Spatial and functional arrangement of Ebola virus polymerase inside phase-separated viral factories

Abstract

Ebola virus (EBOV) infection induces formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and from which diverse viral biogenesis arises. Key to viral factory function is recruitment of the EBOV polymerase, a multifunctional machine that mediates replication and expression of the viral RNA genome. Here we show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal dynamics of exchange that likely facilitates viral replication. We report that EBOV viral factories display either droplet-like or network-like morphology, which could be influenced by multivalent intermolecular interactions between viral proteins. Within the viral factory, we found EBOV polymerase is not uniformly distributed, but instead clusters into foci. The distance between these foci increases when viral replication is enabled. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence and regulate viral biogenesis.

Reentrant DNA shells tune polyphosphate condensate size

“…Bleached regions in polyP-Mg 2+ condensates reached just under 80% recovery within 50 minutes in fluorescence recovery a fter p hotobleaching (FRAP) experiments (Fig 1c, Fig S1a). Compared to some other protein-RNA systems which can recover within seconds to a few minutes for a similar size of bleached region 39–41 , polyP recovery in polyP-Mg 2+ condensates is relatively slow.…”

“…For the imaging studies, polyP was labeled with AlexaFluor 647 using a reported procedure 25,38 . Compared to some other protein-RNA systems which can recover within seconds to a few minutes for a similar size of bleached region [39][40][41] , polyP recovery in polyP-Mg 2+ condensates is relatively slow. The absorbance data also reveal reentrant behavior with a reduction in scattering observed for Mg 2+ concentrations above 100 mM.…”

Nucleocapsid condensation drives Ebola viral factory maturation and dispersion

“…Although we occasionally observed loosely coiled NCs interspacing the condensed NC, in the majority of cases the volume between condensed NCs is occupied by material lacking high-order organization. Recently, it was shown that L is not homogeneously distributed within the VF in cells transfected with an EBOV minigenome system lacking VP24 [9]. Since VP35 interacts with L [15], we hypothesize that L could be present in the interspaced volume and stays associated with NCs for incorporation into budding virions.…”

“…6A), indicating that the viscosity of VFs progressively and dramatically increases during VF maturation. For EBOV, polymerase L, VP35 as well as RNA-binding by NP have been shown to influence the fluidity of reconstituted and infection induced viral condensates [8,9]. Since the presence of condensed NCs is a main distinguishing feature between the early and late VFs, we hypothesized that NC condensation is responsible for the observed reduction in fluidity of late VFs.…”

“…Recent work showed that EBOV VFs are bona fide liquid organelles [8][9][10] harnessing viral polymerase activity [7] and maintaining integrity through NP-NP interactions even in the absence of the viral RNA genome [8]. EBOV NP is considered the main driving force for liquid organelle formation and facilitates the recruitment of VP35 [11,12], VP30 [13], VP24 [14] and L [15,16] into the VFs.…”