15 research outputs found
Regulation of Hepatitis C Virion Production via Phosphorylation of the NS5A Protein
Hepatitis C virus (HCV) is a significant pathogen, infecting some 170 million people worldwide. Persistent virus infection often leads to cirrhosis and liver cancer. In the infected cell many RNA directed processes must occur to maintain and spread infection. Viral genomic RNA is constantly replicating, serving as template for translation, and being packaged into new virus particles; processes that cannot occur simultaneously. Little is known about the regulation of these events. The viral NS5A phosphoprotein has been proposed as a regulator of events in the HCV life cycle for years, but the details have remained enigmatic. NS5A is a three-domain protein and the requirement of domains I and II for RNA replication is well documented. NS5A domain III is not required for RNA replication, and the function of this region in the HCV lifecycle is unknown. We have identified a small deletion in domain III that disrupts the production of infectious virus particles without altering the efficiency of HCV RNA replication. This deletion disrupts virus production at an early stage of assembly, as no intracellular virus is generated and no viral RNA and nucleocapsid protein are released from cells. Genetic mapping has indicated a single serine residue within the deletion is responsible for the observed phenotype. This serine residue lies within a casein kinase II consensus motif, and mutations that mimic phosphorylation suggest that phosphorylation at this position regulates the production of infectious virus. We have shown by genetic silencing and chemical inhibition experiments that NS5A requires casein kinase II phosphorylation at this position for virion production. A mutation that mimics phosphorylation at this position is insensitive to these manipulations of casein kinase II activity. These data provide the first evidence for a function of the domain III of NS5A and implicate NS5A as an important regulator of the RNA replication and virion assembly of HCV. The ability to uncouple virus production from RNA replication, as described herein, may be useful in understanding HCV assembly and may be therapeutically important
A Deletion in NS5A Domain III Disrupts the Production of Infectious Virus.
<p>A). Schematic representation of the domain organization of NS5A indicating the locations of the three domains and low complexity domain connectors (LCS). The location of the membrane anchoring helix is also shown. Numbers indicate amino acid positions on the Con1 genotype 1b NS5A sequence. Sequences of the C terminus of NS5A indicated by the black box on the schematic are shown below for representative sequences of major HCV genotypes. The sequences shown are 1a H77 (AF011753), 1b Con1 (AJ238799), 2a JFH-1 (AB047639), 2b HC-J8 (D10988), 3a HCVCEN (X76918), 4a ED43 (Y11604), 5a EUH1480 (Y13184), and 6a 6a33 (AY859526). B). IHC analysis of cells electroporated with indicated viral RNAs (top of image rows) for NS5A 48 hours post electroporation (top row of panels) or 48 hours post infection (bottom row of panels). C). Western blot of wild type (WT) and deletion B (del B) NS5A from cells electroporated with replication competent full-length genomes shown in B and D. D). RNA replication 48 hour time course of J6/JFH-1 (blue line), J6/JFH-1 deletion B (red line), or J6/JFH-1 pol- (black line) as determined by real time PCR analysis. E). RNA replication 48 hour time course of indicated subgenomic replicon RNAs. pSGR-JFH-1 (red line), pSGR-JFH-1 deletion B (blue line), and pSGR-JFH-1 pol- (black line). F). Infectivity release 48 hours (gray bars) and 6 days post (black bars) electroporation from indicated viral RNA electroporated cells in units of tissue culture infectious dose 50% value per milliliter. G). Infectivity released from cells via freeze-thaw treatment 48 hours post electroporation from indicated viral RNA electroporated cells. H). Relative HCV RNA release from cells electroporated with the indicated HCV constructs. Values reported are arbitrary units normalized to the RNA release by a non-replicating J6/JFH-1 pol-.</p
Single amino acid mutations that mimic phosphorylation of serine 457 alter particle assembly.
<p>A). IHC analysis of cells electroporated with indicated point mutation bearing viral RNAs. AEED indicates mutation of serine 457 to alanine, DEED is the same residue changed to aspartic acid, SEEDcc is a change of the codon of serine 457 while retaining serine at this position. Rows of images are as described in 1B, 2B and 2D. B). RNA replication 48 hour time course of J6/JFH-1 (red line), J6/JFH-1 deletion B (blue line), J6/JFH-1 pol- (black line), AEED RNA (green line), DEED RNA (purple line), and SEEDcc RNA (light blue line) as determined by real time PCR analysis. C). Infectivity release 48 hours post electroporation from indicated viral RNA electroporated cells in units of tissue culture infectious dose 50% value per milliliter. D). Western blot analysis of NS5A from cells electroporated with replication competent full-length viral genomes as shown in panel A. Nomenclature used is as described in A.</p
The casein kinase II inhibitor DMAT disrupts the production of infectious virus.
