Disruption of influenza virus packaging signals
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ABSTRACT:
PROVIDER: PRJEB64123 | ENA |
REPOSITORIES: ENA
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Project description:Influenza A packaging mutants
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Project description:We isolated pgRNA and characterized its protein interactome in cells transfected with packaging competent and packaging incompetent HBV plasmids to identify proteins potentially playing a role in viral packaging. We identified over 250 proteins preferentially associated with pgRNA from the packaging competent version of the virus. These included proteins known to support capsid formation, enhance viral gene expression, catalyze nucleocapsid dephosphorylation, and bind to N6-methylations on the viral genome.
2024-03-10 | MSV000094286 | MassIVE
Project description:Sequential disruption of SPLASH-identified RNA-RNA interactions challenges their role in influenza A virus genome packaging
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Project description:Influenza A virus segment 2 mRNA expresses three polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs 1, 4 and 5 respectively. Two short open reading frames (sORFs) initiated by AUGs 2 and 3 are also present. To understand translational regulation in this system, we systematically mutated AUGs 1-4 and monitored polypeptide synthesis from plasmids and recombinant viruses. This identified sORF2 as a key regulatory element with opposing effects on PB1-F2 and PB1-N40 expression. We propose a model in which AUGs 1-4 are accessed by leaky ribosomal scanning, with sORF2 repressing synthesis of downstream PB1-F2. However, sORF2 also up-regulates PB1-N40 expression, most likely by a reinitiation mechanism that permits skipping of AUG4. Surprisingly, we also found that in contrast to plasmid-driven expression, viruses with improved AUG1 initiation contexts produced less PB1 in infected cells and replicated poorly, producing virions with elevated particle:PFU ratios. Analysis of the genome content of virus particles showed reduced packaging of the mutant segment 2 vRNAs. Overall, we conclude that segment 2 mRNA translation is regulated by a combination of leaky ribosomal scanning and reinitiation, and that the sequences surrounding the PB1 AUG codon are multifunctional, containing overlapping signals for translation initiation and for segment-specific packaging.
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Project description:During mammalian spermiogenesis, the majority of the nucleosomes packaging the male haploid genome are replaced by protamines to produce a highly compact chromatin architecture that is critical to male fertility. We have carried out a genomewide survey of murine spermatozoal chromatin using a micrococal nuclease approach to characterise the DNA sequences that remain packaged by histones.
2009-06-09 | GSE15941 | GEO
Project description:During mammalian spermiogenesis, the majority of the nucleosomes packaging the male haploid genome are replaced by protamines to produce a highly compact chromatin architecture that is critical to male fertility. We have carried out a genomewide survey of human spermatozoal chromatin using both a salt and micrococal nuclease approach to characterise the DNA sequences that remain packaged by histones.
2009-06-09 | GSE12088 | GEO
Project description:The influenza A virus genome is composed of eight single-stranded negative-sense viral RNA segments (vRNAs). The eight vRNAs are selectively packaged into each progeny virion. This process likely involves specific interactions between the vRNAs via segment-specific packaging signals located in both the 3'- and 5'-terminal regions of the respective vRNAs. To assess the importance of vRNA-vRNA interactions via packaging signals for selective genome packaging, we generated mutant viruses possessing silent mutations in the packaging signal region of the hemagglutinin (HA) vRNA. A mutant virus possessing silent mutations in nucleotides (nt) 1664 to 1676 resulted in defects in HA vRNA incorporation and showed a reduction in viral growth. After serial passage, the mutant virus acquired additional mutations in the 5'-terminal packaging signal regions of both the HA and polymerase basic 2 (PB2) vRNAs. These mutations contributed to the recovery of viral growth and HA vRNA packaging efficiency. In addition, an RNA-RNA interaction between the 5' ends of HA and PB2 vRNAs was confirmed in vitro, and this interaction was disrupted following the introduction of silent mutations in the HA vRNA. Thus, our results demonstrated that RNA-RNA interactions between the packaging signal regions of HA vRNA and PB2 vRNA are important for selective genome packaging. IMPORTANCE While numerous viral genomes comprise a single genome segment, the influenza A virus possesses eight segmented genomes. Influenza A virus can benefit from having a segmented genome because the segments can reassort with other strains of the influenza virus to create new genetically distinct strains. The influenza A virus efficiently incorporates one copy of each of its eight genomic segments per viral particle. However, the mechanism by which each segment is specifically selected is poorly understood. The genome segments contain RNA signals that facilitate the incorporation of segments into virus particles. These regions may facilitate specific interactions between the genome segments, creating an eight-segment complex, which can then be packaged into individual particles. In this study, we provide evidence that RNA signals contribute to specific interactions between two of the influenza virus genome segments.
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Project description:Viruses package host RNAs in their virions which are associated with a range of functions in the viral life cycle. Previous transcriptomic profiling on host RNA packaging were mostly focused on retroviruses. Which host RNAs are packaged in other viruses at the transcriptome level has not been thoroughly examined. Here we apply both small RNA and large RNA sequencing of SARS-CoV-2 virions from six individual isolates in Vero cell cultures to profile packaging of host RNAs in a coronavirus and to explore SARS-CoV-2 genomic RNA modifications. We find selective packaging of specific tRNAs, tRNA fragments and signal recognition particle RNA into SARS-CoV-2 virions. Virions from all individual clones package the same set of host RNAs, suggesting a common mechanism of selective packaging. We estimate that a SARS-CoV-2 virion contains up to 1 SRP RNA and 4 tRNA molecules. We identify tRNA modification differences between the virion-packaged tRNAs and those in the uninfected host cells. Furthermore, we find subgenomic viral RNAs in the virions, and uncharacterized candidate modifications in the SARS-CoV-2 genomic RNA. Our results reveal an under-explored aspect of viral-host interaction that may be explored for viral therapeutics.
2022-03-06 | GSE182883 | GEO
Project description:The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5' and 3' packaging sequences of HA(MO) into an otherwise HA(EN) backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.
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