Topoisomerase Inhibitors Increase Episomal DNA Expression by Inducing the Integration of Episomal DNA in Hepatic Cells.

Gene therapy is a promising strategy to treat and cure most inherited metabolic liver disorders. Viral vectors such as those based on adeno-associated viruses (AAVs) and lentiviruses (LVs) are used as vehicles to deliver functional genes to affected hepatocytes. Adverse events associated with the use of high vector doses have motivated the use of small molecules as adjuvants to reduce the dose. In this study, we showed that a one-hour treatment with topoisomerase inhibitors (camptothecin and etoposide) prior to viral transduction is enough to increase AAV and LV reporter expression in non-dividing hepatic cells in culture. Topoisomerase inhibitors increased both integration-competent (ICLV) and integration-deficient (IDLV) LV-derived expression, with a much stronger increase in the IDLV transduction system. In agreement with that, topoisomerase inhibitors increased viral genome integration in both strains, with a greater impact on the IDLV strain, supporting the idea that topoisomerase inhibitors increased episomal DNA integration, especially when viral integrase activity is abolished. These effects correlated with an increase in the DNA damage response produced by the treatments. Our study highlights the need to monitor DNA damage and undesired integration of viral episomal DNAs into the host genome when studying chemical compounds that increase viral transduction.

SARS-CoV-2 nsp13 Restricts Episomal DNA Transcription without Affecting Chromosomal DNA.

Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. We were initially interested in the interplay between nsp13 and interferon (IFN) signaling, and found that nsp13 inhibited reporter signal in an IFN-ß promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-ß promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted the expression of foreign genes originating from plasmid transfection, but failed to inhibit them after chromosome integration. These data, together with results from a runoff transcription assay and RNA sequencing, suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to the inhibition of episomal DNA transcription, and that this restriction required direct interaction with episomal DNA. Based on these findings, we developed an economical and convenient high-throughput drug screening method targeting nsp13. We evaluated the inhibitory effects of various compounds on nsp13 by the expression of reporter gene plasmid after co-transfection with nsp13. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. IMPORTANCE To combat COVID-19, we need to understand SARS-CoV-2 and develop effective antiviral drugs. In our study, we serendipitously found that SARS-CoV-2 nsp13 could suppress episomal DNA transcription without affecting chromosomal DNA. Detailed characterization revealed that nsp13 suppresses episomal gene expression through its NTPase and helicase functions following DNA binding. Furthermore, we developed a high-throughput drug screening system targeting SARS-CoV-2 nsp13. Compared to traditional SARS-CoV-2 drug screening methods, our system is more economical and convenient, facilitating the development of more potent and selective nsp13 inhibitors and enabling the discovery of new antiviral therapies.

The essential role of the quasi-long terminal repeat sequence for replication and gene expression of an endogenous pararetrovirus, petunia vein clearing virus.

Petunia vein clearing virus (PVCV) is a type member of the genus Petuvirus within the Caulimoviridae family and is defined as one viral unit consisting of a single open reading frame (ORF) encoding a viral polyprotein and one quasi-long terminal repeat (QTR) sequence. Since some full-length PVCV sequences are found in the petunia genome and a vector for horizontal transmission of PVCV has not been identified yet, PVCV is referred to as an endogenous pararetrovirus. Molecular mechanisms of replication, gene expression and horizontal transmission of endogenous pararetroviruses in plants are elusive. In this study, agroinfiltration experiments using various PVCV infectious clones indicated that the replication (episomal DNA synthesis) and gene expression of PVCV were efficient when the QTR sequences are present on both sides of the ORF. Whereas replacement of the QTR with another promoter and/or terminator is possible for gene expression, it is essential for QTR sequences to be on both sides for viral replication. Although horizontal transmission of PVCV by grafting and biolistic inoculation was previously reported, agroinfiltration is a useful and convenient method for studying its replication and gene expression.

Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization.

