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
- Cell Nucleus metabolism virology MeSH
- DEAD-box RNA Helicases metabolism genetics MeSH
- Genome, Viral MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Mason-Pfizer monkey virus * genetics metabolism physiology MeSH
- Virus Replication genetics physiology MeSH
- RNA, Viral * metabolism genetics MeSH
- RNA Helicases metabolism genetics MeSH
- Virus Assembly * genetics physiology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.
- MeSH
- Fullerenes metabolism pharmacology MeSH
- Genome, Viral drug effects MeSH
- gag Gene Products, Human Immunodeficiency Virus metabolism MeSH
- HEK293 Cells MeSH
- HIV-1 drug effects genetics metabolism physiology MeSH
- Anti-HIV Agents metabolism pharmacology MeSH
- Humans MeSH
- Nucleocapsid Proteins metabolism MeSH
- Reverse Transcription MeSH
- RNA, Viral metabolism MeSH
- Virus Uncoating drug effects MeSH
- Protein Binding MeSH
- Virion metabolism MeSH
- Viral Genome Packaging drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Rotavirus genomes are distributed between 11 distinct RNA molecules, all of which must be selectively copackaged during virus assembly. This likely occurs through sequence-specific RNA interactions facilitated by the RNA chaperone NSP2. Here, we report that NSP2 autoregulates its chaperone activity through its C-terminal region (CTR) that promotes RNA-RNA interactions by limiting its helix-unwinding activity. Unexpectedly, structural proteomics data revealed that the CTR does not directly interact with RNA, while accelerating RNA release from NSP2. Cryo-electron microscopy reconstructions of an NSP2-RNA complex reveal a highly conserved acidic patch on the CTR, which is poised toward the bound RNA. Virus replication was abrogated by charge-disrupting mutations within the acidic patch but completely restored by charge-preserving mutations. Mechanistic similarities between NSP2 and the unrelated bacterial RNA chaperone Hfq suggest that accelerating RNA dissociation while promoting intermolecular RNA interactions may be a widespread strategy of RNA chaperone recycling.
- MeSH
- Cryoelectron Microscopy MeSH
- Genome, Viral genetics MeSH
- Molecular Chaperones metabolism MeSH
- Models, Molecular MeSH
- RNA-Binding Proteins metabolism MeSH
- Ribonucleoproteins metabolism MeSH
- RNA, Viral genetics MeSH
- Rotavirus genetics growth & development metabolism MeSH
- RNA Folding genetics MeSH
- Viral Nonstructural Proteins metabolism MeSH
- Viral Genome Packaging genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription.IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse transcription. Mutations that change the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, an enhancement of the basic character of this region increased both the efficiency of gRNA packaging and the infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. In summary, the basic region in M-PMV Gag plays a key role in the packaging of genomic RNA and, consequently, in assembly and reverse transcription.
- MeSH
- Cell Line MeSH
- Gene Products, gag genetics MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Mason-Pfizer monkey virus genetics physiology MeSH
- Mutation genetics MeSH
- Nucleocapsid Proteins genetics MeSH
- Reverse Transcription genetics MeSH
- RNA, Viral genetics MeSH
- Amino Acid Sequence genetics MeSH
- Virus Assembly genetics MeSH
- Zinc Fingers genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Pathway analysis methods, in which differentially expressed genes are mapped to databases of reference pathways and relative enrichment is assessed, help investigators to propose biologically relevant hypotheses. The last generation of pathway analysis methods takes into account the topological structure of a pathway, which helps to increase both specificity and sensitivity of the findings. Simultaneously, the RNA-Seq technology is gaining popularity and becomes widely used for gene expression profiling. Unfortunately, majority of topological pathway analysis methods remains without implementation and if an implementation exists, it is limited in various factors. RESULTS: We developed a new R/Bioconductor package ToPASeq offering uniform interface to seven distinct topology-based pathway analysis methods, of which three we implemented de-novo and four were adjusted from existing implementations. Apart this, ToPASeq offers a set of tailored visualization functions and functions for importing and manipulating pathways and their topologies, facilitating the application of the methods on different species. The package can be used to compare the differential expression of pathways between two conditions on both gene expression microarray and RNA-Seq data. The package is written in R and is available from Bioconductor 3.2 using AGPL-3 license. CONCLUSION: ToPASeq is a novel package that offers seven distinct methods for topology-based pathway analysis, which are easily applicable on microarray as well as RNA-Seq data, both in human and other species. At the same time, it provides specific tools for visualization of the results.
