Sites Of Replication Research Articles

Mutational spectra are associated with bacterial niche

As observed in cancers, individual mutagens and defects in DNA repair create distinctive mutational signatures that combine to form context-specific spectra within cells. We reasoned that similar processes must occur in bacterial lineages, potentially allowing decomposition analysis to detect both disruption of DNA repair processes and exposure to niche-specific mutagens. Here we reconstruct mutational spectra for 84 clades from 31 diverse bacterial species and find distinct mutational patterns. We extract signatures driven by specific DNA repair defects using hypermutator lineages, and further deconvolute the spectra into multiple signatures operating within different clades. We show that these signatures are explained by both bacterial phylogeny and replication niche. By comparing mutational spectra of clades from different environmental and biological locations, we identify niche-associated mutational signatures, and then employ these signatures to infer the predominant replication niches for several clades where this was previously obscure. Our results show that mutational spectra may be associated with sites of bacterial replication when mutagen exposures differ, and can be used in these cases to infer transmission routes for established and emergent human bacterial pathogens.

Replisome loading reduces chromatin motion independent of DNA synthesis.

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.

Replisome loading reduces chromatin motion independent of DNA synthesis

Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.

ORI-Explorer: a unified cell-specific tool for origin of replication sites prediction by feature fusion.

The origins of replication sites (ORIs) are precise regions inside the DNA sequence where the replication process begins. These locations are critical for preserving the genome's integrity during cell division and guaranteeing the faithful transfer of genetic data from generation to generation. The advent of experimental techniques has aided in the discovery of ORIs in many species. Experimentation, on the other hand, is often more time-consuming and pricey than computational approaches, and it necessitates specific equipment and knowledge. Recently, ORI sites have been predicted using computational techniques like motif-based searches and artificial intelligence algorithms based on sequence characteristics and chromatin states. In this article, we developed ORI-Explorer, a unique artificial intelligence-based technique that combines multiple feature engineering techniques to train CatBoost Classifier for recognizing ORIs from four distinct eukaryotic species. ORI-Explorer was created by utilizing a unique combination of three traditional feature-encoding techniques and a feature set obtained from a deep-learning neural network model. The ORI-Explorer has significantly outperformed current predictors on the testing dataset. Furthermore, by employing the sophisticated SHapley Additive exPlanation method, we give crucial insights that aid in comprehending model success, highlighting the most relevant features vital for forecasting cell-specific ORIs. ORI-Explorer is also intended to aid community-wide attempts in discovering potential ORIs and developing innovative verifiable biological hypotheses. The used datasets along with the source code are made available through https://github.com/Z-Abbas/ORI-Explorer and https://zenodo.org/record/8358679.

Structural insights on the nucleoprotein C-terminal domain of Měnglà virus.

Filoviruses depend on the nucleoprotein (NP) to accomplish multiple functions during the viral life cycle. NP is the most abundantly expressed viral protein in infected cells and the main component of the viral nucleocapsid. It can be structurally divided into amino- and carboxy- terminal domains (NTD and CTD). The NTD can homo-oligomerize to interact and protect the (-) ssRNA genome, forming long helical structures. The flexible CTD is responsible for the binding of other nucleocapsid proteins and is involved in the formation of inclusion bodies (IBs)-the cytoplasmic sites of nucleocapsid formation and genome replication. The CTD ends in a ~100-residue globular tail. Měnglà virus (MLAV) is the only member of the new Dianlovirus genus within the Filoviridae family. Their differential characteristics and the possibility of becoming a threat for human health justify the interest in better understanding of its structure and function. In this work, we present the structure of the globular tail of the MLAV NP CTD, showing an overall conformation closely related to that previously reported for the equivalent NP region in MARV. Moreover, analyses of the CTD-CTD interactions in the crystal asymmetric unit revealed a higher-order helicoidal structure. Mutational studies underscore the crucial role of a number of residues, located at the CTD-CTD contact interface, for IB formation. Site-directed mutagenesis of amino acids L653 and F687, involved in the formation of these helicoidal assemblies, abrogate the growth of IBs when the full-length MLAV NP was ectopically expressed in cells. Our findings confirm the role of NP CTD in IB formation, also for MLAV, and focus the attention on particular residues as starting point for further analysis.IMPORTANCEFiloviruses are the causative agents of severe and often fatal hemorrhagic disease in humans. Měnglà virus (MLAV) is a recently reported filovirus, isolated from fruit bats that is capable to replicate in human cells, representing a potential risk for human health. An in-depth structural and functional knowledge of MLAV proteins is an essential step for antiviral research on this virus that can also be extended to other emerging filoviruses. In this study, we determined the first crystal structures of the C-terminal domain (CTD) of the MLAV nucleoprotein (NP), showing important similarities to the equivalent domain in MARV. The structural data also show that the NP CTD has the ability to form large helical oligomers that may participate in the control of cytoplasmic inclusion body formation during viral replication.

