The induction of IFN-beta by the paramyxovirus PIV5 (formerly known as SV5) is limited by the action of the viral V protein that targets the cellular RNA helicase mda-5. Here we show that 12 other paramyxoviruses also target mda-5 by a direct interaction between the conserved cysteine-rich C-terminus of their V proteins and the helicase domain of mda-5. The inhibition of IFN-beta induction is not species-restricted, being observed in a range of mammalian cells as well as in avian cells, and we show that the inhibition of mda-5 function is also not restricted to mammalian cells. In contrast, the V proteins do not bind to the related RNA helicase RIG-I and do not inhibit its activity. The relative contributions of mda-5 and RIG-I to IFN-beta induction are discussed.
Influenza A viruses (IAV) are subject to species barriers that prevent frequent zoonotic transmission and pandemics. One of these barriers is the poor activity of avian IAV polymerases in human cells. Differences between avian and mammalian ANP32 proteins underlie this host range barrier. Human ANP32A and ANP32B homologues both support function of human-adapted influenza polymerase but do not support efficient activity of avian IAV polymerase which requires avian ANP32A. We show here that the gene currently designated as avian ANP32B is evolutionarily distinct from mammalian ANP32B, and that chicken ANP32B does not support IAV polymerase activity even of human-adapted viruses. Consequently, IAV relies solely on chicken ANP32A to support its replication in chicken cells. Amino acids 129I and 130N, accounted for the inactivity of chicken ANP32B. Transfer of these residues to chicken ANP32A abolished support of IAV polymerase. Understanding ANP32 function will help develop antiviral strategies and aid the design of influenza virus resilient genome edited chickens.
It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system.
c Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-V⌬C (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus. The interferon (IFN) response is extremely potent at restricting virus replication and spread prior to activation of the adaptive immune system. IFN-␣ and - are synthesized and secreted from cells in response to virus infection, and this leads to the establishment of an antiviral state in the infected cell and neighboring uninfected cells through the upregulation of hundreds of IFNstimulated genes (ISGs) that together function to make the cell a hostile environment for virus replication. Triggering of the IFN- promoter during infection with paramyxoviruses and other negative-sense viruses occurs through activation of cytosolic pattern recognition receptors (PRRs). The best characterized of these are RIG-I and mda-5, which become activated upon binding of viral pathogen-associated molecular patterns (PAMPs). The viral PAMPs that activate RIG-I and mda-5 are viral RNA molecules; RIG-I is thought to recognize primarily short double-stranded RNAs (dsRNAs) with an uncapped triphosphate moiety, while mda-5 is activated by longer dsRNAs (1-6). Following their activation by PAMP binding, RIG-I and mda-5 elicit a downstream signaling cascade that culminates in the nuclear translocation of the IFN regulatory factor 3 (IRF3) and NF-B transcription factors and subsequent transcription from the IFN- gene. In order to circumvent the powerful IFN response, most viruses have evolved mechanisms to evade it, by encoding viral factors that inhibit IFN induction, the ability of IFN to upregulate ISGs, or the function of certain ISG products (reviewed in reference 7).Parainfluenza virus 5 (PIV5; formerly simian virus 5 [SV5]) is a prototype member of the genus Rubulavirus in the Paramyxoviridae family. The ϳ15-kb negative-sense genomic RNA encodes eight gene products from its seven genes and carries noncoding leader (Le) and trailer (Tr...
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