Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Viruses
2.2. Generating the TB40E Δ1.2 Half and TB40E Δ1.2 Viral Mutants
2.3. Immunofluorescent Imaging
2.4. Growth Curve
2.5. qPCR Analysis
2.6. Immunoblotting
2.7. Biotinylated RNA1.2 In Vitro Transcription
2.8. Isolating RNA-Protein Complexes
2.9. Mass Spectrometry and Data Analysis
2.10. RNA-Immunoprecipitation (RNA-IP)
2.11. Statistical Analysis
3. Results
3.1. Construction and Characterization of RNA1.2 Mutants
3.2. Deletion of RNA1.2 Does Not Disrupt Viral Gene Expression or Protein Production
3.3. RNA1.2 Is Not Required for Productive Lytic Replication in Cell Culture
3.4. Capturing RNA1.2–Protein Complexes Using In Vitro Transcribed, Biotinylated RNA1.2
3.5. Identifying Viral and Cellular Proteins Associated with RNA1.2
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
HCMV | Human Cytomegalovirus |
lncRNA | Long Non-Coding RNA |
BAC | Bacterial Artificial Chromosome |
NF-κB | Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells |
IL-6 | Interleukin 6 |
DRS | Direct RNA Sequencing |
ORFs | Open Reading Frames |
GCV | Ganciclovir |
VGV | Valganciclovir |
MBV | Maribavir |
IE | Immediate-Early |
E | Early |
L | Late |
MIEP | Major Immediate-Early Promoter |
PCR | Polymerase Chain Reaction |
LC-MS | Liquid Chromatography-Mass Spectrometry |
MOI | Multiplicity of Infection |
ATP | Adenosine Triphosphate |
DAPI | 4′,6-Diamidino-2-Phenylindole |
TE | Tris-EDTA Buffer |
hpi | Hours Post-Infection |
PBS | Phosphate Buffered Saline |
RIP | RNA Immunoprecipitation |
TBST | Tris Buffered Saline with Tween-20 |
FDR | False Discovery Rate |
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Name | Type | Sequence |
---|---|---|
∆1.2 +KAN | gBlock | 5′-AGATTTCATCAGGTTTATTTTGGCTGCTGCTAGTCTTTTGCTTCGGACACTCGCGTCCGGTTGGGCCG ATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATC ATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAAC GGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGG GCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAG AGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAA CTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTT ACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAA AATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTA ACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAG TGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTG CCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGG AAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCT ATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATA ATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATT GGTTGTAACACTGGCATTACCCTGTTATCCCTAGATCGATGTACGGGCCAGATATACGCGGCTGCTG CTAGTCTTTTGCTTCGGACACTCGCGTCCGGTTGGGCATTGCCCACAGGAAGATGAGTC-3′ |
∆1.2 half+KAN | gBlock | 5′-CAGACCACTGGGAGTTCAGTTAAAGATTTCATCAGGTTTATTTTGAGGGCGCGGTCATCTTTTACTC GATTTATTCAACAAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATA TCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCA ACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAAT GGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCC AGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTA AACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATG GTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGT GAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCT TTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATG CGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCT TTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAG GGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCA TCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATT GATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTT AATTGGTTGTAACACTGGCATTACCCTGTTATCCCTAGATCGATGTACGGGCCAGATATACGCGAAGA TTTCATCAGGTTTATTTTGAGGGCGCGGTCATCTTTTACTTTTCGGTTTTCTCATTGGCGGG-3′ |
