The structure and mechanism of action of a distinct class of dicistrovirus intergenic region IRESs.

Internal ribosomal entry sites (IRESs) engage with the eukaryotic translation apparatus to promote end-independent initiation. We identified a conserved class of ∼150 nt long intergenic region (IGR) IRESs in dicistrovirus genomes derived from members of the phyla Arthropoda, Bryozoa, Cnidaria, Echinodermata, Entoprocta, Mollusca and Porifera. These IRESs, exemplified by Wenling picorna-like virus 2, resemble the canonical cricket paralysis virus (CrPV) IGR IRES in comprising two nested pseudoknots (PKII/PKIII) and a 3'-terminal pseudoknot (PKI) that mimics a tRNA anticodon stem-loop base-paired to mRNA. However, they are ∼50 nt shorter than CrPV-like IRESs, and PKIII is an H-type pseudoknot that lacks the SLIV and SLV stem-loops that are primarily responsible for the affinity of CrPV-like IRESs for the 40S ribosomal subunit and that restrict initial binding of PKI to its aminoacyl (A) site. Wenling-class IRESs bound strongly to 80S ribosomes but only weakly to 40S subunits. Whereas CrPV-like IRESs must be translocated from the A site to the peptidyl (P) site by elongation factor 2 for elongation to commence, Wenling-class IRESs bound directly to the P site of 80S ribosomes, and decoding begins without a prior translocation step. A chimeric CrPV clone containing a Wenling-class IRES was infectious, confirming that the IRES functioned in cells.

Antibacterial and Antiviral Coating on Surfaces through Dopamine-Assisted Codeposition of an Antifouling Polymer and In Situ Formed Nanosilver.

Bacteria and viruses can adhere onto diverse surfaces and be transmitted in multiple ways. A bifunctional coating that integrates both antibacterial and antiviral activities is a promising approach to mitigate bacterial and viral infections arising from a contaminated surface. However, current coating approaches encounter a slow reaction, limited activity against diverse bacteria or viruses, short-term activity, difficulty in scaling-up, and poor adaptation to diverse material surfaces. Here, we report a new one-step strategy for the development of a polydopamine-based nonfouling antibacterial and antiviral coating by the codeposition of various components. The in situ formed nanosilver in the presence of polydopamine was incorporated into the coating and served as both antibacterial and antiviral agents. In addition, the coassembly of polydopamine and a nonfouling hydrophilic polymer was constructed to prevent the adhesion of bacteria and viruses on the coating. The coating was prepared on model surfaces and thoroughly characterized using various surface analytical techniques. The coating exhibited strong antifouling properties with a reduction of nonspecific protein adsorption up to 90%. The coating was tested against both Gram-positive and Gram-negative bacteria and showed long-term antibacterial effectiveness, which correlated with the composition of the coating. The antiviral activity of the coating was evaluated against human coronavirus 229E. A possible mechanism of action of the coating was proposed. We anticipate that the optimized coating will have applications in the development of infection prevention devices and surfaces.

Expansion of viral genomes with viral protein genome linked copies.

Virus protein-linked genome (VPg) proteins are required for replication. VPgs are duplicated in a subset of RNA viruses however their roles are not fully understood and the extent of viral genomes containing VPg copies has not been investigated in detail. Here, we generated a novel bioinformatics approach to identify VPg sequences in viral genomes using hidden Markov models (HMM) based on alignments of dicistrovirus VPg sequences. From metagenomic datasets of dicistrovirus genomes, we identified 717 dicistrovirus genomes containing VPgs ranging from a single copy to 8 tandem copies. The VPgs are classified into nine distinct types based on their sequence and length. The VPg types but not VPg numbers per viral genome followed specific virus clades, thus suggesting VPgs co-evolved with viral genomes. We also identified VPg duplications in aquamavirus and mosavirus genomes. This study greatly expands the number of viral genomes that contain VPg copies and indicates that duplicated viral sequences are more widespread than anticipated.

Multiple Viral Protein Genome-Linked Proteins Compensate for Viral Translation in a Positive-Sense Single-Stranded RNA Virus Infection.

Viral protein genome-linked (VPg) protein plays an essential role in protein-primed replication of plus-stranded RNA viruses. VPg is covalently linked to the 5' end of the viral RNA genome via a phosphodiester bond typically at a conserved amino acid. Whereas most viruses have a single VPg, some viruses have multiple VPgs that are proposed to have redundant yet undefined roles in viral replication. Here, we use cricket paralysis virus (CrPV), a dicistrovirus that has four nonidentical copies of VPg, as a model to characterize the role of VPg copies in infection. Dicistroviruses contain two main open reading frames (ORFs) that are driven by distinct internal ribosome entry sites (IRESs). We systematically generated single and combinatorial deletions and mutations of VPg1 to VPg4 within the CrPV infectious clone and monitored viral yield in Drosophila S2 cells. Deletion of one to three VPg copies progressively decreased viral yield and delayed viral replication, suggesting a threshold number of VPgs for productive infection. Mass spectrometry analysis of CrPV VPg-linked RNAs revealed viral RNA linkage to either a serine or threonine in VPg, mutations of which in all VPgs attenuated infection. Mutating serine 4 in a single VPg abolished viral infection, indicating a dominant negative effect. Using viral minigenome reporters that monitor dicistrovirus 5' untranslated (UTR) and IRES translation revealed a relationship between VPg copy number and the ratio of distinct IRES translation activities. We uncovered a novel viral strategy whereby VPg copies in dicistrovirus genomes compensate for the relative IRES translation efficiencies to promote infection. IMPORTANCE Genetic duplication is exceedingly rare in small RNA viral genomes, as there is selective pressure to prevent RNA genomes from expanding. However, some small RNA viruses encode multiple copies of a viral protein, most notably an unusual viral protein that is linked to the viral RNA genome. Here, we investigate a family of viruses that contains multiple viral protein genome-linked proteins and reveal a novel viral strategy whereby viral protein copy number counterbalances differences in viral protein synthesis mechanisms.

Dicistrovirus-Host Molecular Interactions.

Members of the family Dicistroviridae are small RNA viruses containing a monopartite positive-sense RNA genome. Dicistroviruses mainly infect arthropods, causing diseases that impact agriculture and the economy. In this chapter, we provide an overview of current and past research on dicistroviruses including the viral life cycle, viral translational control mechanisms, virus structure, and the use of dicistrovirus infection in Drosophila as a model to identify insect antiviral responses. We then delve into how research on dicistrovirus mechanisms has yielded insights into ribosome dynamics, RNA structure/function and insect innate immunity signaling. Finally, we highlight the diseases caused by dicistroviruses, their impacts on agriculture including the shrimp and honey bee industries, and the potential use of dicistroviruses as biopesticides. Although knowledge of the mechanisms underlying dicistrovirus virus-host interactions is limited, the establishment of the first infectious clone should accelerate the discovery of new mechanistic insights into dicistrovirus infections and pathogenesis.

2C-ChIP: measuring chromatin immunoprecipitation signal from defined genomic regions with deep sequencing.

BACKGROUND: Understanding how transcription occurs requires the integration of genome-wide and locus-specific information gleaned from robust technologies. Chromatin immunoprecipitation (ChIP) is a staple in gene expression studies, and while genome-wide methods are available, high-throughput approaches to analyze defined regions are lacking. RESULTS: Here, we present carbon copy-ChIP (2C-ChIP), a versatile, inexpensive, and high-throughput technique to quantitatively measure the abundance of DNA sequences in ChIP samples. This method combines ChIP with ligation-mediated amplification (LMA) and deep sequencing to probe large genomic regions of interest. 2C-ChIP recapitulates results from benchmark ChIP approaches. We applied 2C-ChIP to the HOXA cluster to find that a region where H3K27me3 and SUZ12 linger encodes HOXA-AS2, a long non-coding RNA that enhances gene expression during cellular differentiation. CONCLUSIONS: 2C-ChIP fills the need for a robust molecular biology tool designed to probe dedicated genomic regions in a high-throughput setting. The flexible nature of the 2C-ChIP approach allows rapid changes in experimental design at relatively low cost, making it a highly efficient method for chromatin analysis.