The Experts below are selected from a list of 297 Experts worldwide ranked by ideXlab platform
Bryony C. Bonning - One of the best experts on this subject based on the ideXlab platform.
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corrigendum to infectious genomic rna of rhopalosiphum padi virus transcribed in vitro from a full length cdna clone virology 375 2 2008 410 411
Virology, 2014Co-Authors: Sandhya Boyapalle, Randy J Beckett, Narinder Pal, Allen W Miller, Bryony C. BonningAbstract:This article has been corrected at the request of the Authors. Reason: The acquisition of a full length infectious clone of Rhopalosiphum padi virus (RhPV; Dicistroviridae) was reported. It was determined that the sequence reported in Table 1 is incorrect. In particular, a mutation in the clone at 2185 nt (deletion of a cytosine) results in a frame shift and an early stop codon in ORF1. The helicase, protease, and RNA-dependent RNA polymerase (RdRP), which is required for virus replication, are not expected to be produced. Therefore, the clone RhPV6-1 is highly unlikely to be infectious. However, the results presented in the manuscript can be explained on the basis of the following: The GWSS-Z10 line used in this study was lost. Virus-like particles observed by TEM in the GWSS-Z15 cell line suggest that covert viruses are present. The same may have been true for the GWSS-Z10 line. In addition, sequences similar to Aphid lethal paralysis virus (Dicistroviridae) and Big Sioux river virus (of which the reported partial structural polyprotein is 70% identical to RhPV at the amino acid level) were detected in the aphid host (R. padi).
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The Dicistroviridae: An emerging family of invertebrate viruses
Virologica Sinica, 2009Co-Authors: Bryony C. BonningAbstract:Dicistroviruses comprise a newly characterized and rapidly expanding family of small RNA viruses of invertebrates. Several features of this virus group have attracted considerable research interest in recent years. In this review I provide an overview of the Dicistroviridae and describe progress made toward the understanding and practical application of dicistroviruses, including (i) construction of the first infectious clone of a dicistrovirus, (ii) use of the baculovirus expression system for production of an infectious dicistrovirus, (iii) the use of Drosophila C virus for analysis of host response to virus infection, and (iv) correlation of the presence of Israeli acute paralysis virus with honey bee colony collapse disorder. The potential use of dicistroviruses for insect pest management is also discussed. The structure, mechanism and practical use of the internal ribosome entry site (IRES) elements has recently been reviewed elsewhere.
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infectious genomic rna of rhopalosiphum padi virus transcribed in vitro from a full length cdna clone
Virology, 2008Co-Authors: Sandhya Boyapalle, Randy J Beckett, Narinder Pal, Allen W Miller, Bryony C. BonningAbstract:Availability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses.
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a glassy winged sharpshooter cell line supports replication of rhopalosiphum padi virus Dicistroviridae
Journal of Invertebrate Pathology, 2007Co-Authors: Sandhya Boyapalle, Narinder Pal, Allen W Miller, Bryony C. BonningAbstract:Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.
Sandhya Boyapalle - One of the best experts on this subject based on the ideXlab platform.
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corrigendum to infectious genomic rna of rhopalosiphum padi virus transcribed in vitro from a full length cdna clone virology 375 2 2008 410 411
Virology, 2014Co-Authors: Sandhya Boyapalle, Randy J Beckett, Narinder Pal, Allen W Miller, Bryony C. BonningAbstract:This article has been corrected at the request of the Authors. Reason: The acquisition of a full length infectious clone of Rhopalosiphum padi virus (RhPV; Dicistroviridae) was reported. It was determined that the sequence reported in Table 1 is incorrect. In particular, a mutation in the clone at 2185 nt (deletion of a cytosine) results in a frame shift and an early stop codon in ORF1. The helicase, protease, and RNA-dependent RNA polymerase (RdRP), which is required for virus replication, are not expected to be produced. Therefore, the clone RhPV6-1 is highly unlikely to be infectious. However, the results presented in the manuscript can be explained on the basis of the following: The GWSS-Z10 line used in this study was lost. Virus-like particles observed by TEM in the GWSS-Z15 cell line suggest that covert viruses are present. The same may have been true for the GWSS-Z10 line. In addition, sequences similar to Aphid lethal paralysis virus (Dicistroviridae) and Big Sioux river virus (of which the reported partial structural polyprotein is 70% identical to RhPV at the amino acid level) were detected in the aphid host (R. padi).
