functional genomics and screening
Recorded terms: alanine scanning mutagenesis; site-directed mutagenesis; site-directed promoter mutagenesis; site-directed splice site mutagenesis
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.
Kinase substrate specificity profiling assigns the phosphorylation cluster in the SARS-CoV-2 nucleocapsid SR-rich domain to a cascade initiated by SRPK1 and SRPK2 and extended by GSK-3 and casein kinase 1, whose inhibition suppresses coronavirus replication.
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.
Isolating the SARS-CoV-2 Spike D614G substitution from the ORF1b P314L variant it travels with, through pseudotyped lentiviral particles and a trans-complementation assay with replication-competent virus, shows that the single Spike change raises entry efficiency across human lung, liver and colon cell lines without altering S1 affinity for ACE2.
Sustained IRF7 activity induces the kinase MAP3K8, which phosphorylates the proline-rich hinge of IRF3 and redirects it from homodimers into IRF3 and IRF7 heterodimers, broadening the antiviral transcriptome and scaling the cellular response to the persistence of the viral threat.
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.
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.
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.
Mice lacking IKKε produce normal interferon-β but fail to induce roughly a third of interferon-stimulated genes, because interferon activates IKKε, which phosphorylates STAT1 at Ser708 and thereby determines whether ISGF3 occupies a subset of response elements.