Integrase-defective lentiviral vectors (IDLVs) represent an attractive platform for vaccine development as a result of the ability to induce persistent humoral- and cellular-mediated immune responses against the encoded transgene. Compared with the parental integrating vector, the main advantages for using IDLV are the reduced hazard of insertional mutagenesis and the decreased risk for vector mobilization by wild-type viruses. Here we report on the development and use in the mouse immunogenicity model of simian immunodeficiency virus (SIV)-based IDLV containing a long deletion in the U3 region and with the 3' polypurine tract (PPT) removed from the transfer vector for improving safety and/or efficacy. Results show that a safer extended deletion of U3 sequences did not modify integrase-mediated or -independent integration efficiency. Interestingly, 3' PPT deletion impaired integrase-mediated integration but did not reduce illegitimate, integrase-independent integration efficiency, contrary to what was previously reported in the HIV system. Importantly, although the extended deletion in the U3 did not affect expression or immunogenicity from IDLV, deletion of 3' PPT considerably reduced both expression and immunogenicity of IDLV.

Transcriptionally inactive hepatitis B virus episome DNA preferentially resides in the vicinity of chromosome 19 in 3D host genome upon infection.

The hepatitis B virus (HBV) infects 257 million people worldwide. HBV infection requires establishment and persistence of covalently closed circular (ccc) DNA, a viral episome, in nucleus. Here, we study cccDNA spatial localization in the 3D host genome by using chromosome conformation capture-based sequencing analysis and fluorescence in situ hybridization (FISH). We show that transcriptionally inactive cccDNA is not randomly distributed in host nucleus. Rather, it is preferentially accumulated at specialized areas, including regions close to chromosome 19 (chr.19). Activation of the cccDNA is apparently associated with its re-localization, from a pre-established heterochromatin hub formed by 5 regions of chr.19 to transcriptionally active regions formed by chr.19 and nearby chromosomes including chr.16, 17, 20, and 22. This active versus inactive positioning at discrete regions of the host genome is primarily controlled by the viral HBx protein and by host factors including the structural maintenance of chromosomes protein 5/6 (SMC5/6) complex.

Rapid electrophoretic recovery of DNA from dried blood spots.

Large-scale genetic screening of neonatal dried blood spots for episomal DNA has a great potential to lower patient mortality and morbidity through early diagnosis of primary immunodeficiencies. However, DNA extraction from the surface of dried blood spots remains one of the most time consuming, costly, and labor-intensive parts of DNA analysis. In the present study, we developed and optimized a rapid methodology using only 50 V and heat to extract episomal DNA from dried blood spots prepared from diagnostic cord blood samples. This electric field DNA extraction is the first methodology to use an electric field to extract episomal DNA from a dried blood spot. This 25-minute procedure has one of the lowest times for the extraction of episomal DNA found within the literature and this novel procedure not only negates the need for costly treatment and wash steps, but reduces the time of manual procedures by more than 30 min while retaining the 75-80% of the yield. Combined with real-time PCR, this novel method of electric field extraction not only provides an effective tool for the large scale genetic analysis of neonates, but a key step forward in the simplification and standardization of diagnostic testing.

PJA1 Coordinates with the SMC5/6 Complex To Restrict DNA Viruses and Episomal Genes in an Interferon-Independent Manner.