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infectious genomic rna of rhopalosiphum padi virus transcribed in vitro from a full length cdna clone
Virology, 2008Co-Authors: Sandhya Boyapalle, Randy J Beckett, Narinder Pal, Allen W Miller, Bryony C. BonningAbstract:Availability of a cloned genome from which infectious RNA can be transcribed is essential for investigating RNA virus molecular mechanisms. To date, no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven expression of a cloned Rhopalosiphum padi virus (RhPV; Dicistroviridae) genome that was infectious to aphids, and we identified a cell line (GWSS-Z10) from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious. Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions, consistent with virus infection. Virions from transcript-infected cells were infectious in aphids. This infectious transcript of a cloned RhPV genome provides a valuable tool, and a more tractable system without interference from baculovirus infection, for investigating replication and pathogenesis of dicistroviruses.
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a glassy winged sharpshooter cell line supports replication of rhopalosiphum padi virus Dicistroviridae
Journal of Invertebrate Pathology, 2007Co-Authors: Sandhya Boyapalle, Narinder Pal, Allen W Miller, Bryony C. BonningAbstract:Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10kb positive-sense RNA genome. To study the molecular biology of RhPV, identification of a cell line that supports replication of the virus is essential. We screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA transfection. We observed cytopathic effects (CPE) only in cell lines derived from hemipterans, specifically GWSS-Z10 cells derived from the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10 cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell derived-virus particles to the bird cherry-oat aphid, R. padi, was confirmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.
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A cell culture system and production of an infectious clone of Rhopalosiphum padi virus (Dicistroviridae)
1Co-Authors: Sandhya BoyapalleAbstract:Rhopalosiphum padi virus (RhPV) was first isolated from the bird cherry-oat aphid, Rhopalosiphum padi. RhPV, an icosahedral virus, belongs to the family Dicistroviridae. It has a 10 kb positive-sense RNA genome, with two viral open reading frames (ORFs). We have identified two cell lines, Z10-2 and DMII derived from the homopterans, Homalodisca coagulata and Dalbulus maidis that are permissive for RhPV. Infection, viral replication and production of virions was confirmed by northern blot hybridization, RT-PCR, western blot analysis and immunoelectron microscopy. A cell culture system that allows replication of the virus is crucial for further study of the biology of RhPV. The long term goal of this project is to produce aphid resistant transgenic plants that express RhPV. Acquisition of an infectious clone of RhPV is of primary importance for development of such an aphid resistance technology. A full-length cDNA clone of RhPV was constructed using overlapping reverse transcription PGR products. RNA transcribed from the full-length cDNA clone was infectious upon transfection into Z10-2 and DMII cells, resulting in production of progeny virus phenotypically indistinguishable from the parent virus. The in vitro transcript caused the same cytopathic effects as those caused by transfection of cells with the viral RNA. The virus-like particles (VLPs) purified from the cells transacted with the full-length transcript were infectious to Z10-2 cells and also to R. padi. The infectious cDNA clone of RhPV, together with the cell culture system, will provide valuable experimental tools for the study of replication and pathogenesis of RhPV. The recombinant baculovirus AcRhPV6, which contains the cDNA of RhPV under the control of the polyhedrin promoter, was constructed using the Bac-to-Bac baculovirus
Diego M. A. Guérin - One of the best experts on this subject based on the ideXlab platform.
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ICTV Virus Taxonomy Profile: Dicistroviridae.
Journal of General Virology, 2017Co-Authors: Steven M Valles, Diego M. A. Guérin, Andrew E Firth, Yanping Chen, Yoshifumi Hashimoto, Salvador Herrero, J. R De Miranda, Eugene V. RyabovAbstract:Dicistroviridae is a family of small non-enveloped viruses with monopartite, linear, positive-sense RNA genomes of approximately 8–10 kb. Viruses of all classified species infect arthropod hosts, with some having devastating economic consequences, such as acute bee paralysis virus in domesticated honeybees and taura syndrome virus in shrimp farming. Conversely, the host specificity and other desirable traits exhibited by several members of this group make them potential natural enemies for intentional use against arthropod pests, such as triatoma virus against triatomine bugs that vector Chagas disease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Dicistroviridae which is available at www.ictv.global/report/Dicistroviridae.