The feather epithelium contributes to the dissemination and ecology of clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in ducks

ABSTRACT Immature feathers are known replication sites for high pathogenicity avian influenza viruses (HPAIVs) in poultry. However, it is unclear whether feathers play an active role in viral transmission. This study aims to investigate the contribution of the feather epithelium to the dissemination of clade 2.3.4.4b goose/Guangdong/1996 lineage H5 HPAIVs in the environment, based on natural and experimental infections of domestic mule and Muscovy ducks. During the 2016–2022 outbreaks, H5 HPAIVs exhibited persistent and marked feather epitheliotropism in naturally infected commercial ducks. Infection of the feather epithelium resulted in epithelial necrosis and disruption, as well as the production and environmental shedding of infectious virions. Viral and feather antigens colocalized in dust samples obtained from poultry barns housing naturally infected birds. In summary, the feather epithelium contributes to viral replication, and it is a likely source of environmental infectious material. This underestimated excretion route could greatly impact the ecology of HPAIVs, facilitating airborne and preening-related infections within a flock, and promoting prolonged viral infectivity and long-distance viral transmission between poultry farms.

G-quadruplexes of KSHV oriLyt play important roles in promoting lytic DNA replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) utilizes distinct origins of DNA replication (origin of replications) during the latent and lytic phases of its life cycle. Lytic DNA replication requires the participation of cellular and viral proteins to initiate replication at a specific region in the KSHV genome, oriLyt. These regions contain multiple secondary structures and binding sites for viral and cellular proteins. We discovered the presence of G-quadruplex (G4) sites in the oriLyt region. G-quadruplexes are secondary structures in nucleic acid sequences that are considered regulators of multiple biological processes, such as transcription, translation, and replication initiation. Importantly, herpesviruses have a high density of putative quadruplex sequence or formation sites in their genome, including in the regulatory regions, which control replication and transcription. The binding of RecQ1 to the oriLyt region of KSHV and its ability to unwind the G4 structures led us to speculate that G-quadruplex sites play an important role in lytic DNA replication. In this study, we confirmed the formation of stable G-quadruplexes through biochemical and biophysical assays. We further demonstrated that RecQ1, a helicase, associates with the G-quadruplex forming sites of the oriLyt. The functional significance of G4 sites and RecQ1 was confirmed through the stabilization of G4 and depletion of RecQ1 levels through shRNA. Furthermore, the detection of replication initiation through single-molecule analysis of the replicated DNA demonstrated that G4 stabilization leads to subdued replication initiation at the oriLyt. This confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be used to control virus growth.IMPORTANCEBiological processes originating from the DNA and RNA can be regulated by the secondary structures present in the stretch of nucleic acids, and the G-quadruplexes are shown to regulate transcription, translation, and replication. In this study, we identified the presence of multiple G-quadruplex sites in the region (oriLyt) of Kaposi's sarcoma-associated herpesvirus (KSHV) DNA, which is essential for DNA replication during the lytic cycle. We demonstrated the roles of these G-quadruplexes through multiple biochemical and biophysical assays in controlling replication and efficient virus production. We demonstrated that KSHV achieves this by recruiting RecQ1 (helicase) at those G-quadruplex sites for efficient viral DNA replication. Analysis of the replicated DNA through nucleoside labeling and immunostaining showed a reduced initiation of DNA replication in cells with a pharmacologic stabilizer of G-quadruplexes. Overall, this study confirmed the role of the G-quadruplex in regulating viral DNA replication, which can be exploited for controlling viral DNA replication.

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.

Gastrointestinal symptoms among COVID-19 patients presenting to a primary health care center of Nepal: A cross-sectional study.

