Core for "Identification and Characterization of Varicella Zoster Virus Circular RNA in Lytic Infection"
This study investigates the role of circular RNAs (circRNAs) in the context of Varicella-Zoster Virus (VZV) lytic infection. We employed two sequencing technologies, short-read sequencing and long-read sequencing after RNase R treatment on VZV-infected neuroblastoma cells to identify and characterize both cellular and viral circRNAs. Our large scanning analysis identified and subsequent experiments confirmed 200 VZV circRNAs. Importantly, these VZV circRNAs were also detected in tissues from herpes zoster patients. Moreover, we discovered numerous VZV latency-associated transcripts (VLTs)-like circRNAs (circVLTslytic), which contain multiple exons and different isoforms within the same back-splicing breakpoint. To understand the functional significance of these circVLTslytic, we utilized the Bacteria Artificial Chromosome system to disrupt the expression of viral circRNAs in genomic DNA location. We revealed that the sequence flanking circVLTs’ 5’ splice donor played a pivotal role as a cis-acting element in the formation of circVLTslytic. The circVLTslytic was dispensable for VZV replication, but the mutation of downstream of circVLTslytic exon 5 led to increased acyclovir sensitivity in VZV infection models. These models include in vitro culture of human skin tissue and human DRG, as well as in vivo xenografts of human skin tissue in SCID mice. This suggests that circVLTslytic may have a role in modulating the sensitivity to antiviral treatment. Overall, the study highlights the functional importance of VZV-derived circRNAs and their potential contribution to viral pathogenesis. The findings shed new insight into the regulation of cellular and viral transcription during VZV lytic infection, emphasizing the intricate interplay between circRNAs and viral processes.
- 1.Bash (Ubuntu, version 18.04)
- 2.Perl (https://www.perl.org)
- 3.Java (https://javadl.oracle.com)
- 4.BWA (http://bio-bwa.sourceforge.net)
- 5.Bowtie2 (https://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
- 6.SAMtools (http://www.htslib.org/)
- 7.Trim Galore (http://www.bioinformatics.bbsrc.ac.uk/projects/trim_galore/)
- 8.Unicycler (https://github.com/rrwick/Unicycler)
- 9.CIRI2(version v2.0.6) (https://sourceforge.net/projects/ciri/files/CIRI2/)
- 10.CIRI-full (version 2.0) (https://sourceforge.net/projects/ciri/files/CIRI-full/)
- 11.vircircRNA (https://github.com/jiwoongbio/vircircRNA)
- 12.find_circ (https://github.com/marvin-jens/find_circ)
- 13.CIRI-long (https://github.com/bioinfo-biols/CIRI-long)
- 1.VZV pOka_GFP_Luc strain (https://www.ncbi.nlm.nih.gov/nuccore/PP054841)
All sequencing data used in this study is available via NCBI SRA and Gene Expression Omnibus database, accession numbers PRJNA1056528, GSE223870, GSE252124 and GSE223957.
- For whole-genome sequencing https://www.ncbi.nlm.nih.gov/sra/PRJNA1056528
- For circRNA short reads sequencing https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223870
