Kaposi's sarcoma-associated herpesvirus forms and maintains R-loops at origins of lytic replication
- 1University of Calgary
Abstract
GC-rich sequences are abundant in human herpesviruses genomes. GC-rich regions can form three-stranded RNA:DNA hybrid structures called R-loops. Though these hybrid structures serve important biological roles at telomeres or during cellular DNA synthesis, unscheduled or prolonged R-loop formation causes DNA damage and genome instability. For this reason, several mechanisms exist to resolve R-loops including endoribonucleases RNaseH1 (constitutively expressed) and RNaseH2A (cell cycle-regulated) which degrade the RNA portion of the R-loop. The Kaposi's sarcoma-associated herpesvirus (KSHV) origins of lytic replication (OriLyts) contain multiple cis-acting elements that are required for viral DNA replication including the production of GC-rich and repetitive transcripts, T1.4 (OriLyt-L) and kaposin (OriLyt-R). We previously showed that R-loops form at both OriLyts and that deleting kaposin repeats or decreasing their GC-rich content prevented R-loop formation at OriLyt-R, reduced genome amplification after primary infection and caused defects in latency establishment. To define the contribution that R-loops play in KSHV replication, we overexpressed RNaseH1, reasoning that excess RNaseH1 would resolve both OriLyt R-loops. However, RNaseH1 protein levels decreased following KSHV reactivation in both iSLK and BCBL-1 cell lines. Using co-transfection, we discovered that the KSHV viral replication and transcription activator protein, RTA, mediated RNaseH1 protein decreases in a E3 ligase domain-dependent manner without impacting levels of its cognate RNA transcript. We attempted to construct an RTA-resistant yet functional version of RNaseH1 by site-directed mutagenesis of lysine residues individually or in combination, yet these constructs remain susceptible to RTA-mediated protein decreases. An amino terminally tagged RNaseH1 displayed reduced susceptibility to RTA, suggesting that RTA may target the N-terminus of RNaseH1 for ubiquitination. However, overexpression of the cell-cycle regulated endonuclease, RNaseH2, exhibited RTA resistance, suggesting RNaseH2 may be a tool that will effectively resolve R-loops during KSHV infection. KSHV is not the only herpesvirus to encode a protein that reduces RNaseH1 levels, as co-expression of RTA homologs from the related gamma-herpesviruses EBV and MHV-68 likewise decreased steady-state levels of RNaseH1 protein. We propose that RTA-mediated RNaseH1 degradation is conserved strategy to ensure R-loop persistence during gamma-herpesvirus infection, underscoring the importance of these structures.
Related
- Kinetochore Proteins CENPB and LIC2 are critical for Episome Persistence and Replication of Kaposis's Sarcoma-Associated Herpesvirus
- Kaposi sarcoma herpesvirus vFLIP induces lipid droplet formation in Tert-immortalized microvascular endothelial cells via NF-κB activation
- Reproductive tract herpes-like lesions associated with bovine herpesvirus 4 in dairy cows
- HNF4A maintains proximal tubule identity and limits injury-associated cell states in the adult mouse kidney
- A Plasmid-Derived Machinery Drives Chromosomal Replication Initiation in the Colorectal Cancer-Associated Bacterium Fusobacterium nucleatum and Its Close Relatives
BibTeX
@misc{kleer2026kaposis,
title = {{Kaposi's sarcoma-associated herpesvirus forms and maintains R-loops at origins of lytic replication}},
author = {M. Kleer and J. Fox and J. Corcoran},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.31.748444},
url = {https://www.biorxiv.org/content/10.64898/2026.08.31.748444v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Kaposi's sarcoma-associated herpesvirus forms and maintains R-loops at origins of lytic replication
AU - M. Kleer
AU - J. Fox
AU - J. Corcoran
PY - 2026
DA - 2026/08/31
PB - bioRxiv
DO - 10.64898/2026.08.31.748444
UR - https://www.biorxiv.org/content/10.64898/2026.08.31.748444v1
AB - GC-rich sequences are abundant in human herpesviruses genomes. GC-rich regions can form three-stranded RNA:DNA hybrid structures called R-loops. Though these hybrid structures serve important biological roles at telomeres or during cellular DNA synthesis, unscheduled or prolonged R-loop formation causes DNA damage and genome instability. For this reason, several mechanisms exist to resolve R-loops including endoribonucleases RNaseH1 (constitutively expressed) and RNaseH2A (cell cycle-regulated) which degrade the RNA portion of the R-loop. The Kaposi's sarcoma-associated herpesvirus (KSHV) origins of lytic replication (OriLyts) contain multiple cis-acting elements that are required for viral DNA replication including the production of GC-rich and repetitive transcripts, T1.4 (OriLyt-L) and kaposin (OriLyt-R). We previously showed that R-loops form at both OriLyts and that deleting kaposin repeats or decreasing their GC-rich content prevented R-loop formation at OriLyt-R, reduced genome amplification after primary infection and caused defects in latency establishment. To define the contribution that R-loops play in KSHV replication, we overexpressed RNaseH1, reasoning that excess RNaseH1 would resolve both OriLyt R-loops. However, RNaseH1 protein levels decreased following KSHV reactivation in both iSLK and BCBL-1 cell lines. Using co-transfection, we discovered that the KSHV viral replication and transcription activator protein, RTA, mediated RNaseH1 protein decreases in a E3 ligase domain-dependent manner without impacting levels of its cognate RNA transcript. We attempted to construct an RTA-resistant yet functional version of RNaseH1 by site-directed mutagenesis of lysine residues individually or in combination, yet these constructs remain susceptible to RTA-mediated protein decreases. An amino terminally tagged RNaseH1 displayed reduced susceptibility to RTA, suggesting that RTA may target the N-terminus of RNaseH1 for ubiquitination. However, overexpression of the cell-cycle regulated endonuclease, RNaseH2, exhibited RTA resistance, suggesting RNaseH2 may be a tool that will effectively resolve R-loops during KSHV infection. KSHV is not the only herpesvirus to encode a protein that reduces RNaseH1 levels, as co-expression of RTA homologs from the related gamma-herpesviruses EBV and MHV-68 likewise decreased steady-state levels of RNaseH1 protein. We propose that RTA-mediated RNaseH1 degradation is conserved strategy to ensure R-loop persistence during gamma-herpesvirus infection, underscoring the importance of these structures.
ER -