Viral Episomes Research Articles

Repression of CADM1 transcription by HPV type 18 is mediated by three-dimensional rearrangement of promoter-enhancer interactions.

Upon infection, human papillomavirus (HPV) manipulates host cell gene expression to create an environment that is supportive of a productive and persistent infection. The virus-induced changes to the host cell's transcriptome are thought to contribute to carcinogenesis. Here, we show by RNA-sequencing that oncogenic HPV18 episome replication in primary human foreskin keratinocytes (HFKs) drives host transcriptional changes that are consistent between multiple HFK donors. We have previously shown that HPV18 recruits the host protein CTCF to viral episomes to control the differentiation-dependent viral transcriptional programme. Since CTCF is an important regulator of host cell transcription via coordination of epigenetic boundaries and long-range chromosomal interactions, we hypothesised that HPV18 may also manipulate CTCF to contribute to host transcription reprogramming. Analysis of CTCF binding in the host cell genome by ChIP-Seq revealed that while the total number of CTCF binding sites is not altered by the virus, there are a sub-set of CTCF binding sites that are either enriched or depleted of CTCF. Many of these altered sites are clustered within regulatory elements of differentially expressed genes, including the tumour suppressor gene cell adhesion molecule 1 (CADM1), which supresses epithelial cell growth and invasion. We show that HPV18 establishment results in reduced CTCF binding at the CADM1 promoter and upstream enhancer. Loss of CTCF binding is coincident with epigenetic repression of CADM1, in the absence of CpG hypermethylation, while adjacent genes including the transcriptional regulator ZBTB16 are activated. These data indicate that the CADM1 locus is subject to topological rearrangement following HPV18 establishment. We tested this hypothesis using 4C-Seq (circular chromosome confirmation capture-sequencing) and show that HPV18 establishment causes a loss of long-range chromosomal interactions between the CADM1 transcriptional start site and the upstream transcriptional enhancer. These data show that HPV18 manipulates host cell promoter-enhancer interactions to drive transcriptional reprogramming that may contribute to HPV-induced disease progression.

A Conserved Di-Lysine Motif in the E2 Transactivation Domain Regulates MmuPV1 Replication and Disease Progression.

The papillomavirus E2 protein regulates the transcription, replication, and segregation of viral episomes within the host cell. A multitude of post-translational modifications have been identified which control E2 functions. A highly conserved di-lysine motif within the transactivation domain (TAD) has been shown to regulate the normal functions of the E2 proteins of BPV-1, SfPV1, HPV-16, and HPV-31. This motif is similarly conserved in the E2 of the murine papillomavirus, MmuPV1. Using site-directed mutagenesis, we show that the first lysine (K) residue within the motif, K112, is absolutely required for E2-mediated transcription and transient replication in vitro. Furthermore, mutation of the second lysine residue, K113, to the potential acetyl-lysine mimic glutamine (Q) abrogated E2 transcription and decreased transient replication in vitro, while the acetylation defective arginine (R) mutant remained functional. Both K113 mutants were able to induce wart formation in vivo, though disease progression appeared to be delayed in the K113Q group. These findings suggest that acetylation of K113 may act as a mechanism for repressing MmuPV1 E2 activity.

Targeting EBV Episome for Anti-Cancer Therapy: Emerging Strategies and Challenges.

As a ubiquitous human pathogen, the Epstein-Barr virus (EBV) has established lifelong persistent infection in about 95% of the adult population. The EBV infection is associated with approximately 200,000 human cancer cases and 140,000 deaths per year. The presence of EBV in tumor cells provides a unique advantage in targeting the viral genome (also known as episome), to develop anti-cancer therapeutics. In this review, we summarize current strategies targeting the viral episome in cancer cells. We also highlight emerging technologies, such as clustered regularly interspersed short palindromic repeat (CRISPR)-based gene editing or activation, which offer promising avenues for selective targeting of the EBV episome for anti-cancer therapy. We discuss the challenges, limitations, and future perspectives associated with these strategies, including potential off-target effects, anti-cancer efficacy and safety.

USP7 Inhibitors Destabilize EBNA1 and Suppress Epstein-Barr Virus Tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV+ cells. Finally, we show that GNE6776 selectively inhibited EBV+ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.

Fibroblasts regulate the transcriptional signature of human papillomavirus-positive keratinocytes.

