Viral PIC-pocketing: RSV sequestration of translational preinitiation complexes into bi-phasic biomolecular condensates.

Orthopneumoviruses characteristically form membrane-less cytoplasmic inclusion bodies (IBs) wherein RNA replication and transcription occur. Here, we report a strategy whereby the orthopneumoviruses sequester various components of the translational preinitiation complex machinery into viral inclusion bodies to facilitate translation of their own mRNAs-PIC-pocketing. Electron microscopy of respiratory syncytial virus (RSV)-infected cells revealed bi-phasic organization of IBs, specifically, spherical "droplets" nested within the larger inclusion. Using correlative light and electron microscopy, combined with fluorescence in situ hybridization, we showed that the observed bi-phasic morphology represents functional compartmentalization of the inclusion body and that these domains are synonymous with the previously reported inclusion body-associated granules (IBAGs). Detailed analysis demonstrated that IBAGs concentrate nascent viral mRNA, the viral M2-1 protein as well as components of eukaryotic translation initiation factors (eIF), eIF4F and eIF3, and 40S complexes involved in translation initiation. Interestingly, although ribopuromycylation-based imaging indicates that the majority of viral mRNA translation occurs in the cytoplasm, there was some evidence for intra-IBAG translation, consistent with the likely presence of ribosomes in a subset of IBAGs imaged by electron microscopy. Mass spectrometry analysis of sub-cellular fractions from RSV-infected cells identified significant modification of the cellular translation machinery; however, interestingly, ribopuromycylation assays showed no changes to global levels of translation. The mechanistic basis for this pathway was subsequently determined to involve the viral M2-1 protein interacting with eIF4G, likely to facilitate its transport between the cytoplasm and the separate phases of the viral inclusion body. In summary, our data show that these viral organelles function to spatially regulate early steps in viral translation within a highly selective bi-phasic biomolecular condensate. IMPORTANCE: Respiratory syncytial viruses (RSVs) of cows and humans are a significant cause of morbidity and mortality in their respective populations. These RNA viruses replicate in the infected cells by compartmentalizing the cell's cytoplasm into distinct viral microdomains called inclusion bodies (IBs). In this paper, we show that these IBs are further compartmentalized into smaller structures that have significantly different density, as observed by electron microscopy. Within smaller intra-IB structures, we observed ribosomal components and evidence for active translation. These findings highlight that RSV may additionally compartmentalize translation to favor its own replication in the cell. These data contribute to our understanding of how RNA viruses hijack the cell to favor replication of their own genomes and may provide new targets for antiviral therapeutics in vivo.

Detection of pyrethroid resistance mutations and intron variants in the voltage-gated sodium channel of Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus mosquitoes from Lao People's Democratic Republic.

In Lao People's Democratic Republic, Aedes aegypti (Linnaeus 1762) and Aedes albopictus (Skuse 1894) mosquitoes (Diptera: Culicidae) are vectors of arboviral diseases such as dengue. As the treatment for these diseases is limited, control of the vectors with the use of pyrethroid insecticides is still essential. However, mutations in the voltage-gated sodium channel (vgsc) gene giving rise to pyrethroid resistance are threatening vector control programs. Here, we analysed both Ae. aegypti and Ae. albopictus mosquitoes, which were collected in different districts of Laos (Kaysone Phomvihane, Vangvieng, Saysettha and Xaythany), for vgsc mutations commonly found throughout Asia (S989P, V1016G and F1534C). Sequences of the vgsc gene showed that the F1534C mutation was prevalent in both Aedes species. S989P and V1016G mutations were detected in Ae. aegypti from each site and were always found together. In addition, the mutation T1520I was seen in Ae. albopictus mosquitoes from Saysettha district as well as in all Ae. aegypti samples. Thus, mutations in the vgsc gene of Ae. aegypti are prevalent in the four districts studied indicating growing insecticide resistance throughout Laos. Constant monitoring programmes and alternative strategies for controlling Aedes should be utilized in order to prolong the effectiveness of pyrethroids thereby maximizing vector control.