Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Plasmid and In Vitro Transcription
2.3. Infectious HCV in Cell Culture
2.4. Oligonucleotides (Oligos)
- GmR Negative Control A (Neg. ctr. GmR): 5′-AACACGTCTATACGC-3′;
- GmR 1 for lncR 3/LINC00222 (lncR 3-GmR 1): 5′-GCGTGATTAAATGGAT-3′;
- GmR 2 for lncR 3/LINC00222 (lncR 3-GmR 2): 5′-GACGATAAGAGGTAAC-3′;
- GmR 1 for lncR 7/Lnc-SLC12A7-4 (lncR 7-GmR 1): 5′-TGATTAACAGAACGGA-3′;
- GmR 2 for lncR 7/Lnc-SLC12A7-4 (lncR 7-GmR 2): 5′-ATAAGTGTCTAGTTAG-3′;
- GmR 1 for lncR 8/Lnc-ITM2C-1(lncR 8-GmR 1): 5′-GTTACCAGTGAAGCGG-3′;
- GmR 2 for lncR 8/Lnc-ITM2C-1 (lncR 8-GmR 2): 5′-TCGGATTGGTCACATG-3′;
- GmR 1 for lncR 10/ZNF252P-AS1 (lncR 10-GmR 1): 5′-GTTAATCTGATCTTGC-3′;
- GmR 2 for lncR 10/ZNF252P-AS1 (lncR 10-GmR 2): 5′-TCTGAGCTTGATCACT-3′;
- GmR 1 for GPR55 (GPR55-GmR 1): 5′-GGCGAATCAGATTAAT-3′;
- GmR 2 for GPR55 (GPR55-GmR 2): 5′-AGGACCATCTTGAATG-3′;
2.5. Cell Treatment
2.6. RNA Samples, DNA Removal, and cDNA Preparation
2.7. Microarrays
2.8. Quantitative Real Time-PCR (qRT-PCR)
2.9. Immunofluorescence
2.10. Western Blot
2.11. Protein-Coding Potential
2.12. Statistical Analysis
3. Results
3.1. Identification of lncRNAs Deregulated by HCV Replication
3.2. HCV Replication Increases the Expression of Four lncRNAs
3.3. Low Protein Coding Potential and Subcellular Localization of lncRNAs
3.4. LncR 8/Lnc-ITM2C-1 Favors HCV Viral Replication
3.5. LncR 8/Lnc-ITM2C-1 Is a Short-Term Cis-Acting Regulator of Its Neighbor GPR55
3.6. LncR 8/Lnc-ITM2C-1 Is a Negative regulator of the Antiviral Response
3.7. LncR 8/Lnc-ITM2C-1 Is Upregulated by HCVcc Infection and Facilitates HCV Infection
3.8. GPR55 Negatively Regulates ISGs
3.9. LncR 8/Lnc-ITM2C-1 Is Induced by polyIC
3.10. LncR 8/Lnc-ITM2C-1 Is Downregulated by JAK/STAT Pathway
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Target Gene | Primer Sequences (5′-3′) | Amplicon Size (bp) |
---|---|---|
LncR 2 | F: CTCCCAGAACCTATCGGCAT | 130 |
R: CACAAAGCCTGCGTTCATTC | ||
LncR 3 | F: AGGATGTGACTGCCAGGTAATG | 100 |
R: CAGACCCAGCCTAGCACACAG | ||
LncR 33′ | F: GTGACCCAACTAGAGCCAATAGG | 135 |
R: CTCAAATCAGCTCATGACCATAAG | ||
LncR 7-1 | F: AGGCTACAGGAGGCACTGAGGG | 144 |
R: GGAGCCATCTGGGAGAATGAAATAC | ||
LncR 7-2 | F: GAGGCTACAGGAGGCACTCTTTG | 79 |
R: GGAGCCATCTGGGAGAATGAAATAC | ||
LncR 73′ | F: TCGGGTTCTTGATTTGATTCTC | 142 |
R: TGGACCAAGTATCCTCTAAAAATG | ||
LncR 8 | F: GGTTTTTTGACCTTGGCAATG | 102 |
R: GTGACCCTTGGTGGCTGTTTAT | ||
LncR 83′ | F: GATTCTGTCTCATCCAATCAAGACT | 123 |
R: GTTGTGCTGAGGATTCTGGGT | ||
LncR 10 | F: CGGAAATGCCTAATCTGAACTT | 80 |
R: TAGAGCGGACCCACGAAAC | ||