Persistent human papillomavirus (HPV) infection is necessary but insufficient for viral oncogenesis. Additional contributing co-factors, such as immune evasion and viral integration have been implicated in HPV-induced cancer progression. It is widely accepted that HPV+keratinocytes require co-culture with fibroblasts to maintain viral DNA as episomes. How fibroblasts regulate viral episome maintenance is a critical knowledge gap. Here we present comprehensive RNA sequencing and proteomic analysis demonstrating that coculture with fibroblasts is supportive of the viral life cycle, and is confirmatory of previous observations. Novel observations suggest that errors in "cross-talk" between fibroblasts and infected keratinocytes may regulate HPV integration and drive oncogenic progression. Our co-culture models offer new insights into HPV-related transformation mechanisms.

Genome Instability Precedes Viral Integration in Human Papilloma Virus Transformed Tonsillar Keratinocytes.

Approximately 70% of oropharyngeal squamous carcinomas (OPSCC) are associated with human papillomavirus (HPV). While patients with HPV-positive tumors generally have better outcomes than those with HPV-negative tumors, a subset of HPV-positive patients do have poor outcomes. Our previous work suggested that tumors with integrated virus exhibit significantly greater genome wide genomic instability than those with only episomal viral genomes and patients with HPV+ OPSCC with episomal viral genomes had better outcomes. To explore the causal relation between viral integration and genomic instability, we have examined the time course of viral integration and genetic instability in tonsillar keratinocytes transformed with HPV16. HPV-infected human tonsil keratinocyte cell lines were continuously passaged and every fifth passage some cells were retained for genomic analysis. Whole genome sequencing and optical genomic mapping confirmed that virus integrated in five of six cell lines while remaining episomal in the sixth. In all lines genome instability occurred during early passages, but essentially ceased following viral integration but continued to occur later passages in the episomal line. To test tumorigenicity of the cell lines, cells were injected subcutaneously into the flanks of nude mice. A cell line with the integrated virus induced tumors following injection in the nude mouse while that with the episomal virus did not. Implications: Genomic instability in HPV OPSCC tumors is not the result of viral integration but likely promotes integration. Moreover, transformants with episomal virus appear to be less tumorigenic than those with integrated virus.

Fibroblasts Regulate the Transformation Potential of Human Papillomavirus-positive Keratinocytes.

Persistent human papillomavirus (HPV) infection is necessary but insufficient for viral oncogenesis. Additional contributing co-factors, such as immune evasion and viral integration have been implicated in HPV-induced cancer progression. It is widely accepted that HPV+ keratinocytes require co-culture with fibroblasts to maintain viral episome expression, yet the exact mechanisms for this have yet to be elucidated. Here we present comprehensive RNA sequencing and proteomic analysis demonstrating that fibroblasts not only support the viral life cycle, but reduce HPV+ keratinocyte transformation. Our co-culture models offer novel insights into HPV-related transformation mechanisms.

Detection of Hepatitis B Virus Covalently Closed Circular DNA and Intermediates in Its Formation.

Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and ofits reliable detection due to its low abundance and thepresence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA andall DNA species with free 3' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.

Regulation of EBNA1 protein stability and DNA replication activity by PLOD1 lysine hydroxylase.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpesvirus that is causally associated with various malignancies and autoimmune disease. Epstein-Barr Nuclear Antigen 1 (EBNA1) is the viral-encoded DNA binding protein required for viral episome maintenance and DNA replication during latent infection in proliferating cells. EBNA1 is known to be a highly stable protein, but the mechanisms regulating protein stability and how this may be linked to EBNA1 function is not fully understood. Proteomic analysis of EBNA1 revealed interaction with Procollagen Lysine-2 Oxoglutarate 5 Dioxygenase (PLOD) family of proteins. Depletion of PLOD1 by shRNA or inhibition with small molecule inhibitors 2,-2' dipyridyl resulted in the loss of EBNA1 protein levels, along with a selective growth inhibition of EBV-positive lymphoid cells. PLOD1 depletion also caused a loss of EBV episomes from latently infected cells and inhibited oriP-dependent DNA replication. Mass spectrometry identified EBNA1 peptides with lysine hydroxylation at K460 or K461. Mutation of K460, but not K461 abrogates EBNA1-driven DNA replication of oriP, but did not significantly affect EBNA1 DNA binding. Mutations in both K460 and K461 perturbed interactions with PLOD1, as well as decreased EBNA1 protein stability. These findings suggest that PLOD1 is a novel interaction partner of EBNA1 that regulates EBNA1 protein stability and function in viral plasmid replication, episome maintenance and host cell survival.

Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

KSHV infection of endothelial precursor cells with lymphatic characteristics as a novel model for translational Kaposi's sarcoma studies.

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.

Topological implications of DNA tumor viral episomes

A persistent DNA tumor virus infection transforms normal cells into cancer cells by either integrating its genome into host chromosomes or retaining it as an extrachromosomal entity called episome. Viruses have evolved mechanisms for attaching episomes to infected host cell chromatin to efficiently segregate the viral genome during mitosis. It has been reported that viral episome can affect the gene expression of the host chromosomes through interactions between viral episomes and epigenetic regulatory host factors. This mini review summarizes our current knowledge of the tethering sites of viral episomes, such as EBV, KSHV, and HBV, on host chromosomes analyzed by three-dimensional genomic tools. [BMB Reports 2022; 55(12): 587-594].

4C Analysis of EBV-Host DNA Interactome.

EBV persist as multicopy episomes in latently infected cells and alter transcriptional program of host systems. Knowledge of EBV tethering site helps us understand how EBV attaches to and regulates the host chromosome. Here, we introduce a step-by-step protocol for 4C-seq analysis, including cell fixation, 4C-DNA construction, and sequencing library preparation performed with EBV-positive Burkitt's lymphoma cells. The method can be applied in a variety of studies and cell-types to identify target loci associated with bait positions, such as viral episomes.

Effects of Caffeine, a DNA Damage Response Inhibitor, on Papillomavirus Genome Replication.

Epidemiological studies have revealed that caffeinated coffee imparts a reduced risk of oropharyngeal cancer, of which human papillomavirus (HPV) is one of the causative agents. Caffeine is a known inhibitor of the DNA damage response (DDR) pathway. We sought to test the effects of caffeine on the early replication of the HPV31 virus. It has been reported that the inhibition of several factors necessary for the DDR during the differentiation-dependent stage of HPV block genome amplification, while the HPV genome maintenance replication was unaffected. We first studied the effects of caffeine in the earliest stages of viral infection. Using pseudo-virions (PsV) expressing an m-Cherry reporter gene and quasi-virions (QsV) containing HPV31 genomes to mediate the infection, we found no evidence that caffeine impeded the viral entry; however, the infected cells displayed a reduced HPV copy number. In contrast, caffeine exposure increased the copy number of HPV31 episomes in the transient transfection assays and in the CIN612E cells that stably maintain viral episomes. There was a concomitant increase in the steady state levels of the HPV31 E1 and E2 transcripts, along with increased E2 loading at the viral origin of replication (ori). These results suggest that the caffeine-mediated inhibition of the DDR reduces viral genome replication in the early stage of infection, in contrast to the maintenance stage, in which the inhibition of the DDR may lead to an increase in viral amplicon replication.

The Drivers, Mechanisms, and Consequences of Genome Instability in HPV-Driven Cancers.

Simple SummaryCells infected with high-risk human papillomaviruses (HPV) can accumulate DNA damage and eventually transform into HPV-driven cancers. Genome instability, or the progressive accumulation of DNA alterations (e.g., mutations), in HPV-infected cells is directly induced by the HPV genes and indirectly promoted by HPV infection through the consequences of chronic infection maintenance, increased cell growth, and accumulation of damaging mutations in genes that themselves affect genome instability. While the HPV genome typically exists as a separate entity within cells, genome instability increases the chances of HPV integrating within the host (human) genome, which is common in HPV-induced cancers. The DNA regions surrounding HPV integrations are unstable and can undergo complex alterations that affect both human and HPV genes. This review discusses HPV-dependent and -independent drivers and mechanisms of genome instability in HPV-driven cancers, both globally and around sites of HPV integration, and describes the changes induced in the tumour genome.Human papillomavirus (HPV) is the causative driver of cervical cancer and a contributing risk factor of head and neck cancer and several anogenital cancers. HPV’s ability to induce genome instability contributes to its oncogenicity. HPV genes can induce genome instability in several ways, including modulating the cell cycle to favour proliferation, interacting with DNA damage repair pathways to bring high-fidelity repair pathways to viral episomes and away from the host genome, inducing DNA-damaging oxidative stress, and altering the length of telomeres. In addition, the presence of a chronic viral infection can lead to immune responses that also cause genome instability of the infected tissue. The HPV genome can become integrated into the host genome during HPV-induced tumorigenesis. Viral integration requires double-stranded breaks on the DNA; therefore, regions around the integration event are prone to structural alterations and themselves are targets of genome instability. In this review, we present the mechanisms by which HPV-dependent and -independent genome instability is initiated and maintained in HPV-driven cancers, both across the genome and at regions of HPV integration.

