Related Experiment Video
Updated: Apr 12, 2026
Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins
Published on: August 31, 2017
A ribonucleoprotein complex protects the interleukin-6 mRNA from degradation by distinct herpesviral endonucleases
Mandy Muller1, Stephanie Hutin1, Oliver Marigold1
1Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America.
Abstract:
During lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection, the viral endonuclease SOX promotes widespread degradation of cytoplasmic messenger RNA (mRNA). However, select mRNAs escape SOX-induced cleavage and remain robustly expressed. Prominent among these is interleukin-6 (IL-6), a growth factor important for survival of KSHV infected B cells. IL-6 escape is notable because it contains a sequence within its 3' untranslated region (UTR) that can confer protection when transferred to a SOX-targeted mRNA, and thus overrides the endonuclease targeting mechanism. Here, we pursued how this protective RNA element functions to maintain mRNA stability. Using affinity purification and mass spectrometry, we identified a set of proteins that associate specifically with the protective element. Although multiple proteins contributed to the escape mechanism, depletion of nucleolin (NCL) most severely impacted protection. NCL was re-localized out of the nucleolus during lytic KSHV infection, and its presence in the cytoplasm was required for protection. After loading onto the IL-6 3' UTR, NCL differentially bound to the translation initiation factor eIF4H. Disrupting this interaction, or depleting eIF4H, reinstated SOX targeting of the RNA, suggesting that interactions between proteins bound to distant regions of the mRNA are important for escape. Finally, we found that the IL-6 3' UTR was also protected against mRNA degradation by the vhs endonuclease encoded by herpes simplex virus, despite the fact that its mechanism of mRNA targeting is distinct from SOX. These findings highlight how a multitude of RNA-protein interactions can impact endonuclease targeting, and identify new features underlying the regulation of the IL-6 mRNA.
Insights
Kaposi
Area of Science:
- Virology and Molecular Biology
- RNA Biology and Regulation
Background:
- Lytic Kaposi's sarcoma-associated herpesvirus (KSHV) infection involves viral endonuclease SOX degrading cytoplasmic mRNA.
- Certain mRNAs, like interleukin-6 (IL-6), escape SOX-mediated degradation, crucial for KSHV-infected B cell survival.
Purpose of the Study:
- To elucidate the mechanism by which a specific RNA element in IL-6 mRNA confers protection against SOX endonuclease targeting.
- To identify proteins involved in maintaining mRNA stability and understand their role in SOX escape.
Main Methods:
- Affinity purification coupled with mass spectrometry to identify proteins binding to the protective RNA element.
- Depletion studies of identified proteins (e.g., nucleolin) and translation factors (e.g., eIF4H) to assess their role in mRNA protection.
- Cross-reactivity analysis using herpes simplex virus (HSV) vhs endonuclease.
Main Results:
- A set of proteins was identified that specifically associate with the protective RNA element, with nucleolin (NCL) playing a key role.
- NCL relocalizes to the cytoplasm during lytic KSHV infection and is essential for protecting IL-6 mRNA.
- NCL interacts with eIF4H on the IL-6 3' UTR, and disrupting this interaction or depleting eIF4H re-sensitizes the mRNA to SOX degradation.
- The protective element also confers resistance to HSV's vhs endonuclease, indicating a broad protective function.
Conclusions:
- RNA-protein interactions, particularly involving nucleolin and eIF4H, are critical for shielding specific mRNAs from viral endonuclease activity.
- These findings reveal novel mechanisms of mRNA regulation during viral infection and highlight the importance of cytoplasmic protein complexes in maintaining mRNA stability.
- The IL-6 3' UTR element offers protection against distinct viral endonucleases, suggesting conserved strategies for mRNA escape.
More Related Videos
Related Concept Videos
01:30Inhibitors of Viral Protein Synthesis
02:30siRNA - Small Interfering RNAs
In the cytoplasm, siRNA is processed from a double-stranded RNA, which comes from either endogenous DNA transcription or exogenous sources like a virus. This double-stranded RNA is then cleaved by the...
02:28Leaky Scanning
01:29Viruses with RNA Genomes
01:23RNA Interference
This process occurs naturally in cells, often through the activity of genomically-encoded microRNAs. Researchers can take advantage of this mechanism by introducing synthetic RNAs to deactivate specific genes for research or therapeutic purposes. For example, RNAi could be used...
01:23RNA Interference