Coronavirus disease 2019 (COVID-19) is a major public health problem causing significant morbidity and mortality worldwide. Apart from respiratory symptoms, gastrointestinal symptoms like nausea, vomiting, diarrhea, and abdominal discomfort are quite common among COVID-19 patients. The gastrointestinal tract can be a potential site for virus replication and feces a source of transmission. Thus, ignorance of enteric symptoms can hinder effective disease control. The objective of this study is to see the gastrointestinal manifestation of the disease and its effect on morbidity and mortality. This observational cross-sectional retrospective study was carried out among 165 laboratory-confirmed COVID-19 patients in primary health care of Gorkha, Nepal from March 1, 2021 to March 1, 2022. A systematic random sampling method was adopted while data were entered and analyzed by Statistical Package for Social Sciences version 21. Of 165 patients, 97 patients (58.78%) had enteric involvement. Among gastrointestinal symptoms, diarrhea in 67 patients (40.6%) and nausea and/or vomiting in 66 patients (40%) were the most common symptoms, followed by abdominal pain in 27 patients (16.4%) and anorexia in 19 patients (11.5%). Of the majority of cases with gastrointestinal involvement, 63 (63%) were below 50 years of age. Many of the patients who received vaccination had gastrointestinal symptoms (79%). Complications like acute respiratory distress syndrome, shock, and arrhythmia developed in 9.7% of patients, with the death of eight patients. COVID-19 vaccination was associated with 4.32 times higher odds of having gastrointestinal involvement in subsequent COVID-19 infection. Diarrhea followed by nausea/vomiting was among the most common gastrointestinal symptoms affecting younger age groups in our study. Enteric symptoms were more common among vaccinated people rather than among nonvaccinated ones.

P03-15: Screening of genotoxic substances that induce DNA double-strand breaks occurred at DNA replication sites

P03-15: Screening of genotoxic substances that induce DNA double-strand breaks occurred at DNA replication sites

Disruption of Ebola NP0VP35 Inclusion Body-like Structures reduce Viral Infection

Viral inclusion bodies (IBs) are potential sites of viral replication and assembly. How viral IBs form remains poorly defined. Here we describe a combined biophysical and cellular approach to identify the components necessary for IB formation during Ebola virus (EBOV) infection. We find that the eNP0VP35 complex containing Ebola nucleoprotein (eNP) and viral protein 35 (eVP35), the functional equivalents of nucleoprotein (N) and phosphoprotein (P) in non-segmented negative strand viruses (NNSVs), phase separates to form inclusion bodies. Phase separation of eNP0VP35 is reversible and modulated by ionic strength. The multivalency of eVP35, and not eNP, is also critical for phase separation. Furthermore, overexpression of an eVP35 peptide disrupts eNP0VP35 complex formation, leading to reduced frequency of IB formation and limited viral infection. Together, our results show that upon EBOV infection, the eNP0VP35 complex forms the minimum unit to drive IB formation and viral replication.

High rate of adenovirus detection in gastrointestinal biopsies of symptomatic stem cell transplant recipients.

The gastrointestinal (GI) tract is a major human adenovirus (HAdV) replication site in patients undergoing hematopoietic stem cell transplantation (HSCT), yet the prevalence and correlates of HAdV GI infection in this setting have remained poorly recognized, especially among adult HSCT recipients. We retrospectively studied the prevalence and risk factors of HAdV GI-tissue infection in HSCT recipients (73 adults and 15 children) with GI symptoms who underwent GI-tissue biopsy between January-2012 and December-2017. The presence of HAdV in the GI tissues was determined by real-time PCR. HAdV GI-tissue infection was detected in 21 (23.9%) patients, with similar infection rates identified in adults and children. GI-tissue detection was more common at late (>100 days) compared to early times post-transplantation (50%vs. 12.9%, p<.001). The presence of bloody diarrhea, Arab ethnicity (p=.014) and concurrent cytomegalovirus GI-tissue detection (p=.025) were significantly correlated with HAdV GI-tissue infection, while chronic graft versus host disease was of borderline association (p=.055). Our findings reveal a high rate and new clinical-demographic correlates of HAdV GI-tissue infection in adult and pediatric HSCT recipients with GI symptoms. The findings highlight the need for future prospective studies to assess the relatedness of HAdV infection to the GI symptoms, and the prevalence, impact, and treatment of HAdV GI infection in HSCT recipients.

