Nucleic Acids Res

Micro (mi)RNAs are small non-coding RNAs with key regulatory functions. Recent advances in the field allowed researchers to identify their targets. However, much less is known regarding the regulation of miRNAs themselves. The accumulation of these tiny regulators can be modulated at various levels during their biogenesis from the transcription of the primary transcript (pri-miRNA) to the stability of the mature miRNA. Here, we studied the importance of the pri-miRNA secondary structure for the regulation of mature miRNA accumulation. To this end, we used the Kaposi's sarcoma herpesvirus, which encodes a cluster of 12 pre-miRNAs. Using small RNA profiling and quantitative northern blot analysis, we measured the absolute amount of each mature miRNAs in different cellular context. We found that the difference in expression between the least and most expressed viral miRNAs could be as high as 60-fold. Using high-throughput selective 2'-hydroxyl acylation analyzed by primer extension, we then determined the secondary structure of the long primary transcript. We found that highly expressed miRNAs derived from optimally structured regions within the pri-miRNA. Finally, we confirmed the importance of the local structure by swapping stem-loops or by targeted mutagenesis of selected miRNAs, which resulted in a perturbed accumulation of the mature miRNA

Genome Evolution of Two Genetically Homogeneous Infectious Bursal Disease Virus Strains During Passages in vitro and ex vivo in the Presence of a Mutagenic Nucleoside Analog

The avibirnavirus infectious bursal disease virus (IBDV) is responsible for a highly contagious and sometimes lethal disease of chickens (Gallus gallus). IBDV genetic variation is well-described for both field and live-attenuated vaccine strains, however, the dynamics and selection pressures behind this genetic evolution remain poorly documented. Here, genetically homogeneous virus stocks were generated using reverse genetics for a very virulent strain, rvv, and a vaccine-related strain, rCu-1. These viruses were serially passaged at controlled multiplicities of infection in several biological systems, including primary chickens B cells, the main cell type targeted by IBDV in vivo. Passages were also performed in the absence or presence of a strong selective pressure using the antiviral nucleoside analog 7-deaza-2′-C-methyladenosine (7DMA). Next Generation Sequencing (NGS) of viral genomes after the last passage in each biological system revealed that (i) a higher viral diversity was generated in segment A than in segment B, regardless 7DMA treatment and viral strain, (ii) diversity in segment B was increased by 7DMA treatment in both viruses, (iii) passaging of IBDV in primary chicken B cells, regardless of 7DMA treatment, did not select cell-culture adapted variants of rvv, preserving its capsid protein (VP2) properties, (iv) mutations in coding and non-coding regions of rCu-1 segment A could potentially associate to higher viral fitness, and (v) a specific selection, upon 7DMA addition, of a Thr329Ala substitution occurred in the viral polymerase VP1. The latter change, together with Ala270Thr change in VP2, proved to be associated with viral attenuation in vivo. These results identify genome sequences that are important for IBDV evolution in response to selection pressures. Such information will help tailor better strategies for controlling IBDV infection in chickens

Development of a primary cell model derived from porcine dorsal soft palate for foot-and-mouth disease virus research and diagnosis

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals that has a significant socio-economic impact. One concern associated with this disease is the ability of its etiological agent, the FMD virus (FMDV), to persist in its hosts through underlying mechanisms that remain to be elucidated. While persistence has been described in cattle and small ruminants, it is unlikely to occur in pigs. One of the factors limiting the progress in understanding FMDV persistence and, in particular, differential persistence is the lack of suitable in vitro models. A primary bovine cell model derived from the dorsal soft palate, which is the primary site of replication and persistence of FMDV in cattle, has been developed, and it seemed relevant to develop a similar porcine model. Cells from two sites of FMDV replication in pigs, namely, the dorsal soft palate and the oropharyngeal tonsils, were isolated and cultured. The epithelial character of the cells from the dorsal soft palate was then assessed by immunofluorescence. The FMDV-sensitivity of these cells was assessed after monolayer infection with FMDV O/FRA/1/2001 Clone 2.2. These cells were also grown in multilayers at the air-liquid interface to mimic a stratified epithelium susceptible to FMDV infection. Consistent with what has been shown in vivo in pigs, our study showed no evidence of persistence of FMDV in either the monolayer or multilayer model, with no infectious virus detected 28 days after infection. The development of such a model opens up new possibilities for the study and diagnosis of FMDV in porcine cells

