R-loop-forming Sequences Analysis in Thousands of Viral Genomes Identify A New Common Element in Herpesviruses

Abstract

R-loops are RnA-DnA hybrid sequences that are emerging players in various biological processes, occurring in both prokaryotic and eukaryotic cells. in viruses, R-loop investigation is limited and functional importance is poorly understood. Here, we performed a computational approach to investigate prevalence, distribution, and location of R-loop forming sequences (RLfS) across more than 6000 viral genomes. A total of 14637 RLFS loci were identified in 1586 viral genomes. Over 70% of RLfS-positive genomes are dsDnA viruses. in the order Herpesvirales, RLfS were presented in all members whereas no RLfS was predicted in the order Ligamenvirales. Analysis of RLfS density in all RLfS-positive genomes revealed unusually high RLfS densities in herpesvirus genomes, with RLfS densities particularly enriched within repeat regions such as the terminal repeats (tRs). RLfS in tRs are positionally conserved between herpesviruses. Validating the computationally-identified RLFS, R-loop formation was experimentally confirmed in the TR and viral Bcl-2 promoter of Kaposi sarcomaassociated herpesvirus (KSHV). These predictions and validations support future analysis of RLFS in regulating the replication, transcription, and genome maintenance of herpesviruses. Recent advances in the field of genomics have revealed widespread occurrence of non-canonical nucleic acid-forming structures, such as Z-DNA 1 , hairpin loops 2,3 , and G-quadruplexes (guanine-rich sequences that attain specific four-stranded conformations) 4 , in various genomes including viruses 5. These structures likely play important roles in the replication strategies used by a particular virus. For instance, Z-DNA structures in the simian virus 40 enhancer regions activate transcription 1. Hairpin structures at the termini of the adeno-associated virus genome promote persistent DNA circles and concatemers during recombination processes that occur in the infected host cell 2,3. G-quadruplexes are present in several viral genomes e.g., human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), and human papillomavirus (HPV) (reviewed in 4 and function in various aspects of the replication cycles of these viruses. Another non-canonical nucleic acid structure called an R-loop or RNA:DNA structure is preferentially formed within G-rich sequences and possesses greater thermodynamic stability than the original DNA:DNA duplex 6. R-loops have been experimentally observed in a wide range of organisms, from bacteria to mammals 6,7 , where they function in transcription 8 , telomere maintenance 9 , genome instability 10,11 , and epigenetic regulation 12. They are also associated with certain diseases, such as Prader-Willi syndrome 13 , ataxia with oculomotor apraxia 14 , amyotrophic lateral sclerosis 15 , spinal muscular atrophy 16,17 , motor neuron disorders 18 , cancers 19 and many others 20. R-loops, have been predicted by computational approaches in various organisms 21-23 and demonstrated by direct experimental evidence, however, genome-scale identification of R-loops in t...

A Panel of Kaposi’s Sarcoma-Associated Herpesvirus Mutants in the Polycistronic Kaposin Locus for Precise Analysis of Individual Protein Products

“…To determine whether R-loops formed at the kaposin and T1.4 loci (part of OriLytR and OriLytL, respectively), we reactivated WT iSLK cells for the indicated times, and performed the DRIP procedure using primers to detect these viral sequences, ensuring that for the kaposin locus, we used primer sets that binds in the KapA region that remains intact in all the kaposin-deficient mutants. GC-rich sequences from both the T1.4 and kaposin transcripts were precipitated, suggesting that R-loops form at the T1.4 (OriLytL) and kaposin (OriLytR) loci in WT iSLK cells but not at ORF45 (a negative control as predicted earlier [ 85 ]) ( Fig. 8B and C ).…”

“…However, no studies to date have considered the potential of the GC-rich kaposin transcript to be an important feature of the function of OriLytR, despite the proximal location of kaposin DRs to OriLytR ( 84 ). In silico analysis predicted that numerous R-loops would form in the KSHV genome; however, R-loop formation in KSHV DNA was only experimentally confirmed in the TR region ( 85 ). To experimentally determine whether R-loops form at both OriLyt regions in the KSHV genome, we performed an immunoprecipitation procedure for the DNA:RNA hybrids called DNA:RNA immunoprecipitation (DRIP) ( 77 ).…”

“…(B to E) Primer sets for the kaposin locus and T1.4 locus were used to determine and quantify the presence of RNA:DNA hybrids at each locus. Values were plotted as the fold increase over the ORF45 locus as a negative KSHV locus that does not form an R-loop ( 85 ). Statistical significance was determined using a two-way ANOVA with a Tukey’s post hoc test.…”

Mutational Analysis of SARS-CoV-2 Genomes in Key Cities of China

“…We can clearly see that the trend of gene sequence visualization is the same in all regions, but we can also see the existence of differences, indicating that the virus has changed in the process of transmission [5].…”

KITSUNE: A Tool for Identifying Empirically Optimal K-mer Length for Alignment-Free Phylogenomic Analysis

“…We arbitrarily selected and downloaded seven complete viral genomes associated with human diseases from different taxonomic ranks from NCBI: Chikungunya virus (CHIKV), Dengue virus (DENV) ( Kinney et al, 1997 ), human immunodeficiency virus 1 (HIV) ( Martoglio et al, 1997 ), influenza A virus (IAV) ( Fields and Winter, 1982 ), Zika virus (ZIKV) ( Wongsurawat et al, 2018a , b ), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ( Harcourt et al, 2020 ), and Kaposi’s sarcoma-associated human herpes virus (KSHV) strain GK18 (NC_009333) ( Rezaee et al, 2006 ) (a DNA virus that can interact with the host through an RNA/DNA hybrid mechanism) ( Wongsurawat et al, 2020 ).…”