Efficacy of Systemically Administered Retargeted Oncolytic Herpes Simplex Viruses-Clearance and Biodistribution in Naïve and HSV-Preimmune Mice.

We investigated the anticancer efficacy, blood clearance, and tissue biodistribution of systemically administered retargeted oncolytic herpes simplex viruses (ReHVs) in HSV-naïve and HSV-preimmunized (HSV-IMM) mice. Efficacy was tested against lung tumors formed upon intravenous administration of cancer cells, a model of metastatic disease, and against subcutaneous distant tumors. In naïve mice, HER2- and hPSMA-retargeted viruses, both armed with mIL-12, were highly effective, even when administered to mice with well-developed tumors. Efficacy was higher for combination regimens with immune checkpoint inhibitors. A significant amount of infectious virus persisted in the blood for at least 1 h. Viral genomes, or fragments thereof, persisted in the blood and tissues for days. Remarkably, the only sites of viral replication were the lungs of tumor-positive mice and the subcutaneous tumors. No replication was detected in other tissues, strengthening the evidence of the high cancer specificity of ReHVs, a property that renders ReHVs suitable for systemic administration. In HSV-IMM mice, ReHVs administered at late times failed to exert anticancer efficacy, and the circulating virus was rapidly inactivated. Serum stability and in vivo whole blood stability assays highlighted neutralizing antibodies as the main factor in virus inactivation. Efforts to deplete mice of the neutralizing antibodies are ongoing.

Deep spatial profiling of Venezuelan equine encephalitis virus reveals increased genetic diversity amidst neuroinflammation and cell death during brain infection.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.

Persisting effects of seed bed preparation and early grazing on plant communities in grassland restoration

Semi-natural mountain grasslands are species-rich and provide a wide range of ecosystem services, such as biomass production for livestock grazing and carbon sequestration. Land abandonment but also habitat destruction are threatening these ecosystems highlighting the need for restoration when degraded. The transfer of plant material containing seed collected at non-degraded reference sites is increasingly used for restoration but the appropriate methods to prepare the transfer and to manage the grassland afterwards are still under debates. This study aims at testing the effects of seed bed preparation by harrowing and livestock grazing on the establishment of transferred targets and community trajectories towards the reference system. In a previous study, first year seedling counts showed a positive effect of seed bed preparation and a marginally significant negative effect of grazing on the seedling recruitment of the transferred species. We asked whether the results on first year seedling densities translate into significant effects on plant cover and whether these effects on plant cover change in the following three years. Both grazing and harrowing effects on species establishment were tested using a full factorial split-plot experimental design on five replicate sites. Data on species richness and vegetation cover for both the whole plant community and the transferred species were analysed during four years after seed transfer. The cover of transferred species increased whereas their species richness remained unchanged. We found a weak positive effect of harrowing on total cover whereas grazing had no effect. Seed bed preparation by harrowing had a clear positive effect on transferred target species cover. The negative effect of grazing on first-year seedling recruitment of transferred target species turned into a positive effect in the fourth year. However, this effect was only apparent in the harrowed plots resulting in a significant grazing-by-harrowing interaction. The similarity of the plant communities to the reference increased from the first to the fourth year and this increase was stronger in grazed and harrowed plots. In conclusion, the results confirmed the positive effect of seed bed preparation on restoration by seed transfer and demonstrated that initial grazing exclusion is not needed in mountain grasslands of our study area.

SARS-CoV-2 nonstructural protein 6 from Alpha to Omicron: evolution of a transmembrane protein.