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x ray structure of triatoma virus empty capsid insights into the mechanism of uncoating and rna release in dicistroviruses
Acta Crystallographica Section A, 2016Co-Authors: Ruben Sanchezeugenia, Gerardo Anibal Marti, Aritz Durana, Ibai Lopezmarijuan, Diego M. A. GuérinAbstract:In viruses, uncoating and RNA release are two key steps of successfully infecting a target cell. During these steps, the capsid must undergo the necessary conformational changes to allow RNA egress. Despite their importance, these processes are poorly understood in the family Dicistroviridae. Here, we used X-ray crystallography to solve the atomic structure of a Triatoma virus(TrV) empty particle (Protein Data Bank ID 5L7O), which is the resulting capsid after RNA release. It is observed that the overall shape of the capsid and of the three individual proteins is maintained in comparison with the mature virion. Furthermore, no channels indicative of RNA release are formed in the TrV empty particle. However, the most prominent change in the empty particle when compared with the mature virion is the loss of order in the N-terminal domain of the VP2 protein. In mature virions, the VP2 N-terminal domain of one pentamer is swapped with its twofold related copy in an adjacent pentamer, thereby stabilizing the binding between the pentamers. The loss of these interactions allows us to propose that RNA release may take place through transient flipping-out of pentameric subunits. The lower number of stabilizing interactions between the pentamers and the lack of formation of new holes support this model. This model differs from the currently accepted model for rhinoviruses and enteroviruses, in which genome externalization occurs by extrusion of the RNA through capsid channels.
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Inoculation of Triatoma Virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice
Parasites & Vectors, 2013Co-Authors: Jailson F B Querido, Diego M. A. Guérin, Jon Agirre, Gerardo A Marti, Marcelo Sousa SilvaAbstract:Background Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans ( Hemiptera: Reduviidae ), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas’ vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. Methods In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. Results In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. Conclusions We conclude that, under our experimental conditions, TrV is unable to replicate in mice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.
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The cryo-EM Reconstruction of Drosophila C Virus (DCV) at 5.4 Å
Biophysical Journal, 2013Co-Authors: Leandro F. Estrozi, Jon Agirre, Jean-luc Imler, Estelle Santiago, Jorge Navaza, Guy Schoehn, Diego M. A. GuérinAbstract:The Dicistroviridae family, which is currently classified under the Picornavirales order, groups a pool of arthropod-infecting viruses with bicistronic genomes. The interest in this family of viruses has been fueled due to the economical implications of their hosts, which range from beneficial arthropods (bees and shrimps) to insect pests (crickets, ants and triatomines). Two crystallographic structures of dicistroviruses have been reported to date: Cricket Paralysis Virus (CrPV, type species of the Cripavirus genus) and Triatoma Virus (TrV). their structures revealed that dicistroviruses share a core archetypal organization, which is complemented by external and internal capsid-wide differences that likely have arisen from unique host adaptation. In this work we report the cryoEM reconstruction at 5.4 A resolution, and C-alpha trace of Drosophila C Virus (DCV), a viral pathogen that infects Drosophila melanogaster, among other Drosophila species. This virus holds a 65.8% sequence identity with CrPV and, given the ability of the latter to replicate in Drosophila hosts, a detailed comparison can give insight into the infective cycle of dicistroviruses.Keywords: cryoEM, reconstruction, Dicistroviridae, DCV
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Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles.
Virology, 2011Co-Authors: Jon Agirre, Rubén Sánchez-eugenia, Kerman Aloria, Jesus M. Arizmendi, Ibon Iloro, Felix Elortza, Gerardo Anibal Marti, Emmanuelle Neumann, Félix A. Rey, Diego M. A. GuérinAbstract: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.
Eric Jan - One of the best experts on this subject based on the ideXlab platform.