- For circRNA long reads sequencing https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE252124
- For mRNA sequencing https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE223957
conda activate assembly
time unicycler -t 100 \
-1 trim_galore_out_dir/VZV_BAC_WT_short_1_val_1.fq.gz -2 trim_galore_out_dir/VZV_BAC_WT_short_2_val_2.fq.gz \
-o unicycler_VZV_BAC_WT
##Generating genome contained human and VZV
cat chrALL.fa PP054841.fasta > chrALL.fabwa index chrALL.fafor i in mock_1 mock_2 mock_3 vzv_24h_1 vzv_24h_2 vzv_24h_3 vzv_48h_1 vzv_48h_2 vzv_48h_3
do
mkdir ${i}_ciri2_output
bwa mem -t 100 chrALL.fa ${i}_1.fq.gz ${i}_2.fq.gz > ${i}_ciri2_output/${i}.sam
perl /kydata/ysm/CIRI/CIRI_v2.0.6/CIRI2.pl -I ${i}_ciri2_output/${i}.sam -O ${i}_ciri2_output/${i}.ciri -F chrALL.fa -A mRNA.gtf -T 56
perl /kydata/ysm/CIRI/CIRI-AS/CIRI_AS_v1.2.pl -S ${i}_ciri2_output/${i}.sam -C ${i}_ciri2_output/${i}.ciri -F chrALL.fa -A mRNA.gtf -O ${i}_ciri2_output/${i} -D yes
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-full.jar RO1 -1 ${i}_1.fastq -2 ${i}_2.fastq -o ${i}_ciri2_output/${i}
bwa mem -t 42 chrALL.fa ${i}_ciri2_output/${i}_ro1.fq > ${i}_ciri2_output/${i}_ro1.sam
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-full.jar RO2 -r chrALL.fa -s ${i}_ciri2_output/${i}_ro1.sam -l 300 -o ${i}_ciri2_output/${i}RO2
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-full.jar Merge -c ${i}_ciri2_output/${i}.ciri -as ${i}_ciri2_output/${i}_jav.list -ro ${i}_ciri2_output/${i}RO2_ro2_info.list -r chrALL.fa -o ${i}_ciri2_output/${i}
unset DISPLAY
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-vis.jar -o ${i}_ciri2_output/${i}_stdir -i ${i}_ciri2_output/${i}_merge_circRNA_detail.anno -l ${i}_ciri2_output/${i}_library_length.list -a mRNA.gtf -r chrALL.fa -min 1
donebowtie2-build chrALL.fa chrALL.fafor i in mock_1 mock_2 mock_3 vzv_24h_1 vzv_24h_2 vzv_24h_3 vzv_48h_1 vzv_48h_2 vzv_48h_3
do
bowtie2 -p 150 --very-sensitive --score-min=C,-15,0 --mm -x chrALL.fa -q -1 ${i}_1.fq.gz -2 ${i}_2.fq.gz | /kydata/ysm/samtools-1.13/bin/samtools view -hbuS - | /kydata/ysm/samtools-1.13/bin/samtools sort -o ${i}_output.bam
/kydata/ysm/samtools-1.13/bin/samtools view -hf 4 ${i}_output.bam | /kydata/ysm/samtools-1.13/bin/samtools view -Sb - > unmapped.bam
python2 /kydata/ysm/find_circ-1.2/unmapped2anchors.py unmapped.bam | gzip > anchors.fq.gz
bowtie2 -p 150 --reorder --mm --score-min=C,-15,0 -q -x chrALL.fa -U anchors.fq.gz | python2 /kydata/ysm/find_circ-1.2/find_circ.py --genome=chrALL.fa --prefix=${i}_ --name=${i} --stats=${i}_stats.txt --reads=${i}_splice_reads.fa > ${i}_spliced_sites.bed
grep CIRCULAR ${i}_spliced_sites.bed | grep -v chrM | gawk '$5>=2' | grep UNAMBIGUOUS_BP | grep ANCHOR_UNIQUE | python2 /kydata/ysm/find_circ-1.2/maxlength.py 100000 > ${i}_find_circ.candidates.bed
donecat mock_1_1.fq.gz mock_2_1.fq.gz mock_3_1.fq.gz vzv_24h_1_1.fq.gz vzv_24h_2_1.fq.gz vzv_24h_3_1.fq.gz vzv_48h_1_1.fq.gz vzv_48h_2_1.fq.gz vzv_48h_3_1.fq.gz > all_1.fq.gz