We recently reported that mutations in both the spike glycoprotein and nonstructural protein 6 (nsp6) were associated with attenuation of the SARS-CoV-2 Omicron BA.1 variant. While mutations in spike allow evasion of neutralizing antibodies and promote specific modes of viral entry, the role of nsp6 mutations in pathogenesis is less clear. Nsp6 is essential for modifying the endoplasmic reticulum and generating double-membrane vesicles, the site of viral RNA replication. To investigate the evolution of nsp6, we evaluated 91,596 high-confidence human SARS-CoV-2 whole-genome sequences across 19 variants and lineages. While nsp6 of early variants of concern, such as Alpha, Beta, and Gamma, carried a triple amino acid deletion (106-108, termed ΔSGF), the Delta, Epsilon, and Mu lineages retained the ancestral nsp6 sequence. For nsp6 in the emerging Omicron variants, we report a transition from an amino acid 105-107 ΔLSG deletion in BA.1 to increased dominance of the ΔSGF in BA.2 and subsequent lineages. Our findings indicate that deletion within nsp6 was independently selected in multiple lineages of SARS-CoV-2, both early and late in the pandemic. Analysis of SARS-CoV-2-related coronaviruses in bats and pangolins revealed nsp6 sequences similar to the ancestral SARS-CoV-2 virus, indicating that the deletion in nsp6 may be an adaptation to replication in humans. Analysis of nsp6 sequences from multiple coronaviruses predicts a multipass transmembrane protein with a conserved C-terminal domain. Monitoring and evaluating changes in nsp6 and other nonstructural proteins will contribute to our understanding of factors associated with the attenuation of pandemic coronaviruses. IMPORTANCE There is an ongoing need to evaluate genetic changes in SARS-CoV-2 for effects on transmission and pathogenesis. We recently reported an unexpected role for replicase component nsp6, in addition to changes in spike, in the attenuation of Omicron BA.1. In this commentary, we document a triple-amino-acid deletion in a predicted lumenal domain of nsp6 that was found in multiple, independent variants of SARS-CoV-2, including all recent Omicron lineages. Furthermore, we modeled the predicted structure of nsp6, implicating a multipass transmembrane architecture as conserved in members of the Coronaviridae family. This information can guide future studies investigating the role of nsp6 in the pathogenesis of existing and emerging coronaviruses.

Piscine Vitamin D Receptors Vdra/Vdrb in the Absence of Vitamin D Are Utilized by Grass Carp Reovirus for Promoting Viral Replication.

The vitamin D receptor (VDR) plays a pivotal role in the biological actions of vitamin D (VitD). However, little is known about the functions of VDR in the production of viral inclusion bodies (VIBs). Using a representative strain of grass carp reovirus (GCRV) genotype I, GCRV-873, we show that GCRV-873 recruits grass carp Vdrs for promoting the production of VIBs in the absence of VitD. Inhibition of cholesterol synthesis by lovastatin impairs the production of VIBs and blocks the effects of grass carp Vdrs in promoting the production of VIBs in the absence of VitD. Furthermore, grass carp Vdrs are found to form the Vdra-Vdrb heterodimer, which is vital for 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgcr)-dependent cholesterol synthesis and GCRV replication. Intriguingly in the presence of VitD, grass carp Vdra but not Vdrb forms the heterodimer with the retinoid X receptor beta b (Rxrbb), which induces the transcription of those genes involved in the RIG-I-like receptor (RLR) antiviral signaling pathway for inhibiting GCRV infection. Furthermore, the VitD-activated Vdra-Vdrb heterodimer attenuates the transcription of the RLR antiviral signaling pathway induced by VitD. In the presence of VitD, a balance between the Vdra-Rxrbb heterodimers as coactivators and Vdra-Vdrb heterodimers as corepressors in affecting the transcriptional regulation of the RLR antiviral signaling pathway may eventually determine the outcome of GCRV infection. Transfection with VitD can abolish the effect of grass carp Vdrs in promoting GCRV replication in a dose-dependent manner. Taken together, these findings demonstrate that GCRV utilizes host Vdrs to increase hmgcr-dependent cholesterol synthesis for promoting its replication, which can be prevented by VitD treatment. IMPORTANCE Grass carp reovirus (GCRV) is the causative agent of grass carp hemorrhagic disease, which seriously harms freshwater fish. Although many positive or negative regulators of GCRV infection have been identified in teleosts, little is known about the molecular mechanisms by which GCRV utilizes host factors to generate its infectious compartments beneficial for viral replication and infection. Here, we show that in the absence of VitD, the GCRV-873 strain utilizes host vitamin D receptors Vdra/Vdrb to increase hmgcr-dependent cholesterol synthesis for promoting the production of VIBs, which are important functional sites for aquareovirus replication and assembly. The negative regulation of Vdrs during viral infection can be prevented by VitD treatment. Thus, this present work broadens understanding of the pivotal roles of Vdrs in the interaction between the host and GCRV in the absence or presence of VitD, which might provide a rational basis for developing novel anti-GCRV strategies.