Viral and episomal DNAs, as signs of infections and dangers, induce a series of immune responses in the host, and cells must sense foreign DNAs to eliminate the invaders. The cell nucleus is not "immune privileged" and exerts intrinsic mechanisms to control nuclear-replicating DNA viruses. Thus, it is important to understand the action of viral DNA sensing in the cell nucleus. Here, we reveal a mechanism of restriction of DNA viruses and episomal plasmids mediated by PJA1, a RING-H2 E3 ubiquitin ligase. PJA1 restricts the DNA viruses hepatitis B virus (HBV) and herpes simplex virus 1 (HSV-1) but not the RNA viruses enterovirus 71 (EV71) and vesicular stomatitis virus (VSV). Similarly, PJA1 inhibits episomal plasmids but not chromosome-integrated reporters or endogenous genes. In addition, PJA1 has no effect on endogenous type I and II interferons (IFNs) and interferon-stimulated genes (ISGs), suggesting that PJA1 silences DNA viruses independent of the IFN pathways. Interestingly, PJA1 interacts with the SMC5/6 complex (a complex essential for chromosome maintenance and HBV restriction) to facilitate the binding of the complex to viral and episomal DNAs in the cell nucleus. Moreover, treatment with inhibitors of DNA topoisomerases (Tops) and knockdown of Tops release PJA1-mediated silencing of viral and extrachromosomal DNAs. Taken together, results of this work demonstrate that PJA1 interacts with SMC5/6 and facilitates the complex to bind and eliminate viral and episomal DNAs through DNA Tops and thus reveal a distinct mechanism underlying restriction of DNA viruses and foreign genes in the cell nucleus.IMPORTANCE DNA viruses, including hepatitis B virus and herpes simplex virus, induce a series of immune responses in the host and lead to human public health concerns worldwide. In addition to cytokines in the cytoplasm, restriction of viral DNA in the nucleus is an important approach of host immunity. However, the mechanism of foreign DNA recognition and restriction in the cell nucleus is largely unknown. This work demonstrates that an important cellular factor (PJA1) suppresses DNA viruses and transfected plasmids independent of type I and II interferon (IFN) pathways. Instead, PJA1 interacts with the chromosome maintenance complex (SMC5/6), facilitates the complex to recognize and bind viral and episomal DNAs, and recruits DNA topoisomerases to restrict the foreign molecules. These results reveal a distinct mechanism underlying the silencing of viral and episomal invaders in the cell nuclei and suggest that PJA1 acts as a potential agent to prevent infectious and inflammatory diseases.

Identification and molecular characterization of Taro bacilliform virus and Taro bacilliform CH virus from East Africa.

Taro (Colocasia esculenta) and tannia (Xanthosoma sp.) are important root crops cultivated mainly by small-scale farmers in sub-Saharan Africa and the South Pacific. Viruses are known to be one of the most important constraints to production, with infections resulting in severe yield reduction. In 2014 and 2015, surveys were conducted in Ethiopia, Kenya, Tanzania and Uganda to determine the identity of viruses infecting taro in East Africa. Screening of 392 samples collected from the region using degenerate badnavirus primers revealed an incidence of 58-74% among the four countries surveyed, with sequence analysis identifying both Taro bacilliform virus (TaBV) and Taro bacilliform CH virus (TaBCHV). TaBCHV was identified from all four countries while TaBV was identified in all except Ethiopia. Full-length sequences from representative TaBV and TaBCHV isolates showed that the genome organization of TaBV isolates from East Africa was consistent with previous reports while TaBCHV isolates from East Africa were found to encode only four ORFs, distinct from a previous report from China. Phylogenetic analysis showed that all East African TaBV isolates form a single subgroup within known TaBV isolates, while TaBCHV isolates form at least two distinct subgroups. To the authors' knowledge, this is the first report describing the occurrence and genome organization of TaBV and TaBCHV isolates from East Africa and the first full-length sequence of the two viruses from tannia.

New therapies for chronic hepatitis B.

Approximately 350 million people worldwide are chronically infected with hepatitis B virus (HBV), representing a significant public health challenge. Nucleos/tide analogues (NUCs) and interferon alpha (IFNα), the current standard of care for chronic infection, aim at preventing progression of the disease to cirrhosis, hepatocellular carcinoma (HCC) and death. However, in contrast to the case of hepatitis C virus infection, in which novel antiviral drugs cure the vast majority of treated patients, in regard to HBV, cure is rare due to the unusual persistence of viral DNA in the form of covalently closed circular DNA (cccDNA) within the nucleus of infected cells. Available therapies for HBV require lifelong treatment and surveillance, as reactivation frequently occurs following medication cessation and the occurrence of HCC is decreased but not eliminated, even after years of successful viral suppression. Progress has been made in the development of new therapeutics, and it is likely that only a combination of immune modulators, inhibitors of gene expression and replication and cccDNA-targeting drugs will eradicate chronic infection. This review aims to summarize the state of the art in HBV drug research highlighting those agents with the greatest potential for success based on in vitro as well as on data from clinical studies.