Cryo-EM Structure and Functional Studies of EBNA1 Binding to the Family of Repeats and Dyad Symmetry Elements of Epstein-Barr Virus oriP.

Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional viral-encoded DNA-binding protein essential for Epstein-Barr virus (EBV) DNA replication and episome maintenance. EBNA1 binds to two functionally distinct elements at the viral origin of plasmid replication (oriP), termed the dyad symmetry (DS) element, required for replication initiation and the family of repeats (FR) required for episome maintenance. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the EBNA1 DNA binding domain (DBD) from amino acids (aa) 459 to 614 and its interaction with two tandem sites at the DS and FR. We found that EBNA1 induces a strong DNA bending angle in the DS, while the FR is more linear. The N-terminal arm of the DBD (aa 444 to 468) makes extensive contact with DNA as it wraps around the minor groove, with some conformational variation among EBNA1 monomers. Mutation of variable-contact residues K460 and K461 had only minor effects on DNA binding but had abrogated oriP-dependent DNA replication. We also observed that the AT-rich intervening DNA between EBNA1 binding sites in the FR can be occupied by the EBNA1 AT hook, N-terminal domain (NTD) aa 1 to 90 to form a Zn-dependent stable complex with EBNA1 DBD on a 2×FR DNA template. We propose a model showing EBNA1 DBD and NTD cobinding at the FR and suggest that this may contribute to the oligomerization of viral episomes important for maintenance during latent infection. IMPORTANCE EBV latent infection is causally linked to diverse cancers and autoimmune disorders. EBNA1 is the viral-encoded DNA binding protein required for episomal maintenance during latent infection and is consistently expressed in all EBV tumors. The interaction of EBNA1 with different genetic elements confers different viral functions, such as replication initiation at DS and chromosome tethering at FR. Here, we used cryo-EM to determine the structure of the EBNA1 DNA-binding domain (DBD) bound to two tandem sites at the DS and at the FR. We also show that the NTD of EBNA1 can interact with the AT-rich DNA sequence between tandem EBNA1 DBD binding sites in the FR. These results provide new information on the mechanism of EBNA1 DNA binding at DS and FR and suggest a higher-order oligomeric structure of EBNA1 bound to FR. Our findings have implications for targeting EBNA1 in EBV-associated disease.

Role of HPV16 E1 in cervical carcinogenesis.

Cervical cancer is the fourth most common cancer in women worldwide. More than 90% of cases are caused by the human papillomavirus (HPV). Vaccines developed only guard against a few HPV types and do not protect people who have already been infected. HPV is a small DNA virus that infects the basal layer of the stratified epithelium of the skin and mucosa through small breaks and replicates as the cells differentiate. The mucosal types of HPV can be classified into low-risk and high-risk groups, based on their association with cancer. Among HPV types in high-risk group, HPV type 16 (HPV-16) is the most common, causing 50% of all cancer cases. HPV infection can occur as transient or persistent infections, based on the ability of immune system to clear the virus. Persistent infection is characterized by the integration of HPV genome. HPV-16 exhibits a different integration pattern, with only 50% reported to be integrated at the carcinoma stage. Replication of the HPV genome depends on protein E1, an ATP-dependent helicase. E1 is essential for the amplification of the viral episome in infected cells. Previous studies have shown that E1 does not only act as a helicase protein but is also involved in recruiting and interacting with other host proteins. E1 has also been deemed to drive host cell proliferation. Recent studies have emphasized the emerging role of HPV E1 in cervical carcinogenesis. In this review, a possible mechanism by which E1 drives cell proliferation and oncogenesis will be discussed.

Human papillomaviruses sensitize cells to DNA damage induced apoptosis by targeting the innate immune sensor cGAS.

