We explored how a simple retrovirus, Mason-Pfizer monkey virus (M-PMV) to facilitate its replication process, utilizes DHX15, a cellular RNA helicase, typically engaged in RNA processing. Through advanced genetic engineering techniques, we showed that M-PMV recruits DHX15 by mimicking cellular mechanisms, relocating it from the nucleus to the cytoplasm to aid in viral assembly. This interaction is essential for the correct packaging of the viral genome and critical for its infectivity. Our findings offer unique insights into the mechanisms of viral manipulation of host cellular processes, highlighting a sophisticated strategy that viruses employ to leverage cellular machinery for their replication. This study adds valuable knowledge to the understanding of viral-host interactions but also suggests a common evolutionary history between cellular processes and viral mechanisms. This finding opens a unique perspective on the export mechanism of intron-retaining mRNAs in the packaging of viral genetic information and potentially develop ways to stop it.

• MeSH
• buněčné jádro metabolismus   virologie   MeSH
• DEAD-box RNA-helikasy metabolismus   genetika   MeSH
• genom virový MeSH
• HEK293 buňky MeSH
• lidé MeSH
• Masonův-Pfizerův opičí virus * genetika   metabolismus   fyziologie   MeSH
• replikace viru genetika   fyziologie   MeSH
• RNA virová * metabolismus   genetika   MeSH
• RNA-helikasy metabolismus   genetika   MeSH
• sestavení viru * genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

In this study, we employed short- and long-read sequencing technologies to delineate the transcriptional architecture of the human monkeypox virus and to identify key regulatory elements that govern its gene expression. Specifically, we conducted a transcriptomic analysis to annotate the transcription start sites (TSSs) and transcription end sites (TESs) of the virus by utilizing Cap Analysis of gene expression sequencing on the Illumina platform and direct RNA sequencing on the Oxford Nanopore technology device. Our investigations uncovered significant complexity in the use of alternative TSSs and TESs in viral genes. In this research, we also detected the promoter elements and poly(A) signals associated with the viral genes. Additionally, we identified novel genes in both the left and right variable regions of the viral genome.IMPORTANCEGenerally, gaining insight into how the transcription of a virus is regulated offers insights into the key mechanisms that control its life cycle. The recent outbreak of the human monkeypox virus has underscored the necessity of understanding the basic biology of its causative agent. Our results are pivotal for constructing a comprehensive transcriptomic atlas of the human monkeypox virus, providing valuable resources for future studies.

• MeSH
• genom virový MeSH
• lidé MeSH
• počátek transkripce * MeSH
• promotorové oblasti (genetika) MeSH
• RNA virová genetika   MeSH
• sekvenční analýza RNA * metody   MeSH
• stanovení celkové genové exprese MeSH
• transkriptom * MeSH
• virus opičích neštovic genetika   MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

We currently lack antivirals for most human viruses. In a quest for new molecules, focusing on viral RNA, instead of viral proteins, can represent a promising strategy. In this study, new inhibitors were identified starting from a published crystal structure of the tertiary SARS-CoV-2 RNA involved in the -1 programmed ribosomal frameshift. The pseudoknot structure was refined, and a virtual screening was performed using the repository of binders to the nucleic acid library, taking into consideration RNA flexibility. Hit compounds were validated against the wild-type virus and with a dual-luciferase assay measuring the frameshift efficiency. Several active molecules were identified. Our study reveals new inhibitors of SARS-CoV-2 but also highlights the feasibility of targeting RNA starting from virtual screening, a strategy that could be broadly applied to drug development.

• MeSH
• antivirové látky * farmakologie   chemie   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• molekulární modely MeSH
• preklinické hodnocení léčiv * metody   MeSH
• RNA virová * metabolismus   antagonisté a inhibitory   genetika   MeSH
• SARS-CoV-2 * účinky léků   MeSH
• simulace molekulového dockingu MeSH
• uživatelské rozhraní počítače MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The single-stranded RNA genome of SARS-CoV-2 is highly structured. Numerous helical stem-loop structures interrupted by mismatch motifs are present in the functionally important 5'- and 3'-UTRs. These mismatches modulate local helical geometries and feature unusual arrays of hydrogen bonding donor and acceptor groups. However, their conformational and dynamical properties cannot be directly inferred from chemical probing and are difficult to predict theoretically. A mismatch motif (SL1-motif) consisting of three consecutive U•U base pairs is located in stem-loop 1 of the 3'-UTR. We combined NMR-spectroscopy and MD-simulations to investigate its structure and dynamics. All three U•U base pairs feature two direct hydrogen bonds and are as stable as Watson-Crick A:U base pairs. Plasmodium falciparum 25S rRNA contains a triple U•U mismatch motif (Pf-motif) differing from SL1-motif only with respect to the orientation of the two closing base pairs. Interestingly, while the geometry of the outer two U•U mismatches was identical in both motifs the preferred orientation of the central U•U mismatch was different. MD simulations and potassium ion titrations revealed that the potassium ion-binding mode to the major groove is connected to the different preferred geometries of the central base pair in the two motifs.