Where and when to start: Regulating DNA replication origin activity in eukaryotic genomes

ABSTRACT In eukaryotic genomes, hundreds to thousands of potential start sites of DNA replication named origins are dispersed across each of the linear chromosomes. During S-phase, only a subset of origins is selected in a stochastic manner to assemble bidirectional replication forks and initiate DNA synthesis. Despite substantial progress in our understanding of this complex process, a comprehensive ‘identity code’ that defines origins based on specific nucleotide sequences, DNA structural features, the local chromatin environment, or 3D genome architecture is still missing. In this article, we review the genetic and epigenetic features of replication origins in yeast and metazoan chromosomes and highlight recent insights into how this flexibility in origin usage contributes to nuclear organization, cell growth, differentiation, and genome stability.

IODINE AS A POTENTIAL FRONT-LINE DEFENSE AGAINST COVID-19: A LITERATURE REVIEW

The novel coronavirus disease, first identified in 2019 known as COVID-19, is caused by a new strain of severe acute respiratory syndrome coronavirus (SARS-CoV or SARS-CoV-1), named SARS-CoV-2. Recent studies showed that the virus may be airborne and spreads through small respiratory droplets of saliva in aerosols, indirect or direct physical contact with the affected individual, in a similar way to the cold and influenza. Emerging studies also demonstrate the importance of the throat along with salivary glands as sites of viral replication and transmission in early COVID-19 infection. The most common route of entry of SARS-CoV-2 is the upper respiratory tract (nasopharynx) that slowly reaches the lower respiratory tract to infect the epithelial cells within the lungs which can cause lung damage and severe respiratory symptoms, if not treated immediately. Averting colonization of the virus in the nasopharynx could be one of the best options to reduce the incidence of severe infection. It has been well-documented that iodine is one of the most effective of all antimicrobials available. Hospitals and medical facilities worldwide use povidone-iodine (PVP-I) as a standard of care in infection control. Several research studies during the ongoing COVID-19 pandemic showed the in vitro and in vivo efficacy of iodine-containing solutions such as PVP-I (Betadine), Iodine-V (Essential Iodine Drops) etc. and other iodine complexes to effectively kill the SARS-CoV-2 virus within few seconds to hours. Few commercially available iodine-containing gargling, mouthwash, and nasal spray solutions have been recommended to use in humans against SARS-CoV-2 infection by experts to prevent viral spread, especially among health workers. The present article aims to summarize these studies and highlights the rationale, safety and recommendations of use of iodine as an effective method to decrease the viral load during the early COVID-19 infection.

Exploration of the yadokari/yadonushi nature of YkV3 and RnMBV3 in the original host and a model filamentous fungus

The yadokari/yadonushi nature is a recently discovered virus lifestyle; “yadokari” refers to the ability of capsidless positive-sense (+) RNA viruses (yadokariviruses) to utilize the capsids of phylogenetically distant double-stranded RNA (dsRNA) viruses possibly as the replication site, while “yadonushi” refers to the ability of dsRNA viruses to provide capsids to yadokariviruses. This virus–virus interaction, however, has been only studied with limited pathosystems. Here, we established a new study model with a capsidless (+)RNA yadokarivirus YkV3 (family Yadokariviridae) and its capsid donor RnMBV3 (family Megabirnaviridae) in the original host fungus Rosellinia necatrix and a model filamentous fungal host Cryphonectria parasitica. YkV3 has a simple genome structure with one open reading frame of 4305 nucleotides encoding a single polyprotein with an RNA-dependent RNA polymerase and a 2A-like self-cleavage peptide domain. Reverse genetics of YkV3 in R. necatrix showed that YkV3 tolerates a nucleotide substitution in the extreme 5′-terminus. The insertion of two termination codons immediately downstream of the 2A-like cleavage site abolished YkV3 viability, suggesting the importance of the C-terminal portion of the polyprotein of unknown function. Transfection of RnMBV3 and YkV3 into an RNA silencing-deficient mutant Δdcl2 of C. parasitica showed the replication competency of both viruses. Comparison between the wild-type and Δdcl2 strains of C. parasitica in virus accumulation suggested that RnMBV3 and YkV3 are susceptible to RNA silencing in C. parasitica. Taken together, we have established a platform to further explore the yadokari/yadonushi nature using genetically manipulable host fungal and virus strains.