LncR 103′ | F: CCCCTGATGCTTCATAATGG | 111 |
R: AGTTCTAACCTAATTTCCCATCAC |
Target Gene | Primer Sequences (5′-3′) | Amplicon Size (bp) |
---|---|---|
GAPDH | F: GAGTCAACGGATTTGGTCGT | 224 |
R: GATCTCGCTCCTGGAAGATG (= RT) | ||
U6 | F: CTCGCTTCGGCAGCACA | 94 |
R: AACGCTTCACGAATTTGCGT | ||
U99 | F: CCTCCTTTTCTTGGCGGGGA | 138 |
R: CGTTTGAGGATAGAACCAGC | ||
β-actin | F: CATGTACGTTGCTATCCAGGC | 250 |
R: CTCCTTAATGTCACGCACGAT | ||
Jc1-NS3 | RT: GTATGCCACGGCATTCAAG | 190 |
F: GATATAGGTCGACGGCTCCA | ||
R: TTCCTCGGAACAACCATCTC | ||
GAS5 | F: CCTGTGAGGTATGGTGCTGG | 383 |
R: GGTCCAGGCAAGTTGGACTC | ||
ITM2C | F: GTGGTGTGCTGTATGAGGACT | 93 |
R: CGTAGTTCTCGTCGAGGTAGAT | ||
GPR55 | F: GAAAACCCTACAGTTTGCAGTCC | 123 |
R: GAGGTGGCAGCATAATCGGG | ||
CXCL10 | F: GTGGCATTCAAGGAGTACCTC R: TGATGGCCTTCGATTCTGGATT | 198 |
ISG15 | F: ACTCATCTTTGCCAGTACAGGAG R: CAGCATCTTCACCGTCAGGTC | 88 |
Mx1 | F: TGCATCGACCTCATTGACTC R: ACCTTGCCTCTCCACTTATC | 218 |
IFITM1 | F: ACTCCGTGAAGTCTAGGGACA R: AGAGCCGAATACCAGTAACAG | 149 |
MDA5 | F:TCGAATGGGTATTCCACAGACG | 152 |
R:GTGGCGACTGTCCTCTGAA | ||
IFN-β | F:GCTTGGATTCCTACAAAGAAGCA | 166 |
R:ATAGATGGTCAATGCGGCGTC | ||
IFN-α | F: GGAGGTTGTCAGAGCAGA | 150 |
R: AATGACAGAATTCATGAAAGCGT | ||
IL28A | F: CAGCCTCAGAGTGTTTCTTCT | 117 |
R: TCCAGTCACGGTCAGCA |
lncRNA | Ref. | Chr | Length(bp) | Gene Symbol | Name |
---|---|---|---|---|---|
lncR 3 | NR_033376.1 | 6 | 1753 | lincRNA 222 | LINC00222 |
lncR 7-2 | NR_104615.1 | 5 | 3451 | LOC100506688 | Lnc-SLC12A7-4:5 |
lncR 8 | NR_038238.1 | 2 | 1893 | LOC151484 | Lnc-ITM2C-1 |
lncR 10 | NR_026974.1 | 8 | 3250 | ZNF252P antisense RNA 1 | ZNF252P-AS1 |
Metric | lncR 3 | lncR 7-2 | lncR 8 | lncR 10 |
---|---|---|---|---|
CPAT coding probability | 1.33% | 10.83% | 69.31% | 80.45% |
PhyloCSF score | −67.4569 | 13.6639 | −112.1426 | 11.7381 |
PRIDE reprocessing 2.0 | 0 | 0 | 0 | 0 |
Lee translation initiation sites | 0 | 0 | 0 | 0 |
Bazzini small ORFs | 0 | 0 | 0 | 0 |
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Hu, P.; Wilhelm, J.; Gerresheim, G.K.; Shalamova, L.A.; Niepmann, M. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses 2019, 11, 549. https://doi.org/10.3390/v11060549
Hu P, Wilhelm J, Gerresheim GK, Shalamova LA, Niepmann M. Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus. Viruses. 2019; 11(6):549. https://doi.org/10.3390/v11060549
Chicago/Turabian StyleHu, Pan, Jochen Wilhelm, Gesche K. Gerresheim, Lyudmila A. Shalamova, and Michael Niepmann. 2019. "Lnc-ITM2C-1 and GPR55 Are Proviral Host Factors for Hepatitis C Virus" Viruses 11, no. 6: 549. https://doi.org/10.3390/v11060549