• MeSH
• 3' nepřekládaná oblast * MeSH
• chybné párování bází * MeSH
• COVID-19 virologie   MeSH
• genom virový MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• nukleotidové motivy * MeSH
• párování bází MeSH
• Plasmodium falciparum genetika   MeSH
• RNA virová * chemie   genetika   MeSH
• SARS-CoV-2 * genetika   chemie   MeSH
• simulace molekulární dynamiky MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Influenza A viruses, causing seasonal epidemics and occasional pandemics, rely on interactions with host proteins for their RNA genome transcription and replication. The viral RNA polymerase utilizes host RNA polymerase II (Pol II) and interacts with the serine 5 phosphorylated (pS5) C-terminal domain (CTD) of Pol II to initiate transcription. Our study, using single-particle electron cryomicroscopy (cryo-EM), reveals the structure of the 1918 pandemic influenza A virus polymerase bound to a synthetic pS5 CTD peptide composed of four heptad repeats mimicking the 52 heptad repeat mammalian Pol II CTD. The structure shows that the CTD peptide binds at the C-terminal domain of the PA viral polymerase subunit (PA-C) and reveals a previously unobserved position of the 627 domain of the PB2 subunit near the CTD. We identify crucial residues of the CTD peptide that mediate interactions with positively charged cavities on PA-C, explaining the preference of the viral polymerase for pS5 CTD. Functional analysis of mutants targeting the CTD-binding site within PA-C reveals reduced transcriptional function or defects in replication, highlighting the multifunctional role of PA-C in viral RNA synthesis. Our study provides insights into the structural and functional aspects of the influenza virus polymerase-host Pol II interaction and identifies a target for antiviral development.IMPORTANCEUnderstanding the intricate interactions between influenza A viruses and host proteins is crucial for developing targeted antiviral strategies. This study employs advanced imaging techniques to uncover the structural nuances of the 1918 pandemic influenza A virus polymerase bound to a specific host protein, shedding light on the vital process of viral RNA synthesis. The study identifies key amino acid residues in the influenza polymerase involved in binding host polymerase II (Pol II) and highlights their role in both viral transcription and genome replication. These findings not only deepen our understanding of the influenza virus life cycle but also pinpoint a potential target for antiviral development. By elucidating the structural and functional aspects of the influenza virus polymerase-host Pol II interaction, this research provides a foundation for designing interventions to disrupt viral replication and transcription, offering promising avenues for future antiviral therapies.

• MeSH
• chřipka lidská virologie   MeSH
• elektronová kryomikroskopie * MeSH
• fosforylace MeSH
• genetická transkripce MeSH
• lidé MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• replikace viru MeSH
• RNA virová metabolismus   genetika   MeSH
• RNA-dependentní RNA-polymerasa * metabolismus   chemie   MeSH
• RNA-polymerasa II * metabolismus   chemie   MeSH
• vazba proteinů MeSH
• virové proteiny * metabolismus   chemie   genetika   MeSH
• virus chřipky A * metabolismus   genetika   enzymologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Global impact of COVID-19 pandemic has heightened the urgency for efficient virus detection and identification of variants such as the Q57H mutation. Early and efficient detection of SARS-CoV-2 among densely populated developing countries is paramount objective. Although RT-PCR assays offer accuracy, however, dependence on expansive kits and availability of allied health resources pose an immense challenge for developing countries. In the current study, RT-LAMP based detection of SARS-Cov-2 with subsequent confirmation of Q57H variant through ARMS-PCR was performed. Among the 212 collected samples, 134 yielded positive results, while 78 tested negative using RT-LAMP. Oropharyngeal swabs of suspected individuals were collected and processed for viral RNA isolation. Isolated viral RNA was processed further by using either commercially available WarmStart Master Mix or our in house developed LAMP master mix separately. Subsequently, the end results of each specimen were evaluated by colorimetry. For LAMP assays, primers targeting three genes (ORF1ab, N and S) were designed using PrimerExplorer software. Interestingly, pooling of these three genes in single reaction tube increased sensitivity (95.5%) and specificity (93.5%) of LAMP assay. SARS-CoV-2 positive specimens were screened further for Q57H mutation using ARMS-PCR. Based on amplicon size variation, later confirmed by sequencing, our data showed 18.5% samples positive for Q57H mutation. Hence, these findings strongly advocate use of RT-LAMP-based assay for SARS-CoV-2 screening within suspected general population. Furthermore, ARMS-PCR also provides an efficient mean to detect prevalent mutations against SARS-Cov-2.

