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.

Cryo-electron Microscopy Structures of Novel Viruses from Mud Crab Scylla paramamosain with Multiple Infections.

Viruses associated with sleeping disease (SD) in crabs cause great economic losses to aquaculture, and no effective measures are available for their prevention. In this study, to help develop novel antiviral strategies, single-particle cryo-electron microscopy was applied to investigate viruses associated with SD. The results not only revealed the structure of mud crab dicistrovirus (MCDV) but also identified a novel mud crab tombus-like virus (MCTV) not previously detected using molecular biology methods. The structure of MCDV at a 3.5-Å resolution reveals three major capsid proteins (VP1 to VP3) organized into a pseudo-T=3 icosahedral capsid, and affirms the existence of VP4. Unusually, MCDV VP3 contains a long C-terminal region and forms a novel protrusion that has not been observed in other dicistrovirus. Our results also reveal that MCDV can release its genome via conformation changes of the protrusions when viral mixtures are heated. The structure of MCTV at a 3.3-Å resolution reveals a T= 3 icosahedral capsid with common features of both tombusviruses and nodaviruses. Furthermore, MCTV has a novel hydrophobic tunnel beneath the 5-fold vertex and 30 dimeric protrusions composed of the P-domains of the capsid protein at the 2-fold axes that are exposed on the virion surface. The structural features of MCTV are consistent with a novel type of virus.IMPORTANCE Pathogen identification is vital for unknown infectious outbreaks, especially for dual or multiple infections. Sleeping disease (SD) in crabs causes great economic losses to aquaculture worldwide. Here we report the discovery and identification of a novel virus in mud crabs with multiple infections that was not previously detected by molecular, immune, or traditional electron microscopy (EM) methods. High-resolution structures of pathogenic viruses are essential for a molecular understanding and developing new disease prevention methods. The three-dimensional (3D) structure of the mud crab tombus-like virus (MCTV) and mud crab dicistrovirus (MCDV) determined in this study could assist the development of antiviral inhibitors. The identification of a novel virus in multiple infections previously missed using other methods demonstrates the usefulness of this strategy for investigating multiple infectious outbreaks, even in humans and other animals.

IRES-dependent ribosome repositioning directs translation of a +1 overlapping ORF that enhances viral infection.

RNA structures can interact with the ribosome to alter translational reading frame maintenance and promote recoding that result in alternative protein products. Here, we show that the internal ribosome entry site (IRES) from the dicistrovirus Cricket paralysis virus drives translation of the 0-frame viral polyprotein and an overlapping +1 open reading frame, called ORFx, via a novel mechanism whereby a subset of ribosomes recruited to the IRES bypasses 37 nucleotides downstream to resume translation at the +1-frame 13th non-AUG codon. A mutant of CrPV containing a stop codon in the +1 frame ORFx sequence, yet synonymous in the 0-frame, is attenuated compared to wild-type virus in a Drosophila infection model, indicating the importance of +1 ORFx expression in promoting viral pathogenesis. This work demonstrates a novel programmed IRES-mediated recoding strategy to increase viral coding capacity and impact virus infection, highlighting the diversity of RNA-driven translation initiation mechanisms in eukaryotes.

Kinetics of initiating polypeptide elongation in an IRES-dependent system.

The intergenic IRES of Cricket Paralysis Virus (CrPV-IRES) forms a tight complex with 80S ribosomes capable of initiating the cell-free synthesis of complete proteins in the absence of initiation factors. Such synthesis raises the question of what effect the necessary IRES dissociation from the tRNA binding sites, and ultimately from all of the ribosome, has on the rates of initial peptide elongation steps as nascent peptide is formed. Here we report the first results measuring rates of reaction for the initial cycles of IRES-dependent elongation. Our results demonstrate that 1) the first two cycles of elongation proceed much more slowly than subsequent cycles, 2) these reduced rates arise from slow pseudo-translocation and translocation steps, and 3) the retarding effect of ribosome-bound IRES on protein synthesis is largely overcome following translocation of tripeptidyl-tRNA. Our results also provide a straightforward approach to detailed mechanistic characterization of many aspects of eukaryotic polypeptide elongation.

RACK1 controls IRES-mediated translation of viruses.

Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.

Modulation of stress granules and P bodies during dicistrovirus infection.

Stress granules (SGs) are dynamic cytosolic aggregates composed of ribonucleoproteins that are induced during cellular stress when protein synthesis is inhibited. The function of SGs is poorly understood, but they are thought to be sites for reorganizing mRNA and protein. Several viruses can modulate SG formation, suggesting that SGs have an impact on virus infection. In this study, we have investigated the relationship of SG formation in Drosophila S2 cells infected by cricket paralysis virus (CrPV), a member of the Dicistroviridae family. Despite a rapid shutoff of host translation during CrPV infection, several hallmark SG markers such as the Drosophila TIA-1 and G3BP (RasGAP-SH3-binding protein) homologs, Rox8 and Rin, respectively, do not aggregate in CrPV-infected cells, even when challenged with potent SG inducers such as heat shock, oxidative stress, and pateamine A treatment. Furthermore, we demonstrate that a subset of P body markers become moderately dispersed at late times of infection. In contrast, as shown by fluorescent in situ hybridization, poly(A)(+) RNA granules still form at late times of infection. These poly(A)(+) RNA granules do not contain viral RNA nor do they colocalize with P body markers. Finally, our results demonstrate that the CrPV viral 3C protease is sequestered to SGs under cellular stress but not during virus infection. In summary, we propose that dicistrovirus infection leads to the selective inhibition of distinct SGs so that viral proteins are available for viral processing.

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles.

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

The Imd pathway is involved in antiviral immune responses in Drosophila.

Cricket Paralysis virus (CrPV) is a member of the Dicistroviridae family of RNA viruses, which infect a broad range of insect hosts, including the fruit fly Drosophila melanogaster. Drosophila has emerged as an effective system for studying innate immunity because of its powerful genetic techniques and the high degree of gene and pathway conservation. Intra-abdominal injection of CrPV into adult flies causes a lethal infection that provides a robust assay for the identification of mutants with altered sensitivity to viral infection. To gain insight into the interactions between viruses and the innate immune system, we injected wild type flies with CrPV and observed that antimicrobial peptides (AMPs) were not induced and hemocytes were depleted in the course of infection. To investigate the contribution of conserved immune signaling pathways to antiviral innate immune responses, CrPV was injected into isogenic mutants of the Immune Deficiency (Imd) pathway, which resembles the mammalian Tumor Necrosis Factor Receptor (TNFR) pathway. Loss-of-function mutations in several Imd pathway genes displayed increased sensitivity to CrPV infection and higher CrPV loads. Our data show that antiviral innate immune responses in flies infected with CrPV depend upon hemocytes and signaling through the Imd pathway.