PLoS Pathog

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis

Kaposi's Sarcoma Herpesvirus microRNAs Target Caspase 3 and Regulate Apoptosis

Kaposi's sarcoma herpesvirus (KSHV) encodes a cluster of twelve micro (mi)RNAs, which are abundantly expressed during both latent and lytic infection. Previous studies reported that KSHV is able to inhibit apoptosis during latent infection; we thus tested the involvement of viral miRNAs in this process. We found that both HEK293 epithelial cells and DG75 cells stably expressing KSHV miRNAs were protected from apoptosis. Potential cellular targets that were significantly down-regulated upon KSHV miRNAs expression were identified by microarray profiling. Among them, we validated by luciferase reporter assays, quantitative PCR and western blotting caspase 3 (Casp3), a critical factor for the control of apoptosis. Using site-directed mutagenesis, we found that three KSHV miRNAs, miR-K12-1, 3 and 4-3p, were responsible for the targeting of Casp3. Specific inhibition of these miRNAs in KSHV-infected cells resulted in increased expression levels of endogenous Casp3 and enhanced apoptosis. Altogether, our results suggest that KSHV miRNAs directly participate in the previously reported inhibition of apoptosis by the virus, and are thus likely to play a role in KSHV-induced oncogenesis

Regulation of the expression of Kaposi's sarcoma associated herpesvirus microRNAs

La dérégulation de l’expression des microARN peut induire des cancers. De plus, ils jouent un rôle crucial dans la pathogénèse et la survie des virus. L’herpès virus humain de type 8 (HHV-8 ou KSHV) est l’agent étiologique du sarcome de Kaposi et est impliqué dans la génération de lymphomes agressifs de type B. De manière intéressante, le génome ce virus code 12 pré-miARN localisés dans la région de latence et exprimés sur un même pri-miARN. Les miARN du KSHV sont importants pour le maintien de la latence, l’inhibition de l’apoptose ou encore la régulation du cycle cellulaire de l’hôte. Nous nous intéressons à leur expression et leur régulation durant l’infection virale. Nous avons résolu la structure secondaire de l’ARN codant ces miARN afin d’identifier les critères structuraux responsables de leur accumulation différentielle. Nous avons initié une analyse cinétique de la première étape de maturation et enfin nous essayons d’identifier des co-facteurs modulant leur expression.It is now well known that modulation of microRNAs expression is linked to the development of cancers. Moreover, they play a crucial role in the pathogenesis and the survival of some viruses. Kaposi’s sarcoma associated herpes virus (KSHV) is the etiologic agent of Kaposi’s sarcoma and is involved in human aggressive B lymphomas generation. Its genome encodes 12 precursor miRNAs that are clustered in a latency region and expressed on a single long primary transcript. KSHV miRNAs are important to maintain the virus latency and to regulate or inhibit the host cell cycle or apoptosis, respectively. Therefore, understanding the regulation of KSHV miRNA accumulation is of prime importance. In this respect, we resolved the secondary structure of them iRNA cluster to identify structural criteria responsible of their differential accumulation. In addition, we started to analyse the mechanism of their maturation by kinetics studies. Finally we tried to identify some cofactors of miRNA expression

Etude de la régulation de l'expression des microARN de l'herpesvirus associé au sarcome de Kaposi

It is now well known that modulation of microRNAs expression is linked to the development of cancers. Moreover, they play a crucial role in the pathogenesis and the survival of some viruses. Kaposi’s sarcoma associated herpes virus (KSHV) is the etiologic agent of Kaposi’s sarcoma and is involved in human aggressive B lymphomas generation. Its genome encodes 12 precursor miRNAs that are clustered in a latency region and expressed on a single long primary transcript. KSHV miRNAs are important to maintain the virus latency and to regulate or inhibit the host cell cycle or apoptosis, respectively. Therefore, understanding the regulation of KSHV miRNA accumulation is of prime importance. In this respect, we resolved the secondary structure of them iRNA cluster to identify structural criteria responsible of their differential accumulation. In addition, we started to analyse the mechanism of their maturation by kinetics studies. Finally we tried to identify some cofactors of miRNA expression.La dérégulation de l’expression des microARN peut induire des cancers. De plus, ils jouent un rôle crucial dans la pathogénèse et la survie des virus. L’herpès virus humain de type 8 (HHV-8 ou KSHV) est l’agent étiologique du sarcome de Kaposi et est impliqué dans la génération de lymphomes agressifs de type B. De manière intéressante, le génome ce virus code 12 pré-miARN localisés dans la région de latence et exprimés sur un même pri-miARN. Les miARN du KSHV sont importants pour le maintien de la latence, l’inhibition de l’apoptose ou encore la régulation du cycle cellulaire de l’hôte. Nous nous intéressons à leur expression et leur régulation durant l’infection virale. Nous avons résolu la structure secondaire de l’ARN codant ces miARN afin d’identifier les critères structuraux responsables de leur accumulation différentielle. Nous avons initié une analyse cinétique de la première étape de maturation et enfin nous essayons d’identifier des co-facteurs modulant leur expression