cat mock_1_2.fq.gz mock_2_2.fq.gz mock_3_2.fq.gz vzv_24h_1_2.fq.gz vzv_24h_2_2.fq.gz vzv_24h_3_2.fq.gz vzv_48h_1_2.fq.gz vzv_48h_2_2.fq.gz vzv_48h_3_2.fq.gz > all_2.fq.gzfor i in all
do
mkdir ${i}_ciri2_output
bwa mem -t 100 chrALL.fa ${i}_1.fq.gz ${i}_2.fq.gz > ${i}_ciri2_output/${i}.sam
perl /kydata/ysm/CIRI/CIRI_v2.0.6/CIRI2.pl -I ${i}_ciri2_output/${i}.sam -O ${i}_ciri2_output/${i}.ciri -F chrALL.fa -A mRNA.gtf -T 56
perl /kydata/ysm/CIRI/CIRI-AS/CIRI_AS_v1.2.pl -S ${i}_ciri2_output/${i}.sam -C ${i}_ciri2_output/${i}.ciri -F chrALL.fa -A mRNA.gtf -O ${i}_ciri2_output/${i} -D yes
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-full.jar RO1 -1 ${i}_1.fastq -2 ${i}_2.fastq -o ${i}_ciri2_output/${i}
bwa mem -t 42 chrALL.fa ${i}_ciri2_output/${i}_ro1.fq > ${i}_ciri2_output/${i}_ro1.sam
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-full.jar RO2 -r chrALL.fa -s ${i}_ciri2_output/${i}_ro1.sam -l 300 -o ${i}_ciri2_output/${i}RO2
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-full.jar Merge -c ${i}_ciri2_output/${i}.ciri -as ${i}_ciri2_output/${i}_jav.list -ro ${i}_ciri2_output/${i}RO2_ro2_info.list -r chrALL.fa -o ${i}_ciri2_output/${i}
unset DISPLAY
java -jar /kydata/ysm/CIRI/CIRI-full_v2.0/CIRI-vis.jar -o ${i}_ciri2_output/${i}_stdir -i ${i}_ciri2_output/${i}_merge_circRNA_detail.anno -l ${i}_ciri2_output/${i}_library_length.list -a mRNA.gtf -r chrALL.fa -min 1
donefor i in all
do
bowtie2 -p 150 --very-sensitive --score-min=C,-15,0 --mm -x chrALL.fa -q -1 ${i}_1.fq.gz -2 ${i}_2.fq.gz | /kydata/ysm/samtools-1.13/bin/samtools view -hbuS - | /kydata/ysm/samtools-1.13/bin/samtools sort -o ${i}_output.bam
/kydata/ysm/samtools-1.13/bin/samtools view -hf 4 ${i}_output.bam | /kydata/ysm/samtools-1.13/bin/samtools view -Sb - > unmapped.bam
python2 /kydata/ysm/find_circ-1.2/unmapped2anchors.py unmapped.bam | gzip > anchors.fq.gz
bowtie2 -p 150 --reorder --mm --score-min=C,-15,0 -q -x chrALL.fa -U anchors.fq.gz | python2 /kydata/ysm/find_circ-1.2/find_circ.py --genome=chrALL.fa --prefix=${i}_ --name=${i} --stats=${i}_stats.txt --reads=${i}_splice_reads.fa > ${i}_spliced_sites.bed
grep CIRCULAR ${i}_spliced_sites.bed | grep -v chrM | gawk '$5>=2' | grep UNAMBIGUOUS_BP | grep ANCHOR_UNIQUE | python2 /kydata/ysm/find_circ-1.2/maxlength.py 100000 > ${i}_find_circ.candidates.bed
done
##Build BWA index
perl vircircRNA_chromosome.pl PP054841.fasta > concatenated_circular_PP054841.fasta
bwa index concatenated_circular_PP054841.fasta
bwa mem -t 100 -Y concatenated_circular_PP054841.fasta all_1.fq.gz all_2.fq.gz > mapped_read.sam
perl vircircRNA_junction.pl -g PP054841.gff3 -A junction.alignment.html mapped_read.sam concatenated_circular_PP054841.fasta > PP054841_junction.txt
perl vircircRNA_diagram.pl -g PP054841.gff3 > PP054841_junction.txt concatenated_circular_PP054841.fasta PP054841.fasta > diagram.pngminimap2 -d chrALL.min chrALL.fa
#######call
CIRI-long call -i SY5Y-pokaA.fq.gz \
-o SY5Y-pokaA_chrALL \
-r chrALL.fa \
-p SY5Y-pokaA_chrALL \
-t 100