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temporal regulation of distinct internal ribosome entry sites of the Dicistroviridae cricket paralysis virus
Viruses, 2016Co-Authors: Anthony Khong, Jennifer M Bonderoff, Ruth V Spriggs, Erik Tammpere, Craig H Kerr, Thomas J Jackson, Anne E Willis, Eric JanAbstract:Internal ribosome entry is a key mechanism for viral protein synthesis in a subset of RNA viruses. Cricket paralysis virus (CrPV), a member of Dicistroviridae, has a positive-sense single strand RNA genome that contains two internal ribosome entry sites (IRES), a 5′untranslated region (5′UTR) and intergenic region (IGR) IRES, that direct translation of open reading frames (ORF) encoding the viral non-structural and structural proteins, respectively. The regulation of and the significance of the CrPV IRESs during infection are not fully understood. In this study, using a series of biochemical assays including radioactive-pulse labelling, reporter RNA assays and ribosome profiling, we demonstrate that while 5′UTR IRES translational activity is constant throughout infection, IGR IRES translation is delayed and then stimulated two to three hours post infection. The delay in IGR IRES translation is not affected by inhibiting global translation prematurely via treatment with Pateamine A. Using a CrPV replicon that uncouples viral translation and replication, we show that the increase in IGR IRES translation is dependent on expression of non-structural proteins and is greatly stimulated when replication is active. Temporal regulation by distinct IRESs within the CrPV genome is an effective viral strategy to ensure optimal timing and expression of viral proteins to facilitate infection.
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insights into factorless translational initiation by the trna like pseudoknot domain of a viral ires
PLOS ONE, 2012Co-Authors: Eric JanAbstract:The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNA(i)-independent IRES translation.
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modular domains of the Dicistroviridae intergenic internal ribosome entry site
RNA, 2010Co-Authors: Christopher J Jang, Eric JanAbstract:The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae viral family can directly assemble 80S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. These functions are directed by two independently folded domains of the IGR IRES. One domain, composed of overlapping pseudoknots II and III (PKII/III), mediates ribosome recruitment. The second domain, composed of PKI, mimics a tRNA anticodon–codon interaction to position the ribosome at the ribosomal A-site. Although adopting a common secondary structure, the dicistrovirus IGR IRESs can be grouped into two classes based on distinct features within each domain. In this study, we report on the modularity of the IGR IRESs and show that the ribosome-binding domain and the tRNA anticodon mimicry domain are functionally interchangeable between the Type I and the Type II IGR IRESs. Using structural probing, ribosome-binding assays, and ribosome positioning analysis by toeprinting assays, we show that the chimeric IRESs fold properly, assemble 80S ribosomes, and can mediate IRES translation in rabbit reticulocyte lysates. We also demonstrate that the chimeric IRESs can stimulate the ribosome-dependent GTPase activity of eEF2, which suggests that the ribosome is primed for a step downstream from IRES binding. Overall, the results demonstrate that the dicistrovirus IGR IRESs are composed of two modular domains that work in concert to manipulate the ribosome and direct translation initiation.
Sunnie R Thompson - One of the best experts on this subject based on the ideXlab platform.
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in vivo functional analysis of the Dicistroviridae intergenic region internal ribosome entry sites
Nucleic Acids Research, 2011Co-Authors: Marla I Hertz, Sunnie R ThompsonAbstract:Some viral and cellular messages use an alternative mechanism to initiate protein synthesis that involves internal recruitment of the ribosome to an internal ribosome entry site (IRES). The Dicistroviridae intergenic regions (IGR) have been studied as model IRESs to understand the mechanism of IRES-mediated translation. In this study, the in vivo activity of IGR IRESs were compared. Our analysis demonstrates that Class I and II IGR IRESs have comparable translation efficiency in yeast and that Class II is significantly more active in mammalian cells. Furthermore, while Class II IGR IRES activity was enhanced in yeast grown at a higher temperature, temperature did not affect IGR IRES activity in mammalian cells. This suggests that Class II IRESs may not function optimally with yeast ribosomes. Examination of chimeric IGR IRESs, established that the IRES strength and temperature sensitivity are mediated by the ribosome binding domain. In addition, the sequence of the first translated codon is also an important determinant of IRES activity. Our findings provide us with a comprehensive overview of IGR IRES activities and allow us to begin to understand the differences between Classes I and II IGR IRESs.
