Latent Infection of a New Alphanodavirus in an Insect Cell Line

Li, Tiancheng; Scotti, Paul D.; Miyamura, Tatsuo; Takeda, Naokazu

doi:10.1128/jvi.00807-07

Cited by 71 publications

(83 citation statements)

References 32 publications

(35 reference statements)

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“…We thus suspect that our Sf21 cell line has a low-level persistent infection with FHV, as has been demonstrated for other cell lines (26). We also built contigs from the NωVVLP-RNAseq reads that bore weak protein homology to other Rhabdoviridae and so may reflect a low-level infection with an unknown insect virus (Fig.…”

Section: Resultsmentioning

confidence: 99%

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Routh

Domitrovic

Johnson

2012

Proc. Natl. Acad. Sci. U.S.A.

106

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Next-generation sequencing is a valuable tool in our growing understanding of the genetic diversity of viral populations. Using this technology, we have investigated the RNA content of a purified nonenveloped single-stranded RNA virus, flock house virus (FHV). We have also investigated the RNA content of virus-like particles (VLPs) of FHV and the related Nudaurelia capensis omega virus. VLPs predominantly package ribosomal RNA and transcripts of their baculoviral expression vectors. In addition, we find that 5.3% of the packaged RNAs are transposable elements derived from the Sf21 genome. This observation may be important when considering the therapeutic use of VLPs. We find that authentic FHV virions also package a variety of host RNAs, accounting for 1% of the packaged nucleic acid. Significant quantities of host messenger RNAs, ribosomal RNA, noncoding RNAs, and transposable elements are readily detected. The packaging of these host RNAs elicits the possibility of horizontal gene transfer between eukaryotic hosts that share a viral pathogen. We conclude that the genetic content of nonenveloped RNA viruses is variable, not just by genome mutation, but also in the diversity of RNA transcripts that are packaged.deep-sequencing | virus evolution | virus assembly N ext-generation sequencing (NGS) is a powerful tool in the investigation of viral diversity from many different perspectives. NGS has been applied in directed approaches to investigate the prevalence of polymorphism or quasispecies present in a population of known viruses within a host, e.g., foot-and-mouth disease virus (1) or to follow the evolution of a viral genome and the frequency of known polymorphisms during the course of an infection, e.g., human rhinovirus (2) and HIV (3). The total genetic content of biological or clinical samples have been sequenced to uncover viruses present even at very low titers, for example, the presence of porcine circovirus-1 in Rotarix samples (4) and the identification of influenza virus subtypes and norovirus from nasopharyngeal and fecal samples collected during outbreaks (5).NGS has also been applied in unbiased approaches aimed at the discovery of new viral species without any advance genetic information. For example, the Merkel cell polyomavirus was discovered to be the causative agent of its eponymous carcinoma by deep sequencing the transcriptome of tumor cells and analyzing nonhost transcripts (6). Similarly, it has been possible to uncover the history of viral pathogens that have plagued an organism through the de novo assembly of the sequenced siRNAs that would have been generated to tackle viral infections. This approach has revealed the viral genomes of familiar miscreants as well as of new viruses, for example, in fruit flies, mosquitoes, and nematode worms (7).In this report, we employ NGS to investigate the total RNA encapsidated by authentic flock house virus (FHV) virions and by virus-like particles (VLPs) of FHV and of the related tetravirus, Nudaurelia capensis omega virus (NωV). Our analysis emplo...

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Section: Resultsmentioning

confidence: 99%

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Routh

Domitrovic

Johnson

2012

Proc. Natl. Acad. Sci. U.S.A.

106

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“…We previously found that relatively large numbers of overlapping baculovirus transcripts occur in the AcMNPV infection cycle (33), potentially resulting in dsRNA and a RNAi response. In addition, it was also previously observed that an alphanodavirus (TNCL virus) that persistently infects Tn5B1-4 cells appears to be released from suppression upon infection of the cell by AcMNPV (78). Previous studies have also documented viral siRNAs directed against specific hotspots in the Helicoverpa armigera SNPV (HaSNPV) genome (77).…”

Section: Assembly and Annotation Of The T Ni Tnms42 Transcriptomementioning

confidence: 95%

Transcriptome Responses of the Host Trichoplusia ni to Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus

Chen

Zhong

Fei

et al. 2014

J Virol

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Productive infection of Trichoplusia ni cells by the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) leads to expression of ϳ156 viral genes and results in dramatic cell remodeling. How the cell transcriptome responds to viral infection was unknown due to the lack of a reference genome and transcriptome for T. ni. We used an ϳ60-Gb RNA sequencing (RNA-seq) data set from infected and uninfected T. ni cells to generate and annotate a de novo transcriptome assembly of approximately 70,322 T. ni unigenes (assembled transcripts), representing the 48-h infection cycle. Using differential gene expression analysis, we found that the majority of host transcripts were downregulated after 6 h postinfection (p.i.) and throughout the remainder of the infection. In contrast, 5.7% (4,028) of the T. ni unigenes were upregulated during the early period (0 to 6 h p.i.), followed by a decrease through the remainder of the infection cycle. Also, a small subset of genes related to metabolism and stress response showed a significant elevation of transcript levels at 18 and 24 h p.i. but a decrease thereafter. We also examined the responses of genes belonging to a number of specific pathways of interest, including stress responses, apoptosis, immunity, and protein trafficking. We identified specific pathway members that were upregulated during the early phase of the infection. Combined with the parallel analysis of AcMNPV expression, these results provide both a broad and a detailed view of how baculovirus infection impacts the host cell transcriptome to evade cellular defensive responses, to modify cellular biosynthetic pathways, and to remodel cell structure. IMPORTANCEBaculoviruses are insect-specific DNA viruses that are highly pathogenic to their insect hosts. In addition to their use for biological control of certain insects, baculoviruses also serve as viral vectors for numerous biotechnological applications, such as mammalian cell transduction and protein expression for vaccine production. While there is considerable information regarding viral gene expression in infected cells, little is known regarding responses of the host cell to baculovirus infection. In these studies, we assembled a cell transcriptome from the host Trichoplusia ni and used that transcriptome to analyze changes in host cell gene expression throughout the infection cycle. The study was performed in parallel with a prior study of changes in viral gene expression. Combined, these studies provide an unprecedented new level of detail and an overview of events in the infection cycle, and they will stimulate new experimental approaches to understand, modify, and utilize baculoviruses for a variety of applications.

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“…For calibration of the transfection efficiency, pEA-polyhedrin-MycHis was cotransfected with an expression vector that encodes the capsid protein of Flock House virus (FHV) fused to a C-terminal MycHis tag (pEA-FHV-CP-MycHis). The capsid protein of FHV was amplified from cDNA of persistently infected Hi5 cells (27) using 5=-AATTGGATCCCAACATGGTCAACAAC AGCAGACCAAAAC-3= (forward) and 5=-AATTGGATCCGTCTAAAA TCCAAAACCTTCAAATAG-3= (reverse) as primers, and the PCR fragment was subcloned into the BamHI site of the pEA-MycHis vector as described for BmCPV polyhedrin.…”

Section: Methodsmentioning

confidence: 99%

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

Zografidis

Nieuwerburgh

Κολλιοπούλου

et al. 2015

J Virol

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The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. Central to the defense of insects against RNA viruses is the triggering of the so-called exogenous RNA interference pathway (exo-RNAi), by which viral double-stranded RNA (dsRNA) is cleaved intracellularly into viral small interfering RNAs (siRNAs) that in turn silence homologous transcripts (1). It is established that Dicer-2, a cytoplasmic RNase III class enzyme, recognizes and cuts successively the dsRNA template to produce small interfering RNA duplexes of ϳ21 nucleotides (nt) characterized by a signature of 2-nt 3=-hydroxyl overhangs (2). These duplexes are subsequently incorporated into the effector complex RISC (RNA-induced silencing complex) via interactions with Argonaute-2, which, upon discarding one of the two strands (passenger strand), is followed by selection and cleavage of viral sequences bearing perfect complementarity to the remaining strand (guide strand) (1, 3). Dicer-2 may act upon the genome of the virus itself-as in the case of dsRNA viruses-and/or on viral replication and transcription intermediates, although structured, single-stranded RNA (ssRNA) may also be utilized (4). Interestingly, several deepsequencing data have revealed that approximately 18-to 30-nt viral small RNAs (vsRNAs) of infected insects are not evenly distributed along the genomes but map preferentially in distinct genomic areas (hot spots) versus genomic stretches with unmapped or less-mapped vsRNAs (cold spots) (4-13). The origin of these profiles, as well as the hypothetical functional distinction between vsRNAs deriving from hot or cold spots, remains substantially elusive, although hot spots near the end of the genome have been attributed to structured regulatory viral regions (14) and an RNAi decoy-like mechanism driven by abundant vsRNAs ...

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Latent Infection of a New Alphanodavirus in an Insect Cell Line

Cited by 71 publications

References 32 publications

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Host RNAs, including transposons, are encapsidated by a eukaryotic single-stranded RNA virus

Transcriptome Responses of the Host Trichoplusia ni to Infection by the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus

Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection

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