Name | Sequence |
---|---|
Primer 1: 5′-GCCGAGAGAATGCCAGTAAG-3′ | |
RNA 1.2 | Primer 2: 5′-CTGCTGTACGTGTGATGGTTAT |
Probe: /56-FAM/CGT ACT GTG/ZEN/TCT GCG ATG GTC GTC/3IABkFQ/ | |
Primer 1: 5′-GTGTGTGCTGGCCGATG-3′ | |
RNA 4.9 | Primer 2: 5′-GGGACGGTGATTGTGGAG-3′ |
Probe: /56-FAM/ACC TCA ATT/ZEN/GTC GTC AGT ACG CCC/3IABkFQ/ | |
Primer 1: 5′-GCGAAGAAGTGCGAGGATAA-3′ | |
Beta 2.7 | Primer 2: 5′ CATCATCATCGGAGACCATCTT-3′ |
Probe: /56-FAM/AAA TGG ATG/ZEN/ACT CCT TCG TGT CCA GG/3IABkFQ/ | |
Primer 1: 5′-GCGAAGAAGTGCGAGGATAA-3′ | |
RNA 5.0 | Primer 2: 5’-AACATCATCGGAGACCATCTT T-3′ |
Probe: /56-FAM/AAA TGG ATG/ZEN/ACT CCT TCG TGT CCA GG/3IABkFQ/ | |
Primer 1: 5′-GAAAGAAGAGGACGAGGATGAC-3′ | |
IE2 ex5 | Primer 2: 5′-GTGCGGGAAAGAGAGAGAAG-3′ |
Probe: /56-FAM/TCT AAC GAG/ZEN/GAT TCT GAC GTG CGC/3IABkFQ/ | |
Primer 1: 5′-GTAGTGGTTGGGCAGGATAAA-3′ | |
UL54 | Primer 2: 5′-TTGCGGCGTGTCATCTT-3′ |
Probe: /56-FAM/TAT CTA CAC/ZEN/CTC GCT GCT GGA CGA/3IABkFQ/ | |
Primer 1: 5′-TGCGTAAAGTCGAAGAAGGG-3′ | |
UL86 | Primer 2: 5′-CGCACGGTGAACGAAATAAAG-3′ |
Probe: /56-FAM/CAA GGT GGG/ZEN/CAA CAT CAC GCT CTA/3IABkFQ/ | |
Primer 1: 5′-TGACTACCCTACGTTCTCCTAC-3′ | |
7sk | Primer 2: 5′-GTCAAGGGTATACGAGTAGCTG-3′ |
Probe: /56-FAM/CCC TGC TAG/ZEN/AAC CTC CAA ACA AGC T/3IABkFQ/ | |
Primer 1: 5′-TGTAGTTGAGGTCAATGAAGGG-3′ | |
GAPDH | Primer 2: 5′-ACATCGCTCAGACACCATG-3′ |
Probe: /56-FAM/AAG GTC GGA/ZEN/GTC AAC GGA TTT GGT C/3IABkFQ/ |
Name | Type | Sequence |
---|---|---|
pGEM-RNA1.2 | g-Block | 5′-CCCGACGTCGCATGCTCCTCTAGAAGTGTCCCATAAAAGCCGGGCGCTCCGGCGAGACCATGCCA TCCTCGCCTTCGGACGCCCCGCTCCTCTTCTCTCTCCTCTCCTCCCCGCTGCCGCGGCCACTGCCGCC GCCGCCCATACCATCGGCATGTCGGCCGACAAATCGCAGCTGTCGTCGTCGCCGCCGCAGCTGTAGC AGTTAACGTCGCCGGCCTTCAGGAGGAGATGGCGCTCTGCGTCGTCTCTTCGTCCCGCCTCCCTCTGT GGTCGTGGGTGGTGCGAGAGTACACGATGGGTGGCTCTCGTCTCGGGGGACCACAGGGGGAGGGGG GTAATTTATTATTCGTATTACTGTAATTTTGTATCGCTTAATTTGTTTAGAGCCGCACGCTTGACAACGC CTTGTATAGCCTTATTTATCCCGATGACTTTTTTCTCCGTACAAGAAATGGACGTCACTTGAGCAGAC ACAGTTTCATCGACCACGACAGTCTCATGATCTGACTACCTCTGACCCGCCAATGAGAAAACCGAA AAGTAAAAGATGACCGCGCCCTCGGAGTCCTTTTTTCCTTTTCAATCATGAAAGCAAGAGGCAGC CGAGAGAATGCCAGTAAGAGACGACCATCGCAGACACAGTACGATACTCATCTTAGAACGAACCA GCGAATAACCATCACACGTACAGCAGAATCTCATGAACTAGTCAACCAACGTCATAAAATCTTCA CACAATCGTTTTTGCGAACTTTTAGGAACCAGCAAGTCAACAAAAGACTAACAAAGAAAAACCA TCTTGGAATTAAAAAAAGTAGCATCGTTACCTTATGAACCAGCAGCATTCAGTATATACACCAGAT ATAATATATTTATTAATGTATCCTCTCTTTCTCCTGATGTAATTTTGTTTTTGTAAATTCAATTGTTGA AAGTCTCTCCCTGGGGGAATTGCATATCTTATTGATGAAGAAGAAATCCCTGCCATATGTGTTGTC AAACTATCATTATTTCTCTATATGGGTATTTTTTTTCTAAGAAGCAAAAGACTAGCAGCAGCCA AAATAAACCTGATGAAATCTTTAACTGCTCGAGGAATTCGGTACCCCGGGT-3′ |
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Manska, S.; Hagemann, A.; Magana, J.; Rossetto, C.C.; Verma, S.C. Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication. Viruses 2025, 17, 149. https://doi.org/10.3390/v17020149
Manska S, Hagemann A, Magana J, Rossetto CC, Verma SC. Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication. Viruses. 2025; 17(2):149. https://doi.org/10.3390/v17020149
Chicago/Turabian StyleManska, Salomé, Andrew Hagemann, Janna Magana, Cyprian C. Rossetto, and Subhash C. Verma. 2025. "Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication" Viruses 17, no. 2: 149. https://doi.org/10.3390/v17020149
APA StyleManska, S., Hagemann, A., Magana, J., Rossetto, C. C., & Verma, S. C. (2025). Characterization of Human Cytomegalovirus (HCMV) Long Non-Coding RNA1.2 During Lytic Replication. Viruses, 17(2), 149. https://doi.org/10.3390/v17020149