The alg-1 Gene Is Necessary for Orsay Virus Replication in Caenorhabditis elegans

All viruses are obligate intracellular parasites that recruit the cellular machinery of the host they infect to support their own proliferation. We used Caenorhabditis elegans and its only known infecting virus, Orsay virus, to identify host proteins relevant for virus infection. ABSTRACT The establishment of the Orsay virus-Caenorhabditis elegans infection model has enabled the identification of host factors essential for virus infection. Argonautes are RNA interacting proteins evolutionary conserved in the three domains of life that are key components of small RNA pathways. C. elegans encodes 27 argonautes or argonaute-like proteins. Here, we determined that mutation of the argonaute-like gene 1, alg-1, results in a greater than 10,000-fold reduction in Orsay viral RNA levels, which could be rescued by ectopic expression of alg-1. Mutation in ain-1, a known interactor of ALG-1 and component of the RNA-induced silencing complex, also resulted in a significant reduction in Orsay virus levels. Viral RNA replication from an endogenous transgene replicon system was impaired by the lack of ALG-1, suggesting that ALG-1 plays a role during the replication stage of the virus life cycle. Orsay virus RNA levels were unaffected by mutations in the ALG-1 RNase H-like motif that ablate the slicer activity of ALG-1. These findings demonstrate a novel function of ALG-1 in promoting Orsay virus replication in C. elegans. IMPORTANCE All viruses are obligate intracellular parasites that recruit the cellular machinery of the host they infect to support their own proliferation. We used Caenorhabditis elegans and its only known infecting virus, Orsay virus, to identify host proteins relevant for virus infection. We determined that ALG-1, a protein previously known to be important in influencing worm life span and the expression levels of thousands of genes, is required for Orsay virus infection of C. elegans. This is a new function attributed to ALG-1 that was not recognized before. In humans, it has been shown that AGO2, a close relative protein to ALG-1, is essential for hepatitis C virus replication. This demonstrates that through evolution from worms to humans, some proteins have maintained similar functions, and consequently, this suggests that studying virus infection in a simple worm model has the potential to provide novel insights into strategies used by viruses to proliferate.

[1]  I. MacRae,et al.  A structured RNA motif locks Argonaute2:miR-122 onto the 5’ end of the HCV genome , 2021, Nature Communications.

[2]  A. Pasquinelli,et al.  Recovery from heat shock requires the microRNA pathway in Caenorhabditis elegans , 2021, PLoS genetics.

[3]  C. Brosnan,et al.  Cell-type-specific profiling of loaded miRNAs from Caenorhabditis elegans reveals spatial and temporal flexibility in Argonaute loading , 2021, Nature Communications.

[4]  Aiming Wang,et al.  The potyviral silencing suppressor HCPro recruits and employs host ARGONAUTE1 in pro-viral functions , 2020, PLoS pathogens.

[5]  David Wang,et al.  Huntingtin-interacting protein family members have a conserved pro-viral function from Caenorhabditis elegans to humans , 2020, Proceedings of the National Academy of Sciences.

[6]  K. Jeffrey,et al.  A Requirement for Argonaute 4 in Mammalian Antiviral Defense , 2020, Cell reports.

[7]  David Wang,et al.  Entry by multiple picornaviruses is dependent on a pathway that includes TNK2, WASL, and NCK1 , 2019, bioRxiv.

[8]  N. Lin,et al.  Nicotiana benthamiana Argonaute10 plays a pro-viral role in Bamboo mosaic virus infection. , 2019, The New phytologist.

[9]  M. Mangone,et al.  ALG-1 Influences Accurate mRNA Splicing Patterns in the Caenorhabditis elegans Intestine and Body Muscle Tissues by Modulating Splicing Factor Activities , 2019, Genetics.

[10]  David Wang,et al.  The Dietary Restriction-Like Gene drl-1, Which Encodes a Putative Serine/Threonine Kinase, Is Essential for Orsay Virus Infection in Caenorhabditis elegans , 2018, Journal of Virology.

[11]  Anton J. Enright,et al.  Terminal uridylyltransferases target RNA viruses as part of the innate immune system , 2018, Nature Structural & Molecular Biology.

[12]  A. Pasquinelli,et al.  Opposing roles of microRNA Argonautes during Caenorhabditis elegans aging , 2018, PLoS genetics.

[13]  David Wang,et al.  An Evolutionarily Conserved Pathway Essential for Orsay Virus Infection of Caenorhabditis elegans , 2017, mBio.

[14]  Kristen C. Brown,et al.  ALG-5 is a miRNA-associated Argonaute required for proper developmental timing in the Caenorhabditis elegans germline , 2017, Nucleic acids research.

[15]  J. Ahringer,et al.  An Alternative STAT Signaling Pathway Acts in Viral Immunity in Caenorhabditis elegans , 2017, mBio.

[16]  Fedor V. Karginov,et al.  Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells , 2016, Nature Microbiology.

[17]  J. Carrington,et al.  Antiviral roles of plant ARGONAUTES. , 2015, Current opinion in plant biology.

