SH2 modified STAT1 induces HLA-I expression and improves IFN-γ signaling in IFN-α resistant HCV replicon cells
Authors: Partha K Chandra
Luis A Balart
Feyza Gunduz
Srikanta Dash
Bret Poat
Xavier Alvarez
Sidhartha Hazari
Journal: PLoS ONE (2010)
Discipline: Multidisciplinary
Keywords: HCV RNA; interferon alpha; STAT1; STAT2; Jak-STAT pathway
Affiliations: Department of Pathology and Laboratory Medicine, New Orleans, Louisiana, United States of America.

Abstract:  Background: We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway. Methodology/principal findings: In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls. Conclusions: These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation. 

Retracted: No

PubPeer comments:
 This article has a November 2010 correction to add two authors: https: journals.plos.org plosone article?id=10.1371 annotation ff8fe1fe-36ae-43fe-9350-8ba2f191331a  *The authors wish to add two authors to the byline, Dr. Yong Zhang and Dr. Michael J. Holtzman. The correct byline is now: Bret Poat, Sidhartha Hazari1, Partha K. Chandra1, Feyza Gunduz, Luis A. Balart, Xavier Alvarez, Yong Zhang, Michael J. Holtzman, Srikanta Dash. The affiliation for Drs. Zhang and Holtzman is: Department of Medicine, Department of Cell Biology Washington University School of Medicine, St. Louis, Missouri, United States of America. The author contributions should now be: Conceived and designed the experiments: BP SH SD. Performed the experiments: BP SH PKC FG XA. Analyzed the data: BP SH PKC FG LAB SD. Contributed reagents materials analysis tools: BP SH PKC XA YZ MJH SD. Wrote the paper: BP SD. Critically read the manuscript: LAB.* , 
 Figure 2. * Red boxes: two areas in the p-Jak2 blot appear to look similar to each other. Image made darker to bring out some more details. ![file] , 
 Figure 5. * Pink boxes: Four lanes look remarkably similar. ![file] , 
 Figure 6. * Orange and blue boxes: The panels shown in Figure 6A also appear in Figure 6B, in a different orientation and different contrast. I am not sure if these depict the same experiment; maybe the authors can clarify. * Cyan boxes: Two panels that do not appear to show the same experiment also look similar. ![file] , 
 Figures 1C and 8: Unexpected similarities with Figure 7A in Poat et al. Virology Journal (2010), DOI: 10.1186 1743-422X-7-265 - https: pubpeer.com publications C35F31B5C0502D6E465655466E5ECA * Red boxes: One of the panels in Figure 8 looks similar to two panels in Figure 7A of the Virology J paper. * Blue boxes: One of the panels in Figure 1C looks similar to a panel in Figure 7A of the Virology J paper. * Green boxes: One of the panels in Figure 1C looks similar to a panel in Figure 7A of the Virology J paper. From looking at the labels, these appear to represent different experiments, but maybe the authors can clarify. ![file] , 
 Expression of Concern, [31 March 2022] . ---------------- After this article [1] was published, concerns were raised about western blots in Figs 2, 5 and 7, and microscopy images in Figs 1C, 6 and 8. Specifically: - In Fig 1C in this article, the GR17-1 IFN-u03b3 (-) panel appears similar to the 15u20133 Cells IFN-u03b1 (+) panel in Fig 7a in [2], and the GR17-1 IFN-u03b3 (+) panel appears similar to the IRF9 panel in Fig 7A in [2]. - In Fig 2 in this article, the backgrounds of lanes one and two in the p-Jak2 blot appear similar to each other. - In Fig 5 in this article, lanes three, four, six, and seven in the GR17-1 blot appear similar to each other. - In Fig 6A in this article, the differential interference contrast image for IFN-u03b3 (-) appears similar to the S9-13 IFN-u03b3 (-) STAT1-CC-GFP panel in Fig 6B with 90u00b0 rotation. The differential interference contrast image in Fig 6A for IFN-u03b3 (+) appears similar to the S9-13 IFN-u03b3 (+) STAT1-GFP panel in Fig 6B with 90u00b0 rotation. - In Fig 6B in this article, the GR17-1 IFN-u03b3 (+) STAT1-GFP panel appears similar to the GR17-1 IFN-u03b3 (-) STAT1-CC-GFP panel. - In Fig 7 in this article, the GADPH blot appears similar to the HCV blot with an additional band in the final lane for the GADPH blot. - In Fig 8 in this article, the S-Huh7 panel appears similar to the Huh-7 Cells IFN-u03b1 (-) panel in Fig 7A in [2] and to the Huh-7 Cells IFN-u03b1 (+) panel in Fig 7A in [2]. Follow-up on these issues is ongoing; in the meantime, the PLOS ONE Editors issue this Expression of Concern. 2.Poat B., Hazari S., Chandra P.K. et al. Intracellular expression of IRF9 Stat fusion protein overcomes the defective Jak-Stat signaling and inhibits HCV RNA replication. Virol J 7, 265 (2010). pmid:20939906 ,