated state have been developed that have proven successful for inhibiting viral replication (5).  Because it is understood how these drugs function, an interesting platform is provided for research to investigate mechanisms involved with NS5A.  In other words and for example, how is NS5A being phosphorylated?  Understanding mechanistic details of the HCV life cycle is essential for developing more efficient treatments and a global vaccine, and thus research is interested in carrying out such experiments.

Phosphorylation via a protein kinase.

     A recent study motivated by these ideas sought to identify the kinases responsible for NS5A phosphorylation.  The researchers previously identified a serine phosphorylation site (S235) shown to be essential for viral replication activity, and use the phosphorylation state of S235 as a proxy for replicative success.  The study investigates three protein kinases already known to or predicted to phosphorylate NS5A: casein kinase I α (CKIα), polo-like kinase I (PlkI) and calmodulin-dependent kinase II (CaMKII).  By treating HCV infected cells with calmodulin and calmodulin-dependent kinases inhibitors to prevent these kinase activities, the researchers show that in all three cases the NS5A S235 phosphorylation is reduced.  The researchers emphasize that this is not a result of having simply less NS5A in the sample, indicating that these kinases are in fact playing a role in NS5A S235 phosphorylation.

     The researchers then sought to investigate CaMKII activity further, since CKIα has already been studied and the PlKI inhibitor has shown toxic effects to cells. Similar experiments to those previously mentioned suggest that HCV RNA levels are also significantly reduced when treating HCV infected cells with a CaMKII inhibitor, indicative of the successfulness of viral replication in the absence of a kinase (now) known to phosphorylate NS5A.  The next question the researchers attempted to answer is whether or not the CaMKII isoforms expressed in this model system are directly phosphorylating NS5A at S235.  Using the power of labelled proteins, the findings conclude that the two CaMKII isoforms expressed can in fact directly phosphorylate NS5A S235 in vitro.  Taking it one step further to understand mechanistic details in vivo, the researchers investigated the effects of CaMKII knockdowns (reduced expression).  Intuitively, one would expect a CaMKII knockdown to reduce NS5A S235 phosphorylation and HCV RNA levels based on the previous inhibitory experiments.  However, quite the opposite is observed.  CaMKII knockdown does not affect the relative levels of NS5A S235 phosphorylation and actually appears to elevate the RNA levels.
 
     To understand this further, a series of knockdown experiments were performed subsequently involving both CaMKII and CKIα.  The data ultimately suggests a predominant role of CKIα in NS5A S235 phosphorylation in vivo, and a potential negative role of CaMKII in the HCV life cycle; CKIα and CaMKII double knockdown show higher HCV RNA levels than the single CKIα knockdown.  Despite the unexpected findings, these findings provide a foundation for understanding the phosphorylation mechanisms of NS5A.  Further research can aim to fine-tune this model and provide a novel model for NS5A phosphorylation.  Once such a model has been developed, research can then seek to develop specific and efficient drugs to combat such activity essential for the HCV life cycle.  Studies like these evidently provide a foundation for then investigating intervention points for developing global vaccines that could prevent the "invisible" HCV infections from occurring in the first place.      

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Primary Article:
Lee, Kuan-Ying et al. (2016).  Phosphorylation of serine 235 of the Hepatitis C virus non-structural protein NS5A by multiple kinases. PLoS ONE, 11(11): e0166763.

Works Cited
1.  Carter, John B. and Saunders, Venetia A. (2013).  Virology: Principles and applications.  John Wiley & Sons Ltd. 2nd edition.  Print.
2.  http://www.cdc.gov/hepatitis/hcv/cfaq.htm#cFAQ71
3.  Scheel TK, Rice CM (2013).  Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.  Nat Med, 19(7):837-849.
4.  Bartenschlager R, Lohmann V, Penin F. (2013).  The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection.  Nat Rev Microbiology, 11(7): 482-496.
5.  Ross-Thriepland D, Harris M (2014).  Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation. Journal of Virology, 88(3): 1421-1432. 

Links found within text
http://www.hope4cancer.com/diseases-we-treat/non-cancer-diseases/chronic-infections.html
http://web.stanford.edu/group/virus/flavi/2008/flavi.html
http://www.virology.ws/2010/07/22/the-virus-and-the-virion/
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/phosphorylation.html
https://www.ebi.ac.uk/interpro/entry/IPR001245
http://rosalind.info/glossary/protein-isoform/
http://www.dictionary.com/browse/in-vitro
http://www.healthline.com/health/hepatitis-c/symptoms#Overview1