Dairy cattle serum and milk factors contributing to the risk of colon and breast cancers.

The analysis of published epidemiological data on colon and breast cancer reveals a remarkable concordance for most regions of the world. A low incidence for both cancers has been recorded in Mongolia and Bolivia. Discrepant data, however, have been reported for India, Japan and Korea. In India, the incidence of breast cancer is significantly higher than for colon cancer, in Japan and Korea colon cancer exceeds by far the rate of breast cancer. Here, studies are summarized pointing to a species-specific risk for colon cancer after consumption of beef originating from dairy cattle. Uptake of dairy products of Bos taurus-derived milk cattle, particularly consumed at early age, is suggested to represent one of the main risk factors for the development of breast cancer. A recent demonstration of reduced breast cancer rates in individuals with lactose intolerance (Ji et al., Br J Cancer 2014; 112:149-52) seems to be in line with this interpretation. Species-specific risk factors for these cancers are compatible with the transmission of different infectious factors transferred via meat or dairy products. Countries with discordant rates of colon and breast cancer reveal a similar discordance between meat and milk product consumption of dairy cattle. The recent isolation of a larger number of novel presumably viral DNAs from serum, meat and dairy products of healthy dairy cows, at least part of them infectious for human cells, deserves further investigation. Systemic infections early in life, resulting in latency and prevention of subsequent infections with the same agent by neutralizing antibodies, would require reconsideration of ongoing prospective studies conducted in the adult population.

Impact of raltegravir on HIV-1 RNA and DNA forms following initiation of antiretroviral therapy in treatment-naive patients.

OBJECTIVES: The rapid early-phase decay of plasma HIV-1 RNA during integrase inhibitor-based therapy is not fully understood. The accumulation of biologically active episomal HIV-1 cDNAs, following aborted integration, could contribute to antiviral potency in vivo. METHODS: This prospective, controlled clinical observation study explored raltegravir's impact on the dynamics of HIV-1 RNA in plasma, and concentrations of total HIV-1 cDNA, episomal 2-long terminal repeat (LTR) circles and HIV-1 integrants in peripheral blood mononuclear cells (PBMC). Individuals starting therapy with two nucleoside reverse transcriptase inhibitors plus either raltegravir (raltegravir group; n = 10 patients) or boosted protease inhibitor/non-nucleoside reverse transcriptase inhibitor (control group; n = 10 patients) were followed for 48 weeks. RESULTS: Suppression of HIV-1 RNA (<50 copies/mL) was reached earlier (5/10 versus 0/10 at week 4; 8/10 versus 4/10 at week 12) on raltegravir. Significant total HIV-1 cDNA reductions in PBMC were reached by day 99 and persisted until day 330, with median factors of decrease of 7.2 and 8.9, respectively. Broad inter-individual variations, yet no treatment-associated differences, were noted for HIV-1 cDNA concentrations. Despite reductions in HIV-1 RNA (∼3 log) and total HIV-1 cDNA (∼1 log), concentrations of integrants and 2-LTR circles remained largely unchanged. CONCLUSIONS: These results extend the previously reported early benefit of raltegravir on the decline of plasma viraemia to treatment-naive patients. The modest treatment-associated, yet group-independent, decline in total HIV-1 cDNA load and the lack of significant changes in integrated and episomal HIV-1 cDNA suggest that most integrated DNA is archival and targeting of HIV reservoirs other than PBMC may underlie beneficial effects of raltegravir.