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mechanism of translation initiation by Dicistroviridae igr iress
Virology, 2011Co-Authors: Marla I Hertz, Sunnie R ThompsonAbstract:The Dicistroviridae is a growing virus family characterized by a dicistronic genome, wherein each open reading frame (ORF) is translated from an independent internal ribosome entry site (IRES). The 5' IRES that translates the first open reading frame (ORF1) is similar to the picornaviral IRESs. However the second IRES, referred to as the intergenic region (IGR) IRES, - translates ORF2 by and uses an unusual mechanism of initiating protein synthesis. It folds into a compact RNA structure that can bind directly to 40S ribosomal subunits and form 80S complexes to initiate translation in the absence of any initiation factors. Despite its unusual mechanism, the IGR IRES has proven to be an elegant model for elucidating initiation mechanisms employed by IRESs, as well as making it a powerful research tool with diverse applications.
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rps25 is essential for translation initiation by the Dicistroviridae and hepatitis c viral iress
Genes & Development, 2009Co-Authors: Dori M Landry, Marla I Hertz, Sunnie R ThompsonAbstract:Most eukaryotic mRNAs are translated using a cap-dependent mechanism of translation. However, approximately 10% of mammalian mRNAs initiate translation using a cap-independent mechanism that is not well understood. These mRNAs contain an internal ribosome entry site (IRES) located in the 5' untranslated region. The cricket paralysis virus (CrPV) intergenic region IRES (IGR IRES) functions in yeast, mammals, and plants, and does not require any translation initiation factors. We used yeast genetics to understand how ribosomes are recruited directly to the mRNA by an IRES. We found that Rps25p has an essential role in CrPV IGR IRES activity in yeast and mammalian cells but not in cap-dependent translation. Purified 40S ribosomal subunits lacking Rps25 are unable to bind to the IGR IRES in vitro. The hepatitis C virus (HCV) IRES also requires Rps25, demonstrating the function of Rps25 is conserved across IRES types. Yeast strains lacking Rps25 exhibit only slight defects in global translation, readthrough, ribosome biogenesis, and programmed ribosomal frameshifting. This work is the first demonstration of a ribosomal protein that is specifically required for IRES-mediated translation initiation. Our findings provide us with the beginnings of a model for the molecular interactions of an IRES with the ribosome.
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translation initiation factors are not required for Dicistroviridae ires function in vivo
RNA, 2009Co-Authors: Nilsa Deniz, Erik M Lenarcic, Dori M Landry, Sunnie R ThompsonAbstract:The cricket paralysis virus (CrPV) intergenic region (IGR) internal ribosome entry site (IRES) uses an unusual mechanism of initiating translation, whereby the IRES occupies the P-site of the ribosome and the initiating tRNA enters the A-site. In vitro experiments have demonstrated that the CrPV IGR IRES is able to bind purified ribosomes and form 80S complexes capable of synthesizing small peptides in the absence of any translation initiation factors. These results suggest that initiation by this IRES is factor-independent. To determine whether the IGR IRES functions in the absence of initiation factors in vivo, we assayed IGR IRES activity in various yeast strains harboring mutations in canonical translation initiation factors. We used a dicistronic reporter assay in yeast to determine whether the CrPV IGR IRES is able to promote translation sufficient to support growth in the presence of various deletions or mutations in translation initiation factors. Using this assay, we have previously shown that the CrPV IGR IRES functions efficiently in yeast when ternary complexes (eIF2dGTPdinitiator tRNA met ) are reduced. Here, we demonstrate that the CrPV IGR IRES activity does not require the eukaryotic initiation factors eIF4G1 or eIF5B, and it is enhanced when eIF2B, the eIF3b subunit of eIF3, or eIF4E are impaired. Taken together, these data support a model in which the CrPV IGR IRES is capable of initiating protein synthesis in the absence of any initiation factors in vivo, and suggests that the CrPV IGR IRES initiates translation by directly recruiting the ribosomal subunits in vivo.