#####collapse
CIRI-long collapse -i ./SY5Y-pokaA_chrALL.lst \
-o ./SY5Y-pokaA_chrALL_collpase \
-p SY5Y-pokaA_chrALL \
-r chrALL.fa \
-t 100
python3 misc/convert_bed.py SY5Y-pokaA_chrALL_collpase/SY5Y-pokaA_chrALL.info SY5Y-pokaA_chrALL_circ.bed ##Build STAR index
STAR --runThreadN 42 --runMode genomeGenerate --genomeDir /kydata/ysm/index/star_index/chrALL_149 \
--genomeFastaFiles chrALL.fa \
--sjdbGTFfile mRNA.gtf \
--sjdbOverhang 149for i in pOka_WT_1 pOka_WT_2 pOka_WT_3 pOka_M1_1 pOka_M1_3 pOka_M1_3 pOka_M2_1 pOka_M2_2 pOka_M2_3
do
STAR --genomeDir /kydata/ysm/index/star_index/chrALL_149 \
--readFilesIn ${i}_1.fq.gz ${i}_2.fq.gz \
--readFilesCommand gunzip -c \
--twopassMode Basic \
--outSAMtype BAM Unsorted \
--chimSegmentMin 12 \
--chimJunctionOverhangMin 12 \
--alignSJDBoverhangMin 10 \
--alignMatesGapMax 100000 \
--alignIntronMax 100000 \
--chimSegmentReadGapMax 3 \
--alignSJstitchMismatchNmax 5 -1 5 5 \
--runThreadN 100 \
--outSAMstrandField intronMotif \
--chimOutJunctionFormat 1 \
--outFileNamePrefix $i
/kydata/ysm/samtools-1.13/bin/samtools sort ${i}Aligned.out.bam -o ${i}_sorted.bam -@ 40
/kydata/ysm/samtools-1.13/bin/samtools index ${i}_sorted.bam
echo ${i}_mapped
rm ${i}Aligned.out.bam
rm *.out
featureCounts -T 50 -p -t exon -g gene_id -a mRNA.gtf -o counts/${i}.counts.txt ${i}_sorted.bam
echo ${i}_Counts_finished
cut -f 1,7 counts/${i}.counts.txt |grep -v '^#' > feacturCounts/${i}_feacturCounts.txt
echo ${i}_featureCounts_finished
done for i in pOka_WT_1 pOka_WT_2 pOka_WT_3
do
bwa mem -t 100 PP054841.fasta ${i}_1.fq.gz ${i}_2.fq.gz > ${i}.sam
/kydata/ysm/samtools-1.13/bin/samtools view -b ${i}.sam > ${i}.bam
/kydata/ysm/samtools-1.13/bin/samtools sort ${i}.bam -o ${i}_sorted.bam -@ 100
/kydata/ysm/samtools-1.13/bin/samtools index ${i}_sorted.bam
/kydata/ysm/samtools-1.13/bin/samtools depth -a -m 0 ${i}_sorted.bam > ${i}_coverage.txt
echo ${i}_mapped
donebwa index PP054841_M1.fasta
for i in pOka_M1_1 pOka_M1_3 pOka_M1_3
do
bwa mem -t 100 PP054841_M1.fasta ${i}_1.fq.gz ${i}_2.fq.gz > ${i}.sam
/kydata/ysm/samtools-1.13/bin/samtools view -b ${i}.sam > ${i}.bam
/kydata/ysm/samtools-1.13/bin/samtools sort ${i}.bam -o ${i}_sorted.bam -@ 100
/kydata/ysm/samtools-1.13/bin/samtools index ${i}_sorted.bam
/kydata/ysm/samtools-1.13/bin/samtools depth -a -m 0 ${i}_sorted.bam > ${i}_coverage.txt
echo ${i}_mapped
donebwa index PP054841_M2.fasta
for i in pOka_M2_1 pOka_M2_2 pOka_M2_3
do
bwa mem -t 100 PP054841_M2.fasta ${i}_1.fq.gz ${i}_2.fq.gz > ${i}.sam
/kydata/ysm/samtools-1.13/bin/samtools view -b ${i}.sam > ${i}.bam
/kydata/ysm/samtools-1.13/bin/samtools sort ${i}.bam -o ${i}_sorted.bam -@ 100
/kydata/ysm/samtools-1.13/bin/samtools index ${i}_sorted.bam
/kydata/ysm/samtools-1.13/bin/samtools depth -a -m 0 ${i}_sorted.bam > ${i}_coverage.txt
echo ${i}_mapped
doneYang, S., Cao, D., Jaijyan, D.K. et al. Identification and characterization of Varicella Zoster Virus circular RNA in lytic infection. Nat Commun 15, 4932 (2024). https://doi.org/10.1038/s41467-024-49112-4