Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

et al. (2009) Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway. PLoS Pathog 5(2): e1000299. doi:10.1371/journal.ppat.1000299 Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway Irma Sa nchez-Vargas 0 Jaclyn C. Scott 0 B. Katherine Poole-Smith 0 Alexander W. E. Franz 0 Vale rie 0 Barbosa-Solomieu 0 Jeffrey Wilusz 0 Ken E. Olson 0 Carol D. Blair 0 Charles M. Rice, The Rockefeller University, United States of America 0 Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University , Fort Collins, Colorado , United States of America A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti. - Funding: This work was supported by NIH/NIAID grants AI034014 and AI063434 and the Grand Challenges in Global Health, Bill and Melinda Gates Foundation, through Foundation of the NIH. The sponsors had no role in design and conduct of the study or in the collection, analysis, and interpretation of the data or in the preparation, review, or approval of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Dengue virus serotypes 14 (DENV1-4; Flavivirus; Flaviviridae) are medically important, positive-sense RNA viruses transmitted to humans by Aedes aegypti mosquitoes during epidemic outbreaks [1,2]. DEN fever and DEN hemorrhagic fever are major public health burdens in many parts of the world [3]; however, although DENVs can cause severe disease in humans, mosquito infections are non-pathogenic and persistent. We hypothesize that the difference in infection outcomes results from host defense (immune) responses. Ae. aegypti is an important vector because it feeds almost exclusively on humans and is well adapted to life in tropical urban environments [4]. We have only a rudimentary understanding of DENV molecular interactions with Ae. aegypti vectors, including the mosquitos innate defense pathways against arboviruses. DENVs infect the mosquito midgut following ingestion of a viremic blood meal from an acutely infected human, replicate, disseminate to the salivary glands where they are further amplified, and emerge into saliva at the time of transmission. Approximately 10 to 14 days are required for the extrinsic incubation period (EIP), the time between initial infection of the mosquito and transmission [5]. The recent release of the Ae. aegypti genome sequence [6] provides an important tool to begin understanding critical virus-vector interactions during the EIP. Identification of mosquito genes that are orthologs of genes known to be part of innate immune pathways in Drosophila [7,8] is an important step in characterizing mosquito defense mechanisms and makes it possible to manipulate putative antiviral pathways during virus infection. Xi et al [8] have recently shown that the Ae. aegypti Toll pathway, which is also implicated in Drosophila defense against certain viruses, has a role in controlling DENV replication after establishment of a persistent infection. Recent studies with Drosophila clearly show that RNA interference (RNAi) is a potent innate antiviral pathway that is presumably triggered by dsRNA formed in virus-infected cells and leads to degradation of the RNA virus genome. Several groups have shown that RNAi can inhibit infection of Drosophila with RNA viruses from the Dicistroviridae, Nodaviridae, and Togaviridae families [911]. Mutant Drosophila lacking functional key RNAi pathway genes such as dcr2 or ago2 are highly susceptible to some RNA virus infections [911]. In Drosophila, dcr2 encodes the RNAi sensor protein Dicer-2 (Dcr2) that recognizes and cleaves long dsRNA, producing 2125 bp short interfering RNAs (siRNAs) [12,13]. siRNAs are duplexes with 39 overhangs of 2 nucleotides and 59 phosphate and 39 hydroxyl ends [14]. With the assistance of Dcr2 and the protein R2D2, one strand of siRNA is incorporated into a nuclease complex called the RNA-induced silencing complex (RISC), to start the effector phase of the pathway [15 18]. The siRNA strand associated with RISC acts as a guide sequence and anneals to target RNA having sequence compleDengue viruses, globally the most prevalent arboviruses, are transmitted to humans by persistently infected Aedes aegypti mosquitoes. Understanding the mechanisms mosquitoes use to modulate infections by these agents of serious human diseases should give us critical insights into virusvector interactions leading to transmission. RNA interference (RNAi) is an innate defense mechanism used by invertebrates to inhibit RNA virus infections; however, little is known about the antiviral role of RNAi in mosquitoes. RNAi is triggered by double-stranded RNA, leading to degradation of RNA with sequence homology to the dsRNA trigger. We show that dengue virus type 2 (DENV2) infection of Ae. aegypti by the natural route generates dsRNA and DENV2-specific small interfering RNAs, hallmarks of the RNAi response; nevertheless, persistent infection of mosquitoes occurs, suggesting that DENV2 circumvents RNAi. We also show that DENV2 infection is modulated by RNAi, since impairment by silencing expression of genes encoding important sensor and effector proteins in the RNAi pathway increases virus replication in the vector and decreases the incubation period before virus transmission. Our findings indicate a significant role for RNAi in determining the mosquito vectors potential for transmitting human diseases. menta (...truncated)