[18]  Scott A. Rifkin,et al.  Ubiquitin-Mediated Response to Microsporidia and Virus Infection in C. elegans , 2014, PLoS pathogens.

[19]  V. Ambros,et al.  Mutations in Conserved Residues of the C. elegans microRNA Argonaute ALG-1 Identify Separable Functions in ALG-1 miRISC Loading and Target Repression , 2014, PLoS genetics.

[20]  S. Asgari,et al.  MicroRNA-like viral small RNA from Dengue virus 2 autoregulates its replication in mosquito cells , 2014, Proceedings of the National Academy of Sciences.

[21]  E. Miska,et al.  A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity , 2013, eLife.

[22]  N. Jafari,et al.  Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection , 2013, PLoS pathogens.

[23]  Daniel Blankenberg,et al.  CloudMap: A Cloud-Based Pipeline for Analysis of Mutant Genome Sequences , 2012, Genetics.

[24]  Samir Bouasker,et al.  The slicing activity of miRNA-specific Argonautes is essential for the miRNA pathway in C. elegans , 2012, Nucleic acids research.

[25]  I. MacRae,et al.  The Crystal Structure of Human Argonaute2 , 2012, Science.

[26]  M. Labouesse,et al.  Developmental Characterization of the MicroRNA-Specific C. elegans Argonautes alg-1 and alg-2 , 2012, PloS one.

[27]  Steffen Schmidt,et al.  The Caenorhabditis elegans GW182 protein AIN-1 interacts with PAB-1 and subunits of the PAN2-PAN3 and CCR4-NOT deadenylase complexes , 2012, Nucleic acids research.

[28]  J. Masson,et al.  The microRNA pathway controls germ cell proliferation and differentiation in C. elegans , 2012, Cell Research.

[29]  S. Lemon,et al.  Stabilization of hepatitis C virus RNA by an Ago2–miR-122 complex , 2012, Proceedings of the National Academy of Sciences.

[30]  G. Pijlman,et al.  West Nile virus encodes a microRNA-like small RNA in the 3′ untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells , 2011, Nucleic acids research.

[31]  Leonard D. Goldstein,et al.  Natural and Experimental Infection of Caenorhabditis Nematodes by Novel Viruses Related to Nodaviruses , 2011, PLoS biology.

[32]  A. Abbott,et al.  Loss of Individual MicroRNAs Causes Mutant Phenotypes in Sensitized Genetic Backgrounds in C. elegans , 2010, Current Biology.

[33]  O. Gascuel,et al.  New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. , 2010, Systematic biology.

[34]  F. Slack,et al.  Ribosomal protein RPS-14 modulates let-7 microRNA function in Caenorhabditis elegans. , 2009, Developmental biology.

[35]  I. MacRae,et al.  The RNA-induced Silencing Complex: A Versatile Gene-silencing Machine* , 2009, The Journal of Biological Chemistry.

[36]  R. Ketting,et al.  RDE-1 slicer activity is required only for passenger-strand cleavage during RNAi in Caenorhabditis elegans , 2009, Nature Structural &Molecular Biology.

[37]  O. Gascuel,et al.  An improved general amino acid replacement matrix. , 2008, Molecular biology and evolution.

[38]  J. Yates,et al.  Systematic identification of C. elegans miRISC proteins, miRNAs, and mRNA targets by their interactions with GW182 proteins AIN-1 and AIN-2. , 2007, Molecular cell.

[39]  Chris Sander,et al.  Cellular cofactors affecting hepatitis C virus infection and replication , 2007, Proceedings of the National Academy of Sciences.

[40]  Pedro J. Batista,et al.  Analysis of the C. elegans Argonaute Family Reveals that Distinct Argonautes Act Sequentially during RNAi , 2006, Cell.

[41]  R. Andino,et al.  The RNA silencing endonuclease Argonaute 2 mediates specific antiviral immunity in Drosophila melanogaster. , 2006, Genes & development.

[42]  O. Gascuel,et al.  Approximate likelihood-ratio test for branches: A fast, accurate, and powerful alternative. , 2006, Systematic biology.

[43]  Min Han,et al.  The developmental timing regulator AIN-1 interacts with miRISCs and may target the argonaute protein ALG-1 to cytoplasmic P bodies in C. elegans. , 2005, Molecular cell.

[44]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[45]  D. Hirsh,et al.  Extrachromosomal DNA transformation of Caenorhabditis elegans , 1985, Molecular and cellular biology.

[46]  Martin J. Simard,et al.  Argonaute proteins: key players in RNA silencing , 2008, Nature Reviews Molecular Cell Biology.

[47]  Leemor Joshua-Tor,et al.  Slicer and the argonautes. , 2007, Nature chemical biology.

[48]  R. Plasterk,et al.  The Caenorhabditis elegans Argonautes ALG-1 and ALG-2: almost identical yet different. , 2006, Cold Spring Harbor symposia on quantitative biology.