viral engineering
Recorded terms: alphavirus reverse genetics; bidirectional plasmid reverse genetics; flavivirus infectious cDNA clone and virus rescue; in vitro transcription and electroporation; influenza A virus reverse genetics; influenza reverse genetics; paramyxovirus reverse genetics; recombinant alphavirus engineering; recombinant poxvirus engineering; recombinant Sindbis virus; recombinant viral vectors; recombinant VSV reverse genetics; reverse genetics
Escape of a microRNA-targeted Sendai virus from engineered antiviral RNA interference comes not from the virus but from host ADAR1, whose adenosine to inosine editing destroys the target sites, and human ADAR1 also suppresses endogenous silencing in a plant.
Archaeal members of the L30 protein family, but not their orthologues from any other domain of life, block production of the spliced influenza A virus, influenza B virus and isavirus transcripts without measurably disturbing host splicing, identifying a shared and apparently noncanonical feature of orthomyxovirus splicing.
Uncoupling the two steps of influenza A virus genome replication with viral promoter mutations shows that ANP32A is required for synthesis of both the complementary RNA intermediate and progeny genomic RNA, and that it acts on the actively replicating polymerase rather than the encapsidating one.
Restricting nucleoprotein availability in negative-sense RNA virus infections both blocks full-length genome replication and increases production of aberrant replication products that are sensed through RIG-I and MAVS, so a strong interferon response accompanies rather than follows successful infection.
Frames influenza A virus as a compact genetic circuit whose modules can be disrupted, tagged or replaced, and organizes two decades of virus engineering into a design vocabulary of tracking, override, positioning and silencing modules that turns virus construction into a method of inquiry.
A library of 107 barcoded influenza A viruses differing only in their NS1 sequence, competed in dog cells, human cells, chicken eggs and mice, resolves NS1-driven fitness as a set of divergent and partly convergent evolutionary trajectories rather than a single ordered adaptation gradient.
Applying one uniform small RNA-based selective pressure to four virus families in vertebrate cells shows that the ability to escape it tracks with the capacity for polymerase template switching rather than with genome polarity as such, since positive-strand viruses excise the targeted sequence while negative-strand viruses are cleared and a recombination-defective poliovirus cannot escape.
Recreating a small RNA antiviral response in mice, using either host microRNAs repurposed as virus-specific guides or a virus-encoded artificial small interfering RNA, attenuates influenza A virus by more than five logs and prevents disease without any requirement for type I interferon signaling.
An attenuated influenza A virus engineered to deliver individual artificial small interfering RNAs enables a fitness-based loss-of-function screen inside an infected mouse, and that screen identifies MDA5 as a contributor to the antiviral response to influenza A virus despite the established role of RIG-I as the sensor that induces interferon beta.
Replication-incompetent influenza-based vectors carrying no DNA intermediate deliver coding messages and functional small RNAs together to primary human and mouse cells and to mouse lung, with output and cytotoxicity tuned by microRNA target sites in the nucleoprotein segment.
Genetically barcoded influenza A virus libraries tracked through cell culture, embryonated eggs, guinea pigs, ferrets and mice show that transmission bottlenecks differ by route and recipient, with airborne transmission reducing a diverse inoculum to as few as two founder clones.
A Cre recombinase-expressing influenza A virus combined with Cre-responsive reporter and ablation mouse strains showed that a subpopulation of directly infected lung cells, predominantly club cells, survives productive infection, sustains elevated interferon-stimulated gene and chemokine expression, and contributes to bronchiolar epithelial damage after virus is cleared.
Engineering vesicular stomatitis virus to eliminate RISC-loaded small RNAs attenuates rather than enhances replication in mice, and confers no replication advantage even when interferon signaling is removed, arguing that small RNA silencing does not contribute to mammalian antiviral defense.
Replication-competent Sindbis viruses, each encoding an artificial microRNA against one murine open reading frame, turn viral fitness in infected mice into a selection-based screen for host restriction factors, identifying the transcription factors Zfx and Mga as maintainers of antiviral capacity.
The inefficient 5 prime splice site of influenza A virus segment 8 functions as a timing device, causing the nuclear export protein to accumulate slowly as a minor product of abundant NS1 transcription, with both raising and lowering that rate attenuating the virus through mistimed ribonucleoprotein export.
Engineering target sites for miR-192, a microRNA abundant in human and mouse respiratory tissue but absent from the ferret respiratory tract, into the influenza A virus hemagglutinin segment attenuates the virus in mice while leaving replication and transmission in ferrets intact, providing a genetic layer of biocontainment for transmission studies.
Argues that chordates, unlike plants, nematodes and arthropods, do not use small RNAs as antiviral defence, and that this absence of interplay between host microRNAs and RNA viruses is what leaves the microRNA machinery available for engineering viral tropism and small RNA delivery.
Influenza A virus small viral RNAs are shown to be synthesized from the complementary RNA intermediate, to load into the RNA binding cleft of the polymerase PA subunit, and to act there as segment-specific allosteric enhancers of full-length genome synthesis.
Poxviruses degrade host microRNAs through the catalytic subunit of their own poly(A) polymerase, VP55, which adds short nontemplated adenosine tails to argonaute-loaded guide strands and thereby marks them for cellular decay, while small RNAs carrying a 3 prime terminal 2 prime O-methyl group are spared.
Primary microRNA transcripts generated in the cytoplasm by a recombinant Sindbis virus are cleaved without any nuclear involvement yet still require Drosha, which relocalises from nucleus to cytoplasm on infection while the endogenous microRNA profile of the cell remains largely unchanged.
Influenza A virus engineered to be silenced by the hematopoietic microRNA miR-142 shows that replication inside antigen presenting cells is dispensable for CD8 T cell priming and clearance but required for much of the RIG-I-dependent type I interferon response in vivo.
A negative-sense cytoplasmic RNA virus, vesicular stomatitis virus, can be engineered to produce mature Dicer-dependent miR-124 that loads into Argonaute 2, silences targets, reaches many tissues in mice, and persists after the vector itself is cleared.
Inserting hematopoietic-specific miR-142 target sites into the dengue virus 3-prime untranslated region excludes replication from macrophages and dendritic cells in vivo and abolishes spread to other tissues, identifying hematopoietic cells as the dominant amplification compartment.
Influenza A virus can be engineered to encode a cellular pri-microRNA inside an artificial intron of segment 8 and to produce mature, silencing-competent miR-124 during infection without measurable loss of replication or genome stability.
Influenza A virus produces a family of 22 to 27 nucleotide small viral RNAs corresponding to the 5 prime end of each genomic segment, which accumulate as the polymerase shifts toward genome synthesis and whose inhibition selectively depletes genomic RNA without comparably affecting messenger or complementary RNA.
Insertion of a primary microRNA locus into the exclusively cytoplasmic Sindbis virus genome yields mature, functional miR-124 through a Dicer-dependent but microprocessor- and Exportin-5-independent route, defining a cytoplasmic hairpin-processing activity in vertebrate cells that the authors term a virtron.
Engineering microRNA response elements for a mammalian-ubiquitous microRNA directly into the influenza A virus nucleoprotein coding sequence attenuates the virus in mice while leaving growth in embryonated chicken eggs intact, yielding live attenuated vaccine candidates for H1N1 and H5N1.