Wednesday, November 23, 2016

Viral Replication: Helping the enemy

            Hepatitis C virus (HCV) is a single-stranded RNA virus that is surrounded by a lipid membrane called an envelope.  The 9600 base pair genome codes for structural proteins (core, E1, and E2) that are part of the proteinaceous shell, the capsid, and non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that are not part of the capsid (1).  These ten proteins permit infection of more than 150 million people across the globe.  Chronic HCV infection can cause cancer of the liver, liver disease and ultimately death (2). 
Due to the limited nature of viral genomes compared to bacteria or humans, viruses are obligate intracellular parasites that must employ host cell factors for replication.  In order to better understand the host proteins involved, previous research examined the replicative ability of HCV when certain kinases, enzymes that modify proteins with a phosphate group, were partially depleted or knocked-down.  The knockdown indicated that human choline kinase-α (hCKα) potentially inhibited HCV replication (3).  hCKα is a kinase involved in the first step of synthesizing a phospholipid destined for the cell membrane.  However, the molecular relationship between hCKα and HCV replication was not investigated until the work of Wong and Chen (4).
            As a kinase with the ability to phosphorylate any number of substrates, hCKα could regulate HCV replication at any stage.  After confirming the results of the previous research, Wong and Chen performed a specific assay designed to test viral entry into host cells using pseudo-HCV particles that only had the HCV envelope.  Targeted knockdown of hCKα did not have a significant effect on entry compared to a control knockdown.  To test the role of hCKα in RNA synthesis, a luciferase assay was utilized (5).  This technique involves the luciferase gene from a firefly that allows it to glow, which is placed after the promoter required for transcription of a gene of interest.  Wong and Chen used the luciferase assay to measure the transcriptional activity of RNA synthesis of HCV.  In the control knockdown, the luciferase activity first decreased and then increased post viral RNA insertion, or transfection (6).  The trend observed was a result of structural and non-structural proteins being transcribed first before a switch to full-length genome replication.  However, when hCKα was knocked-down, the luciferase activity was decreased compared to the control and never completely recovered over the 72 hour time period.  To investigate how hCKα regulates the RNA replication of HCV, two different competitive inhibitors of the enzymatic activity of hCKα were used.  When cells were transfected with the viral RNA conjugated to the luciferase reporter, there was a decrease in RNA transcription in a dose dependent manner.  Furthermore, the effects of the inhibitors were mitigated by a single residue mutation of the hCKα active site, which constitutes a rescue experiment.  Based on the rescue experiment, the authors definitively show that the activity of hCKα plays a role in regulating HCV RNA synthesis. 
To better understand the role of hCKα activity, Wong and Chen employed fluorescence markers for NS5A and hCKα along with a knockdown targeted to hCKα.  Visualization by microscopy revealed co-localization of NS5A and hCKα to the endoplasmic reticulum (ER) for the control knockdown, but a statistically significant decrease in localization when hCKα was knocked-down.  The differences observed in the fluorescent micrographs (shown below) support the notion that hCKα is important for NS5A localization to the ER.  In the ER membrane, the nonstructural HCV proteins, like NS5A, can help form specialized structures required for active viral replication, termed membrane webs (MWs).  MWs are important for efficient viral replication because these compartments increase the concentration of viral and host components for HCV replication.  When hCKα was knocked-down or activity was inhibited, MW formation was diminished.  Therefore, knockdown of hCKα might inhibit RNA synthesis by altering the ability of necessary replication components to localize to the ER in order to form the MWs.
 Figure 9A: shRNA was used for knockdown of hCKα compared to a control.  Fluorescence shows location of indicated protein and CALR is a marker for the ER.  The white box in the merged image designates the Zoom area and white arrows point to ER localization.

            Wong and Chen provided a valuable context in which to understand the role of hCKα in HCV replication.  However, the authors never examined phosphorylation, which is the primary function of a kinase like hCKα.  Testing of phosphorylation states can be accomplished via numerous methods, including western blotting (7).  Western blotting provides information regarding the relative expression of specific proteins and can be utilized for a phosphorylated form.  Such basic research is important because by better understanding the molecular mechanism of HCV replication, novel antiviral drugs can be developed.  Multiple lines of treatment are critical for effective therapy because RNA viruses have high mutation rates, which allows for rapid development of resistant to antivirals (8).  Basic research is the foundation for controlling HCV replication and thereby limiting transmission.  


1. Moradpour, D., Penin, F., & Rice, C.M. Replication of hepatitis C virus. Nature Review Microbioloy 5:453-463 (2007). http://dx.doi.org/10.1038/nrmicro1645.

2. Hepatitis C. World Health Organization (2016). at <http://www.who.int/mediacentre/factsheets/fs164/en/>

3. Reiss S, Rebhan I, Backes P, Romero-Brey I, Erfle H, Matula P, Kaderali L, Poenisch M, Blankenburg H, Hiet MS, Longerich T, Diehl S, Ramirez F, Balla T, Rohr K, Kaul A, Buhler S, Pepperkok R, Lengauer T, Albrecht M, Eils R, Schirmacher P, Lohmann V, Bartenschlager R. 2011. Recruitment and activation of a lipid kinase by hepatitis C virus NS5A is essential for integrity of the membranous replication compartment. Cell Host Microbe. 9:32–45. 10.1016/j.chom.2010.12.002.

4. Wong, M. & Chen, S. Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation. Journal of Virology 90, 9075-9095 (2016).

5. Smale, S. Luciferase Assay. Cold Spring Harbor Protocols (2016).

6. Kim, T. & Eberwine, J. Mammalian cell transfection: the present and the future. Analytical and Bioanalytical Chemistry 397, 3173-3178 (2010).

7. Yang, P., Liu, Z. & Mahmood, T. Western blot: Technique, theory and trouble shooting. North American Journal of Medical Sciences 6, 160 (2014).

8. Presloid, J. & Novella, I. RNA Viruses and RNAi: Quasispecies Implications for Viral Escape. MDPI (2016). at <http://www.mdpi.com/1999-4915/7/6/2768>

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