• MeSH
• COVID-19 * diagnóza   MeSH
• diagnostické techniky molekulární metody   MeSH
• lidé MeSH
• pandemie MeSH
• polymerázová řetězová reakce MeSH
• RNA virová genetika   MeSH
• SARS-CoV-2 * genetika   MeSH
• senzitivita a specificita MeSH
• testování na COVID-19 MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Human papillomaviruses (HPVs) represent a diverse group of double-stranded DNA viruses associated with various types of cancers, notably cervical cancer. High-risk types of HPVs exhibit their oncogenic potential through the integration of their DNA into the host genome. This integration event contributes significantly to genomic instability and the progression of malignancy. However, traditional detection methods, such as immunohistochemistry or PCR-based assays, face inherent challenges, and thus alternative tools are being developed to fasten and simplify the analysis. Our study introduces an innovative biosensing platform that combines loop-mediated amplification with electrochemical (EC) analysis for the specific detection of HPV16 integration. By targeting key elements like the E7 mRNA, a central player in HPV integration, and the E2 viral gene transcript lost upon integration, we show clear distinction between episomal and integrated forms of HPV16. Our EC data confirmed higher E7 expression in HPV16-positive cell lines having integrated forms of viral genome, while E2 expression was diminished in cells with fully integrated genomes. Moreover, we revealed distinct expression patterns in cervical tissue of patients, correlating well with digital droplet PCR, qRT-PCR, or immunohistochemical staining. Our platform thus offers insights into HPV integration in clinical samples and facilitates further advancements in cervical cancer research and diagnostics.

• MeSH
• biosenzitivní techniky metody   MeSH
• DNA vazebné proteiny genetika   MeSH
• DNA virů genetika   MeSH
• elektrochemické techniky * metody   MeSH
• genom virový MeSH
• infekce papilomavirem * virologie   MeSH
• integrace viru * genetika   MeSH
• lidé MeSH
• lidský papilomavirus 16 * genetika   MeSH
• messenger RNA * genetika   MeSH
• nádory děložního čípku * virologie   MeSH
• onkogenní proteiny virové * genetika   MeSH
• Papillomavirus E7 - proteiny * genetika   MeSH
• progrese nemoci MeSH
• RNA virová genetika   MeSH
• techniky amplifikace nukleových kyselin metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2'-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2'-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.

• MeSH
• COVID-19 * MeSH
• infekce virem zika * MeSH
• lidé MeSH
• methyltransferasy metabolismus   MeSH
• RNA virová genetika   MeSH
• RNA MeSH
• SARS-CoV-2 genetika   MeSH
• virové nestrukturální proteiny chemie   MeSH
• virus opičích neštovic genetika   metabolismus   MeSH
• virus zika * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The inability to predict the evolution of the COVID-19 epidemic hampered abilities to respond to the crisis effectively. The cycle threshold (Ct) from the standard SARS-CoV-2 quantitative reverse transcription-PCR (RT-qPCR) clinical assay is inversely proportional to the amount of SARS-CoV-2 RNA in the sample. We were interested to see if population Ct values could predict future increases in COVID-19 cases as well as subgroups that would be more likely to be affected. This information would have been extremely helpful early in the COVID-19 epidemic. We therefore conducted a retrospective analysis of demographic data and Ct values from 2,076,887 nasopharyngeal swab RT-qPCR tests that were performed at a single diagnostic laboratory in the Czech Republic from April 2020 to April 2022 and from 221,671 tests that were performed as a part of a mandatory school surveillance testing program from March 2021 to March 2022. We found that Ct values could be helpful predictive tools in the real-time management of viral epidemics. First, early measurement of Ct values would have indicated the low viral load in children, equivalent viral load in males and females, and higher viral load in older individuals. Second, rising or falling median Ct values and differences in Ct distribution indicated changes in the transmission in the population. Third, monitoring Ct values and positivity rates would have provided early evidence as to whether prevention measures are effective. Health system authorities should thus consider collecting weekly median Ct values of positively tested samples from major diagnostic laboratories for regional epidemic surveillance.

• MeSH
• COVID-19 * epidemiologie   diagnóza   MeSH
• dítě MeSH
• lidé MeSH
• retrospektivní studie MeSH
• RNA virová genetika   analýza   MeSH
• SARS-CoV-2 * genetika   MeSH
• senioři MeSH
• virová nálož MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

The SARS-CoV-2 viral load in a respiratory sample can be inversely quantified using the cycle threshold (Ct), defined as the number of amplification cycles required to detect the viral genome in a quantitative PCR assay using reverse transcriptase (RT-qPCR). It may be classified as high (Ct < 25), intermediate (25-30) and low (Ct > 30). We describe the clinical course of 3 patients with haematological neoplasms who contracted COVID-19. None of them had been vaccinated. Firstly, a 22-year-old male with a refractory acute lymphoblastic leukaemia experienced an oligosymptomatic COVID-19 and had a Ct of 23 with an ascending curve. Another male, aged 23, had recently begun treatment for a promyelocytic leukaemia. He had a subacute course with high oxygen requirements. His Ct dropped from 28, when he only experienced fever, to 14.8, during the most critical period and on the edge of ventilatory support. Viral clearance was documented 126 days after the beginning of the symptoms. Finally, a 60-year-old male had received rituximab as maintenance therapy for a follicular lymphoma 3 months before contracting COVID-19. He had a fulminant course and required mechanical ventilation a few days later. We highlight the association between the course of CoViD-19 and the Ct. Viral shedding was longer than in immunocompetent hosts.

• MeSH
• COVID-19 * MeSH
• dospělí MeSH
• hematologické nádory * komplikace   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• nádory * MeSH
• RNA virová analýza   genetika   MeSH
• SARS-CoV-2 MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH