The Replicase Protein of Potato Virus X Is Able to Recognize and Replicate Its RNA Component.

The replication system explores the concept of separating the viral RNA involved in the translation of the replicase protein from the replication of the viral genome and has been successfully used to study the replication mechanisms of alphaviruses. We tested the feasibility of this system with potato virus X (PVX), an alpha-like virus, . A viral RNA template was designed which does not produce the replicase and prevents virion formation but remains recognizable by the replicase.

Positive-strand RNA viruses-a Keystone Symposia report.

Positive-strand RNA viruses have been the cause of several recent outbreaks and epidemics, including the Zika virus epidemic in 2015, the SARS outbreak in 2003, and the ongoing SARS-CoV-2 pandemic. On June 18-22, 2022, researchers focusing on positive-strand RNA viruses met for the Keystone Symposium "Positive-Strand RNA Viruses" to share the latest research in molecular and cell biology, virology, immunology, vaccinology, and antiviral drug development. This report presents concise summaries of the scientific discussions at the symposium.

Alphavirus RNA replication in vertebrate cells.

Alphaviruses are positive-strand RNA viruses, typically transmitted by mosquitoes between vertebrate hosts. They encode four essential replication proteins, the non-structural proteins nsP1-4, which possess the enzymatic activities of RNA capping, RNA helicase, site-specific protease, ADP-ribosyl removal and RNA polymerase. Alphaviruses have been key models in the study of membrane-associated RNA replication, which is a conserved feature among the positive-strand RNA viruses of animals and plants.

Macrodomain Binding Compound MRS 2578 Inhibits Alphavirus Replication.

Alphaviruses are positive-strand RNA viruses causing febrile disease. Macrodomain-containing proteins, involved in ADP-ribose-mediated signaling, are encoded by both host cells and several virus groups, including alphaviruses. In this study, compound MRS 2578 that targets the human ADP-ribose glycohydrolase MacroD1 inhibited Semliki Forest virus production as well as viral RNA replication and replicase protein expression.

RNA Viruses in Aquatic Unicellular Eukaryotes.

Increasing sequence information indicates that RNA viruses constitute a major fraction of marine virus assemblages. However, only 12 RNA virus species have been described, infecting known host species of marine single-celled eukaryotes. Eight of these use diatoms as hosts, while four are resident in dinoflagellate, raphidophyte, thraustochytrid, or prasinophyte species.

Separate domains of G3BP promote efficient clustering of alphavirus replication complexes and recruitment of the translation initiation machinery.

G3BP-1 and -2 (hereafter referred to as G3BP) are multifunctional RNA-binding proteins involved in stress granule (SG) assembly. Viruses from diverse families target G3BP for recruitment to replication or transcription complexes in order to block SG assembly but also to acquire pro-viral effects via other unknown functions of G3BP. The Old World alphaviruses, including Semliki Forest virus (SFV) and chikungunya virus (CHIKV) recruit G3BP into viral replication complexes, via an interaction between FGDF motifs in the C-terminus of the viral non-structural protein 3 (nsP3) and the NTF2-like domain of G3BP.

The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication.

The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a 'neck' structure opening towards the cytoplasm.

Purification of Highly Active Alphavirus Replication Complexes Demonstrates Altered Fractionation of Multiple Cellular Membranes.

Positive-strand RNA viruses replicate their genomes in membrane-associated structures; alphaviruses and many other groups induce membrane invaginations called spherules. Here, we established a protocol to purify these membranous replication complexes (RCs) from cells infected with Semliki Forest virus (SFV). We isolated SFV spherules located on the plasma membrane and further purified them using two consecutive density gradients.

Polyprotein Processing as a Determinant for in Vitro Activity of Semliki Forest Virus Replicase.

Semliki Forest virus (SFV) is an arthropod-borne alphavirus that induces membrane invaginations (spherules) in host cells. These harbor the viral replication complexes (RC) that synthesize viral RNA. Alphaviruses have four replicase or nonstructural proteins (nsPs), nsP1-4, expressed as polyprotein P1234.

Blockade of the LRP16-PKR-NF-κB signaling axis sensitizes colorectal carcinoma cells to DNA-damaging cytotoxic therapy.

Acquired therapeutic resistance by tumors is a substantial impediment to reducing the morbidity and mortality that are attributable to human malignancies. The mechanisms responsible for the dramatic shift between chemosensitivity and chemoresistance in colorectal carcinoma have not been defined. Here, we report that LRP16 selectively interacts and activates double-stranded RNA-dependent kinase (PKR), and also acts as scaffolds to assist the formation of a ternary complex of PKR and IKKβ, prolonging the polymers of ADP-ribose (PAR)-dependent nuclear factor kappa B (NF-κB) transactivation caused by DNA-damaging agents and confers acquired chemoresistance.

Partially Uncleaved Alphavirus Replicase Forms Spherule Structures in the Presence and Absence of RNA Template.

Alphaviruses are positive-strand RNA viruses expressing their replicase as a polyprotein, P1234, which is cleaved to four final products, nonstructural proteins nsP1 to nsP4. The replicase proteins together with viral RNA and host factors form membrane invaginations termed spherules, which act as the replication complexes producing progeny RNAs. We have previously shown that the wild-type alphavirus replicase requires a functional RNA template and active polymerase to generate spherule structures.

Alphavirus polymerase and RNA replication.

Alphaviruses are typically arthropod-borne, and many are important pathogens such as chikungunya virus. Alphaviruses encode four nonstructural proteins (nsP1-4), initially produced as a polyprotein P1234. nsP4 is the core RNA-dependent RNA polymerase but all four nsPs are required for RNA synthesis.

Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments.

As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus.

Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease's active site cysteine residue.

Chikungunya virus (CHIKV), genus Alphavirus, family Togaviridae, has a positive-stand RNA genome approximately 12 kb in length. In infected cells, the genome is translated into non-structural polyprotein P1234, an inactive precursor of the viral replicase, which is activated by cleavages carried out by the non-structural protease, nsP2. We have characterized CHIKV nsP2 using both cell-free and cell-based assays.

Design and Validation of Novel Chikungunya Virus Protease Inhibitors.

Chikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome.

An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication.

Autophagy is a catabolic process regulated by the orchestrated action of the autophagy-related (ATG) proteins. Recent work indicates that some of the ATG proteins also have autophagy-independent roles. Using an unbiased siRNA screen approach, we explored the extent of these unconventional functions of ATG proteins.

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling.

Unlabelled: Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug.

Identification and Characterization of a Ribose 2'-O-Methyltransferase Encoded by the Ronivirus Branch of Nidovirales.

Unlabelled: The order Nidovirales currently comprises four virus families: Arteriviridae, Coronaviridae (divided into the subfamilies Coronavirinae and Torovirinae), Roniviridae, and the recently recognized Mesoniviridae RNA cap formation and methylation have been best studied for coronaviruses, with emphasis on the identification and characterization of two virus-encoded methyltransferases (MTases) involved in RNA capping, a guanine-N7-MTase and a ribose-2'-O-MTase. Although bioinformatics analyses suggest that these MTases may also be encoded by other nidoviruses with large genomes, such as toroviruses and roniviruses, no experimental evidence has been reported thus far. In this study, we show that a ronivirus, gill-associated virus (GAV), encodes the 2'-O-MTase activity, although we could not detect 2'-O-MTase activity for the homologous protein of a torovirus, equine torovirus, which is more closely related to coronaviruses.

Versatile Trans-Replication Systems for Chikungunya Virus Allow Functional Analysis and Tagging of Every Replicase Protein.

Chikungunya virus (CHIKV; genus Alphavirus, family Togaviridae) has recently caused several major outbreaks affecting millions of people. There are no licensed vaccines or antivirals, and the knowledge of the molecular biology of CHIKV, crucial for development of efficient antiviral strategies, remains fragmentary. CHIKV has a 12 kb positive-strand RNA genome, which is translated to yield a nonstructural (ns) or replicase polyprotein.

Ability of minus strands and modified plus strands to act as templates in Semliki Forest virus RNA replication.

During virus multiplication, the viral genome is recognized and recruited for replication based on specific cis-acting elements. Here, we dissected the important cis-acting sequence elements in Semliki Forest virus RNA by using a trans-replication system. As the viral replicase is expressed from a separate plasmid, the template RNA can be freely modified in this system.

Discovery of berberine, abamectin and ivermectin as antivirals against chikungunya and other alphaviruses.

Chikungunya virus (CHIKV) is an arthritogenic arbovirus of the Alphavirus genus, which has infected millions of people after its re-emergence in the last decade. In this study, a BHK cell line containing a stable CHIKV replicon with a luciferase reporter was used in a high-throughput platform to screen approximately 3000 compounds. Following initial validation, 25 compounds were chosen as primary hits for secondary validation with wild type and reporter CHIKV infection, which identified three promising compounds.

RNA Replication and Membrane Modification Require the Same Functions of Alphavirus Nonstructural Proteins.

The alphaviruses induce membrane invaginations known as spherules as their RNA replication sites. Here, we show that inactivation of any function (polymerase, helicase, protease, or membrane association) essential for RNA synthesis also prevents the generation of spherule structures in a Semliki Forest virus trans-replication system. Mutants capable of negative-strand synthesis, including those defective in RNA capping, gave rise to spherules.