The cyclic GMP-AMP synthase (cGAS) is a critical regulator of the innate immune response acting as a sensor of double-strand DNAs from pathogens or damaged host DNA. Upon activation, cGAS signals through the STING/TBK1/IRF3 pathway to induce interferon expression. Double stranded DNA viruses target the cGAS pathway to facilitate infection. In HPV positive cells that stably maintain viral episomes, the levels of cGAS were found to be significantly increased over those seen in normal human keratinocytes. Furthermore the downstream effectors of the cGAS pathway, STING and IRF3, were fully active in response to signaling from the secondary messenger cGAMP or poly (dA:dT). In HPV positive cells cGAS was detected in both cytoplasmic puncta as well as in DNA damage induced micronuclei. E6 was responsible for increased levels of cGAS that was dependent on inhibition of p53. CRISPR-Cas9 mediated knockout of cGAS prevented activation of STING and IRF3 but had a minimal effect on viral replication. A primary function of cGAS in HPV positive cells was in response to treatment with etoposide or cisplatin which lead to increased levels of H2AX phosphorylation and activation of caspase 3/7 cleavage while having only a minimal effect on activation of homologous recombination repair factors ATM, ATR or CHK2. In HPV positive cells cGAS was found to regulate the levels of the phosphorylated non-homologous end-joining kinase, DNA-PK, which may contribute to H2AX phosphorylation along with other factors. Importantly cGAS was also responsible for increased levels of DNA breaks along with enhanced apoptosis in HPV positive cells but not in HFKs. This study identifies an important and novel role for cGAS in mediating the response of HPV positive cells to chemotherapeutic drugs.

Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo

Persistence of chronic hepatitis B (CHB) is attributed to maintenance of the intrahepatic pool of the viral covalently closed circular DNA (cccDNA), which serves as the transcriptional template for all viral gene products required for replication. Current nucleos(t)ide therapies for CHB prevent virus production and spread but have no direct impact on cccDNA or expression of viral genes. We describe a potential curative approach using a highly specific engineered ARCUS nuclease (ARCUS-POL) targeting the hepatitis B virus (HBV) genome. Transient ARCUS-POL expression in HBV-infected primary human hepatocytes produced substantial reductions in both cccDNA and hepatitis B surface antigen (HBsAg). To evaluate ARCUS-POL invivo, we developed episomal adeno-associated virus (AAV) mouse and non-human primate (NHP) models containing a portion of the HBV genome serving as a surrogate for cccDNA. Clinically relevant delivery was achieved through systemic administration of lipid nanoparticles containing ARCUS-POL mRNA. In both mouse and NHP, we observed a significant decrease in total AAV copy number and high on-target indel frequency. In the case of the mouse model, which supports HBsAg expression, circulating surface antigen was durably reduced by 96%. Together, these data support a gene-editing approach for elimination of cccDNA toward an HBV cure.

Homing in on Endogenous Badnaviral Elements: Development of Multiplex PCR-DGGE for Detection and Rapid Identification of Badnavirus Sequences in Yam Germplasm.

Viruses of the genus Badnavirus (family Caulimoviridae) are double-stranded DNA-reverse transcribing (dsDNA-RT) plant viruses and have emerged as serious pathogens of tropical and temperate crops globally. Endogenous badnaviral sequences are found integrated in the genomes of several economically important plant species. Infection due to activation of replication-competent integrated copies of the genera Badnavirus, Petuvirus and Cavemovirus has been described. Such endogenous badnaviral elements pose challenges to the development of nucleic acid-based diagnostic methods for episomal virus infections and decisions on health certification for international movement of germplasm and seed. One major food security crop affected is yam (Dioscorea spp.). A diverse range of Dioscorea bacilliform viruses (DBVs), and endogenous DBV (eDBV) sequences have been found to be widespread in yams cultivated in West Africa and other parts of the world. This study outlines the development of multiplex PCR-dependent denaturing gradient gel electrophoresis (PCR-DGGE) to assist in the detection and analysis of eDBVs, through the example of analysing yam germplasm from Nigeria and Ghana. Primers targeting the three most prevalent DBV monophyletic species groups in West Africa were designed to improve DGGE resolution of complex eDBV sequence fingerprints. Multiplex PCR-DGGE with the addition of a tailor-made DGGE sequence marker enables rapid comparison of endogenous badnaviral sequence diversity across germplasm, as illustrated in this study for eDBV diversity in yam.