- MeSH
- Gene Regulatory Networks * MeSH
- Humans MeSH
- Computer Graphics * MeSH
- RNA genetics MeSH
- Sequence Analysis, RNA methods MeSH
- Signal Transduction * MeSH
- Software * MeSH
- Gene Expression Profiling methods MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
To maintain genome integrity, segmented double-stranded RNA viruses of the Reoviridae family must accurately select and package a complete set of up to a dozen distinct genomic RNAs. It is thought that the high fidelity segmented genome assembly involves multiple sequence-specific RNA-RNA interactions between single-stranded RNA segment precursors. These are mediated by virus-encoded non-structural proteins with RNA chaperone-like activities, such as rotavirus (RV) NSP2 and avian reovirus σNS. Here, we compared the abilities of NSP2 and σNS to mediate sequence-specific interactions between RV genomic segment precursors. Despite their similar activities, NSP2 successfully promotes inter-segment association, while σNS fails to do so. To understand the mechanisms underlying such selectivity in promoting inter-molecular duplex formation, we compared RNA-binding and helix-unwinding activities of both proteins. We demonstrate that octameric NSP2 binds structured RNAs with high affinity, resulting in efficient intramolecular RNA helix disruption. Hexameric σNS oligomerizes into an octamer that binds two RNAs, yet it exhibits only limited RNA-unwinding activity compared to NSP2. Thus, the formation of intersegment RNA-RNA interactions is governed by both helix-unwinding capacity of the chaperones and stability of RNA structure. We propose that this protein-mediated RNA selection mechanism may underpin the high fidelity assembly of multi-segmented RNA genomes in Reoviridae.
- MeSH
- Genome, Viral genetics MeSH
- Nucleic Acid Conformation MeSH
- Molecular Chaperones chemistry genetics metabolism MeSH
- Models, Molecular MeSH
- RNA-Binding Proteins chemistry genetics metabolism MeSH
- Orthoreovirus, Avian genetics metabolism MeSH
- RNA, Viral chemistry genetics metabolism MeSH
- Protein Structure, Secondary MeSH
- Base Sequence MeSH
- Protein Binding MeSH
- Viral Nonstructural Proteins chemistry genetics metabolism MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
UNLABELLED: The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. IMPORTANCE: Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.
- MeSH
- Cell Line MeSH
- Cryoelectron Microscopy MeSH
- Genome, Viral * MeSH
- Gene Products, gag MeSH
- Humans MeSH
- Mason-Pfizer monkey virus physiology ultrastructure MeSH
- Mutation MeSH
- Recombinant Proteins MeSH
- RNA, Viral metabolism MeSH
- Amino Acid Sequence MeSH
- Virus Assembly * genetics MeSH
- Amino Acid Substitution MeSH
- Protein Transport MeSH
- Protein Binding MeSH
- Capsid Proteins genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
BACKGROUND: Environmental DNA and metabarcoding allow the identification of a mixture of species and launch a new era in bio- and eco-assessment. Many steps are required to obtain taxonomically assigned matrices from raw data. For most of these, a plethora of tools are available; each tool's execution parameters need to be tailored to reflect each experiment's idiosyncrasy. Adding to this complexity, the computation capacity of high-performance computing systems is frequently required for such analyses. To address the difficulties, bioinformatic pipelines need to combine state-of-the art technologies and algorithms with an easy to get-set-use framework, allowing researchers to tune each study. Software containerization technologies ease the sharing and running of software packages across operating systems; thus, they strongly facilitate pipeline development and usage. Likewise programming languages specialized for big data pipelines incorporate features like roll-back checkpoints and on-demand partial pipeline execution. FINDINGS: PEMA is a containerized assembly of key metabarcoding analysis tools that requires low effort in setting up, running, and customizing to researchers' needs. Based on third-party tools, PEMA performs read pre-processing, (molecular) operational taxonomic unit clustering, amplicon sequence variant inference, and taxonomy assignment for 16S and 18S ribosomal RNA, as well as ITS and COI marker gene data. Owing to its simplified parameterization and checkpoint support, PEMA allows users to explore alternative algorithms for specific steps of the pipeline without the need of a complete re-execution. PEMA was evaluated against both mock communities and previously published datasets and achieved results of comparable quality. CONCLUSIONS: A high-performance computing-based approach was used to develop PEMA; however, it can be used in personal computers as well. PEMA's time-efficient performance and good results will allow it to be used for accurate environmental DNA metabarcoding analysis, thus enhancing the applicability of next-generation biodiversity assessment studies.
- MeSH
- Archaea MeSH
- Bacteria MeSH
- DNA, Environmental chemistry genetics MeSH
- Fungi MeSH
- Metagenomics methods standards MeSH
- Reference Standards MeSH
- Electron Transport Complex IV genetics MeSH
- RNA, Ribosomal, 16S genetics MeSH
- RNA, Ribosomal, 18S genetics MeSH
- Plants MeSH
- Sensitivity and Specificity MeSH
- Software MeSH
- DNA Barcoding, Taxonomic methods standards MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The authors wish to make the following corrections to this paper [...].
- Publication type
- Published Erratum MeSH
