Influenza A Virus Ribonucleoproteins Form Liquid Organelles at Endoplasmic Reticulum Exit Sites

Abstract

18Influenza A virus has an eight-partite RNA genome that during viral assembly forms a 19 supramolecular complex containing one copy of each RNA. Genome assembly is a selective 20 process driven by RNA-RNA interactions and is thought to lead to discrete punctate structures 21 scattered through the cytosol. Here, we show that contrary to the accepted view, formation of 22 these structures is not dependent on RNA-RNA interactions among distinct viral 23 ribonucleoproteins (vRNPs), as they assemble in cells expressing only one vRNP type. We 24 demonstrate that these viral inclusions display characteristics of liquid organelles, segregating 25 from the cytosol without a delimitating membrane, dynamically exchanging material, deforming 26 easily and adapting fast to hypotonic shock. We provide evidence that they develop close to the 27 Endoplasmic Reticulum Exit Sites (ERES), being dependent on continuous ER-Golgi vesicular 28cycling. We show that viral inclusions do not promote escape to interferon response, and 29 propose that they facilitate selected RNA-RNA interactions in a liquid environment of 30 concentrated vRNPs. 31 32 33 34 3 MAIN TEXT 35 Influenza A infections are serious threats to human health causing annual epidemics and 36 occasional pandemics 1 . The virus contains an eight-partite RNA genome, and each segment is 37 encapsidated as an individual viral ribonucleoprotein (vRNP) complex. vRNPs are composed of 38 single-stranded negative-sense RNA, with base paired terminal sequences originating a double 39 stranded RNA portion where the trimeric RNA-dependent RNA polymerase (RdRp), composed 40 of PB1, PB2 and PA, binds. The remaining sequence attaches several copies of unevenly-41 bound nucleoprotein (NP) 2 . The advantages of having a segmented genome are evident for 42 viral evolution 3 and for better gene expression control 4 , but increase the complexity of the 43 assembly of fully infectious virions 5-8 . 44 Viral assembly occurs at the plasma membrane and, in 80% of the cases, 8 distinct 45 vRNPs are packaged selectively into a budding membrane 9 . Seminal work established the 46 requirement of cis-acting and intersegment RNA-RNA interactions for the formation of this 47 supra-molecular complex (reviewed in 5-8 ). However, it is under debate if vRNPs reach the 48 plasma membrane already as complete genome bundles. 49Upon exiting the nucleus, where they replicate, vRNPs accumulate around the 50 microtubule organizing centre 10 and, subsequently, distribute throughout the cytoplasm 51 concentrating in discrete puncta that enlarge as infection progresses 10-14 . Each puncta 52 accommodates different vRNP segments with the diversity in vRNPs increasing proportionally to 53 the proximity of the plasma membrane 11,13 . Such observation led to the proposal that genome 54 assembly preceded vRNP packaging in budding virions by a process intimately linked with the 55 formation of the referred vRNP hotspots 6,11,[13][14][15] . Studies on the biogenesis of vRNP hotspots 56 showed that their formation requires...

Murine GFP-Mx1 forms phase-separated nuclear condensates and associates with cytoplasmic intermediate filaments: novel antiviral activity against vesicular stomatitis virus

“…We and others reported previously that membraneless phase-separated condensates of GFP-MxA in the cytoplasm, or IL-6-induced cytoplasmic and nuclear GFP-STAT3 condensates, or influenza A virus (FLUAV) maturation structures in the cytoplasm were rapidly disassembled (within 1-3 minutes) in cells swollen following exposure to hypotonic buffer (9,21,27,49). We further showed that a return of such cells to isotonic buffer caused rapid reassembly of MxA but in new structures different from the original ones (9,27,49).…”

“…In parallel with these insights, it has now been recognized that replication and maturation of negative-strand RNA viruses [e.g. vesicular stomatitis (VSV), rabies (as in Negri bodies), Ebola and measles viruses] occurs in cytoplasmic phase-separated condensates typically involving the viral nucleocapsid (N) protein with or without additional viral proteins (15)(16)(17)(18)(19)(20)(21). Even the replication of a DNA virus such as Epstein-Barr virus involves phase-separated nuclear bodies (22).…”

A Comprehensive Review on the Interaction Between the Host GTPase Rab11 and Influenza A Virus

“…In this latter scenario, Rab11-vesicles would be the final transporters of vRNPs to the plasma membrane, conflicting with the lack of Rab11 in virions and at the plasma membrane, but consistent with a modest role for microtubules during infection. Supporting data for an involvement of the ER includes a preprint manuscript showing that viral inclusions form in the vicinity of ER exit sites (ERES) in a way that is dependent on continuous cycles between the ER and the Golgi (Alenquer et al, 2018).…”

“…Additionally, viral inclusions were shown to be established in cells expressing one single vRNP indicating that the formation of these structures precedes (and is not contingent on) the establishment of RNA–RNA interactions. Furthermore, viral inclusions were shown to have liquid properties segregating from the cytosol without a delimiting membrane, being able to exchange material dynamically, and deform easily as well as to adapt fast to changes in the cellular environment (Alenquer et al, 2018). Therefore, given the liquid properties of viral inclusions and although it is possible that vesicles in these structures collide to establish vRNP intersegment interactions, other unidentified processes could also take place.…”

“…Data supporting this model has been listed and explained in Sections “Rab11 and vRNP Transport” and “Rab11 and IAV Genome Assembly” and is illustrated in Figure 4.2.1. In addition, viral inclusions were shown to display liquid properties, forming membraneless organelles, whose assembly might be spatially regulated. In fact, vRNP/Rab11 crowding occurred in vicinity of ERES and was shown to be dependent on continuous vesicular cycling between ER and Golgi (Figure 4.2.2) (Alenquer et al, 2018). The mechanisms mediating viral inclusion formation or/and Rab11 re-routing to the ER remain to be investigated.…”