Cis regulation within a cluster of viral microRNAs

Correction: Volume49, Issue18 Page10804-10805 Article Number711910 DOI10.1093/nar/gkab806MicroRNAs (miRNAs) are small regulatory RNAs involved in virtually all biological processes. Although many of them are co-expressed from clusters, little is known regarding the impact of this organization on the regulation of their accumulation. In this study, we set to decipher a regulatory mechanism controlling the expression of the ten clustered pre-miRNAs from Kaposi's sarcoma associated herpesvirus (KSHV). We measured in vitro the efficiency of cleavage of each individual pre-miRNA by the Microprocessor and found that pre-miR-K1 and -K3 were the most efficiently cleaved pre-miRNAs. A mutational analysis showed that, in addition to producing mature miRNAs, they are also important for the optimal expression of the whole set of miRNAs. We showed that this feature depends on the presence of a canonical pre-miRNA at this location since we could functionally replace pre-miR-K1 by a heterologous pre-miRNA. Further in vitro processing analysis suggests that the two stem-loops act in cis and that the cluster is cleaved in a sequential manner. Finally, we exploited this characteristic of the cluster to inhibit the expression of the whole set of miRNAs by targeting the premiR-K1 with LNA-based antisense oligonucleotides in cells either expressing a synthetic construct or latently infected with KSHV.Peer reviewe

Porcine Epidemic Diarrhea Virus, Surrogate for Coronavirus Decay Measurement in French Coastal Waters and Contribution to Coronavirus Risk Evaluation

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infected patients mainly displays pulmonary and oronasal tropism; however, the presence of the virus has also been demonstrated in the stools of patients and consequently in wastewater treatment plant effluents, raising the question of the potential risk of environmental contamination (such as seawater contamination) through inadequately treated wastewater spillover into surface or coastal waters even if the environmental detection of viral RNA alone does not substantiate risk of infection. Therefore, here, we decided to experimentally evaluate the persistence of the porcine epidemic diarrhea virus (PEDv), considered as a coronavirus representative model, in the coastal environment of France. Coastal seawater was collected, sterile-filtered, and inoculated with PEDv before incubation for 0 to 4 weeks at four temperatures representative of those measured along the French coasts throughout the year (4, 8, 15, and 24°C). The decay rate of PEDv was determined using mathematical modeling and was used to determine the half-life of the virus along the French coast in accordance with temperatures from 2000 to 2021. We experimentally observed an inverse correlation between seawater temperature and the persistence of infectious viruses in seawater and confirm that the risk of transmission of infectious viruses from contaminated stool in wastewater to seawater during recreational practices is very limited. The present work represents a good model to assess the persistence of coronaviruses in coastal environments and contributes to risk evaluation, not only for SARS-CoV-2 persistence, but also for other coronaviruses, specifically enteric coronaviruses from livestock. IMPORTANCE The present work addresses the question of the persistence of coronavirus in marine environments because SARS-CoV-2 is regularly detected in wastewater treatment plants, and the coastal environment, subjected to increasing anthropogenic pressure and the final receiver of surface waters and sometimes insufficiently depurated wastewater, is particularly at risk. The problem also arises in the possibility of soil contamination by CoV from animals, especially livestock, during manure application, where, by soil impregnation and runoff, these viruses can end up in seawater. Our findings are of interest to researchers and authorities seeking to monitor coronaviruses in the environment, either in tourist areas or in regions of the world where centralized systems for wastewater treatment are not implemented, and more broadly, to the scientific community involved in “One Health” approaches