Quantitative Characterization of Defective Virus Emergence by Deep Sequencing

Timm, Collin M.; Akpinar, Fulya; Yin, John

doi:10.1128/jvi.02675-13

Cited by 29 publications

(28 citation statements)

References 50 publications

(43 reference statements)

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“…These species may possibly reflect truncated genomes lacking the copy-back sequence. Indeed, several kinds of defective replication products have been observed for vesicular stomatitis virus (19,47) and SeV (48,49). The C KO phenotype of MV is dominant: a C-protein-defective virus inhibited the replication efficiency of a C-protein-expressing virus in trans.…”

Section: Discussionmentioning

confidence: 99%

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: 99%

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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“…Infectious virus concentrations were quantified by plaque assay (47). DIPs were produced by fixed-multiplicity serial infections using VSV-rWT-DsRed-Ex at 10 PFU/cell, and the passage with the highest DIP concentration (determined by yield reduction assay [44]) was stored at Ϫ80°C for later infections (30). No RFP expression was observed in cells infected with only DIP and in cells coinfected with DIPs and the Indiana serotype of vesicular stomatitis virus, VSV-N1 (57), reflecting the loss of a functional RFP gene during DIP generation.…”

Section: Methodsmentioning

confidence: 99%

“…industrial-scale production of live-attenuated vaccines, because they readily emerge during live-virus cultures (29)(30)(31).…”

mentioning

confidence: 99%

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

Akpinar

Timm

Yin

2016

J Virol

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T he infection of a cell by a virus produces a mixture of viable and noninfectious progeny particles (1-3). A common class of noninfectious particles has defective genomes, often carrying deletions in essential genes that disable their ability to productively infect cells. However, in coinfections with viable or helper virus, the genomes of these defective particles compete with the viral replication machinery and packaging processes, interfering with infectious virus production in vitro (4, 5), and often reducing virulence in vivo (6, 7). These so-called defective interfering particles (DIPs) have for many decades been observed in laboratory cultures of virtually every class of DNA and RNA virus (4,8). More recently, DIPs have been isolated and characterized from patients infected with influenza virus (9), individuals infected with dengue virus (10, 11), and birds infected with West Nile virus (12). Moreover, sequencing of patient and natural isolates has contributed to a growing list of diverse viral genomes that carry deletions in essential genes or regulatory sequences, including hepatitis C virus (HCV) (13), polyomavirus BK (14), hepatitis B virus (15), human papillomavirus type 16 (16), and baculovirus (17). Notably, for hepatitis C virus, trans-complementation studies showed that defective HCV genomes could be encapsidated and released from cells as infectious virus-like particles (13)

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“…Based on the analysis of six replicate populations, the total-to-infectious particle ratio was found to be 2.9 (Table 2). To explore the potential reasons for high total-to-infectious particle ratio, the presence of defective interfering particles (DIPs) in virus population (Timm et al, 2013) and the limitations of plaque assay were investigated. TEM or interference assay did not detect any DIP in virus samples eliminating the possibility of the contribution of DIPs to total-to-infectious particle ratio in the analyzed virus samples.…”

Section: Resultsmentioning

confidence: 99%

Characterization of vesicular stomatitis virus populations by tunable resistive pulse sensing

Akpinar

Yin

2015

Journal of Virological Methods

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Although transmission electron microscopy (TEM) has historically been the method of choice to estimate concentrations of virus and virus-like particles, these measures can often be time-consuming and labor-intensive to perform. Tunable resistive pulse sensing (TRPS) is an emerging method that applies principles of Coulter counting to nanoscale particles and may provide a simpler and higher-throughput alternative to TEM for the quantitation of virus populations. To assess the performance of TRPS compared to TEM, the samples of polymer spheres at a diameter of 100 nm and vesicular stomatitis virus (VSV) were characterized using both techniques. TRPS was able to quantify concentrations down to 107 particles per ml, providing nearly 50-fold larger measurement range, and more reproducible counts than TEM. Total-to-infectious particle ratio of VSV populations as measured by TRPS and plaque assay suggested that each VSV particle is infectious. In addition to particle counts, TRPS successfully measured particle size distributions based on hundreds of particles. Such high throughput sustained by TRPS can assist quantitative characterization of virus populations.

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Quantitative Characterization of Defective Virus Emergence by Deep Sequencing

Cited by 29 publications

References 50 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

High-Throughput Single-Cell Kinetics of Virus Infections in the Presence of Defective Interfering Particles

Characterization of vesicular stomatitis virus populations by tunable resistive pulse sensing

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