Deep Sequencing Analysis of Defective Genomes of Parainfluenza Virus 5 and Their Role in Interferon Induction

Abstract

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...

Measles Virus C Protein Impairs Production of Defective Copyback Double-Stranded Viral RNA and Activation of Protein Kinase R

“…However, after further passage of the inoculum, we detected copyback DIRNAs, which are known to efficiently trigger innate immune responses: in the case of vesicular stomatitis virus (VSV) DI-011, just a single particle is sufficient to induce a quantum yield of interferon (54,55). DI-RNAs have been observed not only with VSV, SENV, and parainfluenza virus 5 (PIV5) (54,56,57) but also for MV (36,45), where a vaccine strain-derived inoculum able to express the C protein produced high quantities of DI-RNAs after high MOI passage in cell culture. Herein, we show that a single round of C KO passage on Vero cells led to amplification of the DI-RNA.…”

Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

“…Next, the growth of rOROV, rOROVdelNSm, and rOROVdelNSs in A549 cells was compared to their growth in IFN-incompetent A549/V cells. These cells express the V protein of parainfluenza type 5 virus, thereby blocking type I IFN signaling via STAT1 degradation (39). Cells were infected at an MOI of 0.001, and titers were measured at 48 h p.i.…”

Generation of Mutant Uukuniemi Viruses Lacking the Nonstructural Protein NSs by Reverse Genetics Indicates that NSs Is a Weak Interferon Antagonist

“…We investigated the growth of the viruses in an IFN-competent cell line, A549 (a human lung cell line), and a derivative cell line, A549/ PIV5-V (A549/V), which expresses the V protein of parainfluenza virus type 5 to block IFN signaling (31). Both wtUUKV and rUUKV grew to titers of ϳ10 6 FFU/ml by 72 h p.i.…”