<p>A). IHC infectivity assay showing cells 48 hours post infection stained for NS5A expression. Individual images correspond to cells treated with DMSO, 0.5 µM DMAT in DMSO, and 2 µM DMAT 4 hours after electroporation. B). Infectivity release of J6/JFH-1 and J6/JFH-1 DEED 48 hours post electroporation versus dose of DMAT added to cells. Values shown are percent infectivity release of DMSO treated cells receiving identical viral RNAs. Cell viability is percent viable cells of a DMSO only treated control cells as measured by an ATP based cell viability assay. C). HCV RNA replication 48 hours post indicated concentration of DMAT treatment for J6/JFH-1 (light gray bars) and J6/JFH-1 DEED (dark gray bars) RNA electroporations. Bars designated zero are DMSO only treated. D). Treatment of cells with an unrelated kinase inhibitor, 2-aminopurine (2-AP), does not affect infectious virus release. Cells were treated with 2-AP at the indicated concentration or a DMSO control for 1 hour prior to infection. Virus output was then titered 48 hours post infection. In another experiment, 2-AP, was left in the culture medium at 1 mM for the 48 hour course of the experiment (black bars). E). Effect of CKII inhibition with DMAT on NS2 stability. Indicated doses of DMAT were added to cells electroporated with JC1 HCV RNA for a period of 48 hours. Following this treatment, cells were lysed and Western blots for NS2 (top row) and β-actin (bottom row) were performed to look for differences in NS2 stability.</p
Mapping the disruption of infectious virus production.
<p>A). Sequence of JFH-1 NS5A around the region of deletion B (indicated by gray line). Deletions B-1, B-2 and B-3 are indicated by black bars. Numbers refer to amino acid numbering of the mature JFH-1 NS5A protein. Residue serine 452 is shown in red. B). IHC analysis of cells electroporated with indicated deletion bearing viral RNAs (top of image rows) for NS5A 48 hours post electroporation (top row of panels) or 48 hours post infection (bottom row of panels). Numbers beneath each panel are HCV RNA copies per 100 ng of total RNA (top row of images) and infectivity release from titering (bottom row of images). C). Sequence of JFH-1, as described in A, now showing the location of deletion B-4 and B-5 with black bars. D). IHC analysis of deletion B-4 and B-5. Panel layout is as described in B. Numbers below the images are as described in B. E). Western blot analysis of NS5A from cells electroporated with replication competent full-length viral genomes of wild-type (WT) and deletions B-1, B-2, and B-3 (del B1, del B2, and del B3) shown in panel B. F). Western blot analysis of NS5A from cells electroporated with replication competent full-length viral genomes as shown in panel D. Nomenclature is as described in E.</p
Core protein release.
a<p>Values reported as average of three independent experiments in the format “average value (+/−standard deviation)” in units of femptomoles of HCV core protein per liter 24 hours post electroporation.</p
Levels of CKII α and α′ subunits affects infectious virus production.
<p>A). Western blots of cells electroporated with CKI α, CKIα′, or both CKIα and CKIα′ siRNAs are indicated and probed with antibodies recognizing CKI α, CKIα′, or both CKIα and CKIα′. A time course of 72 hours post siRNA delivery is shown. β-actin loading control panels are shown in the lower row of images. B). Cell viability, expressed as percent viability of cells electroporated with no siRNA, for cells electroporated with irrelevant siRNA (IRR), CKI α (CKIA), CKIα′ (CKIA′), or both CKIα and CKIα′ (MIX) as determined by ATP activity assay. C). CKI α and CKIα′ messenger RNA levels, relative to GAPDH in arbitrary units. Dark lines and text cues indicate which real time PCR probe set was used for each set of bars. Designations under bars indicate the siRNA delivered to the cells. Designations of siRNAs are as described in B. D). HCV RNA replication 72 hours post siRNA treatment for cells treated with various siRNAs (labels below columns) and infected with HCV viruses indicated below the dark bars (J6/JFH-1 or J6/JFH-1 with the DEED mutation). Column colors further highlight the different viruses used for these experiments, with light gray for J6/JFH-1 infected samples, and dark gray for J6/JFH-1 DEED infected samples. E). Infectivity release 72 hours post treatment with the indicated siRNAs (column labels) for cells infected with viruses indicated by dark bars (J6/JFH-1 or J6/JFH-1 with the DEED mutation). Column bar colors are as designated in D. F). CKII α and CKIIα′ catalytic subunit over-expression (OE) relative to cells transfected with an empty vector (M). Western blots using CKII α and CKIIα′ antisera as indicated. β-actin Western blot of samples shown in over-expression samples as a loading control.</p