Highly immunostimulatory RNA derived from a Sendai virus defective viral genome

Mercado-López, Xiomara; Cotter, Christopher R.; Kim, Won keun; Sun, Yan; Muñoz, Luis E.; Tapia, Karla; López, Carolina B.

doi:10.1016/j.vaccine.2013.09.040

Cited by 58 publications

(81 citation statements)

References 38 publications

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“…DI-RNAs of negative-strand RNA viruses are potent triggers of innate immune receptors, including PKR, mda-5, and RIG-I (12-14), thereby inducing the expression of type I IFNs in vitro (50) and in vivo (51). The MV DI-RNAs characterized here, if not cotranscriptionally encapsidated, form perfect hairpins throughout the double-stranded structure.…”

Section: Discussionmentioning

confidence: 96%

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

Pfaller

Mastorakos

Matchett

et al. 2015

J Virol

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Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wildtype measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [C KO ]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form doublestrand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The C KO phenotype was dominant: DI-RNAs derived from vac2 with a C KO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and other infectious diseases. The efficacy of MV-based vectors depends on their replication proficiency and immune activation capacity.Here we document that copy-back defective interfering RNAs (DI-RNAs) are generated by recombinant vaccine and wild-type MVs immediately after rescue. The MV C protein interferes with DI-RNA generation and may enhance the processivity of the viral polymerase. We frequently detected clusters of A-to-G or U-to-C transitions and noted that sequences flanking individual mutations contain motifs favoring recognition by the adenosine deaminase acting on RNA-1 (ADAR1). The consistent type of transitions on the DI-RNAs indicates that these are direct substrates for editing by ADAR1. The ADAR1-mediated biased hypermutation events are consistent with the protein kinase R (PKR)-ADAR1 balancing model of innate immunity activation. We show by coinfection that the C-defective phenotype is dominant.

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Section: Discussionmentioning

confidence: 96%

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

Pfaller

Mastorakos

Matchett

et al. 2015

J Virol

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“…While the potent activity of DVGs may be partially explained by a faster accumulation in infected cells (12) due to their strong flanking antigenomic promoters, infection with large amounts of standard virus lacking DVGs does not compensate for their poor immunostimulatory activity (12,14), suggesting that the relative level of PAMPs does not fully account for the more potent recognition of DVGs. Interestingly, treatment of SeV DVGs with phosphatase to eliminate the 5=ppp motif significantly diminishes but does not eliminate their immunostimulatory ability (11,12), suggesting that other RNA motifs play a role in DVG recognition. Using in vitro-transcribed RNA from a highly immunostimulatory SeV DVG, a critical role for secondary structures in the immunostimulatory activity of DVG RNA is beginning to emerge (11,20).…”

Section: Immunostimulatory Molecular Motifs Of Dvgsmentioning

confidence: 99%

“…Interestingly, treatment of SeV DVGs with phosphatase to eliminate the 5=ppp motif significantly diminishes but does not eliminate their immunostimulatory ability (11,12), suggesting that other RNA motifs play a role in DVG recognition. Using in vitro-transcribed RNA from a highly immunostimulatory SeV DVG, a critical role for secondary structures in the immunostimulatory activity of DVG RNA is beginning to emerge (11,20).…”

Section: Immunostimulatory Molecular Motifs Of Dvgsmentioning

confidence: 99%

“…DI particles rendered these cells fully capable of stimulating T cells, thereby promoting the transition from the innate to the adaptive immune response (11)(12)(13)(14). Stocks of SeV lacking DI particles failed to induce a potent response even at a high multiplicity of infection, but potent activation of dendritic cells was restored following supplementation with purified DI particles.…”

Section: Cellular Response To Dvgsmentioning

confidence: 99%

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Defective Viral Genomes: Critical Danger Signals of Viral Infections

López

2014

J Virol

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Viruses efficiently block the host antiviral response in order to replicate and spread before host intervention. The mechanism initiating antiviral immunity during stealth viral replication is unknown, but recent data demonstrate that defective viral genomes generated at peak virus replication are critical for this process in vivo. This article summarizes the supporting evidence and highlights gaps in our understanding of the mechanisms and impact of immunostimulatory defective viral genomes generated during natural infections. BATTLE FOR VIRUS AND HOST COEXISTENCE

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“…The presence of a large excess of genetically defective proviruses has been observed in cells in blood[13,14] and in brain[15] but likely applies to all tissues. Nevertheless, “replication incompetent” viral reservoirs might still contribute to pathogenesis if they were able to support abortive viral expression (either viral RNA or antigen) that could elicit inflammatory or immune responses as encountered with other viral infections[16,17]. We review observations concerning tissue reservoirs by organ system with particular attention to the nature of the infected cell types, evidence for viral compartmentalization if any, and evidence for differences in ARV concentrations.…”

Section: Introductionmentioning

confidence: 99%

Tissue reservoirs of HIV

Wong

Yukl

2016

Current Opinion in HIV and AIDS

195

161

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Purpose Tissue reservoirs of HIV may promote the persistent immunopathology responsible for non-AIDS morbidity and data support multifocal reactivation from tissues as the source of viral rebound during ART interruption. The heterogeneity of tissue reservoirs and incomplete knowledge about their composition are obstacles to an HIV cure. Recent findings In addition to the higher concentration of infected CD4+ T cells found in both central lymphoid tissues and gut, specific subsets of CD4+ T cells appear to play a disproportionate role in HIV persistence. Recently, a subset of central memory T cells enriched in lymphnode germinal centers called T-follicular helper cells have been identified that express more viral RNA and occupy an anatomic niche inaccessible to CTL killing. Additional observations suggest that ARV concentrations may be lower in some tissues raising the possibility for localized, low-level viral replication. Finally, some recent data implicate the persistence of infected, non-CD4+ T cell types in tissues during ART. Summary The retention of infected cells in a wide variety of tissues, often with distinct viral and cellular characteristics, underscores the importance of studying tissue reservoirs in the development and assessment of cure strategies. Both inhibitory ARVs and latency reversing drugs must reach these sites and novel strategies may be needed to attack virus in cells as variable as Tfh and macrophages.

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Highly immunostimulatory RNA derived from a Sendai virus defective viral genome

Cited by 58 publications

References 38 publications

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

Defective Viral Genomes: Critical Danger Signals of Viral Infections

Tissue reservoirs of HIV

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