Detection of Viral RNA Splicing in Diagnostic Virology

Majerčiak, Vladimır; Zheng, Zhiming

doi:10.1007/978-1-4614-3970-7_38

Cited by 4 publications

(3 citation statements)

References 212 publications

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“…RNA splicing plays an essential role in eukaryotic gene expression and almost all human pre-mRNAs are spliced. RNA splicing has been observed in transcripts of numerous viruses, including herpesviruses [ 12 , 13 ]. However, the number of split genes varies among individual herpesvirus family members, from only a few in α-herpesviruses to an estimated ~30% in KSHV [ 9 , 14 ].…”

Section: Introductionmentioning

confidence: 99%

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

et al. 2022

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RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with multiple exons and introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF70-K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70-K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70-K3 locus, we further confirmed ORF57 regulation of ORF70-K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70-K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.

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Section: Introductionmentioning

confidence: 99%

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

et al. 2022

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“…RNA splicing plays an essential role in eukaryotic gene expression and almost all human pre-mRNAs are spliced. RNA splicing has been observed in transcripts of numerous viruses, including herpesviruses [12, 13]. However, the number of split genes varies among individual herpesvirus family members, from only a few in alpha-herpesviruses to an estimated ∼30% in KSHV [9, 14].…”

Section: Introductionmentioning

confidence: 99%

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

Majerčiak

Lobanov

Cam

et al. 2022

Preprint

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RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splicing junctions from RNA-seq identified hundreds of novel viral RNA splicing events, including the splicing events spanning large parts of the viral genome and production of vIRF4 circRNAs. We found that the 57KO alters the viral RNA splicing efficiency of affected RNA splicing events. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with multiple exons and introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF70-K3 RNA bears two introns, of which the first intron is positioned in the ORF70 coding region as an alternative intron and the second intron in the intergenic region as a constitutive intron. In WT cells expressing ORF57, most ORF70-K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out without affecting the splicing efficiency of the second intron. Using a minigene comprising of ORF70-K3 locus, we further confirmed ORF57 regulation of ORF70-K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70-K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.

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“…The gene clusters KSHV ORF50/K8/K8.1 (Tang and Zheng, 2002), ORF73/ORF72/K13 (Ajiro and Zheng, 2014), ORF56/ORF57 and ORF58/ORF59/ORF60/ORF61/ORF62 (Majerciak et al, 2006) are examples of such a gene structure. The use of a single pA site for polyadenylation of multiple transcripts is also found in hepatitis B virus, adenoviruses, human papillomaviruses and others (Majerciak and Zheng, 2013; Wang et al, 2011). As a result, transcripts derived from the upstream genes in a given cluster span over several downstream genes in order to reach a PAS further downstream.…”

Section: Introductionmentioning

confidence: 99%

PA‐seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma–Associated Herpesvirus Transcripts

Majerčiak

Zheng

et al. 2016

CP Microbiology

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Kaposi sarcoma-associated herpesvirus (KSHV) is a human oncovirus linked to the development of several malignancies in immunocompromised patients. Like other herpesviruses, KSHV has a large DNA genome encoding more than 100 distinct gene products. Despite being transcribed and processed by cellular machinery, the structure and organization of KSHV genes in the virus genome differs from that observed in cellular genes from the human genome. A typical feature of KSHV expression is the production of polycistronic transcripts initiated from different promoters but sharing the same polyadenylation site (pA site). This represents a challenge in determination of the 3' end of individual viral transcripts. Such information is critical for generation of a virus transcriptional map for genetic studies. Here we present PA-seq, a high-throughput method for genome-wide analysis of pA sites of KSHV transcripts in B lymphocyte with latent or lytic KSHV infection. Besides identification of all viral pA sites, PA-seq also provides quantitative information about the levels of viral transcripts associated with each pA site enabling to determine the relative expression levels of viral genes at various stages of infection. Due to the indiscriminate nature of PA-seq, the pA sites of host transcripts are also concurrently mapped in the testing samples. Therefore, this technology can simultaneously estimate the expression changes of host genes and RNA polyadenylation upon KSHV infection.

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Detection of Viral RNA Splicing in Diagnostic Virology

Cited by 4 publications

References 212 publications

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57

PA‐seq for Global Identification of RNA Polyadenylation Sites of Kaposi's Sarcoma–Associated Herpesvirus Transcripts

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