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Assembly and transport of filovirus nucleocapsids [1]

['Olga Dolnik', 'Institute Of Virology', 'Philipps-University Marburg', 'Marburg', 'Stephan Becker']

Date: 2022-11 

Abstract Filovirus-infected cells are characterized by typical cytoplasmic inclusion bodies (IBs) located in the perinuclear region. The formation of these IBs is induced mainly by the accumulation of the filoviral nucleoprotein NP, which recruits the other nucleocapsid proteins, the polymerase co-factor VP35, the polymerase L, the transcription factor VP30 and VP24 via direct or indirect protein–protein interactions. Replication of the negative-strand RNA genomes by the viral polymerase L and VP35 occurs in the IBs, resulting in the synthesis of positive-strand genomes, which are encapsidated by NP, thus forming ribonucleoprotein complexes (antigenomic RNPs). These newly formed antigenomic RNPs in turn serve as templates for the synthesis of negative-strand RNA genomes that are also encapsidated by NP (genomic RNPs). Still in the IBs, genomic RNPs mature into tightly packed transport-competent nucleocapsids (NCs) by the recruitment of the viral protein VP24. NCs are tightly coiled left-handed helices whose structure is mainly determined by the multimerization of NP at its N-terminus, and these helices form the inner layer of the NCs. The RNA genome is fixed by 2 lobes of the NP N-terminus and is thus guided by individual NP molecules along the turns of the helix. Direct interaction of the NP C-terminus with the VP35 and VP24 molecules forms the outer layer of the NCs. Once formed, NCs that are located at the border of the IBs recruit actin polymerization machinery to one of their ends to drive their transport to budding sites for their envelopment and final release. Here, we review the current knowledge on the structure, assembly, and transport of filovirus NCs.

Citation: Dolnik O, Becker S (2022) Assembly and transport of filovirus nucleocapsids. PLoS Pathog 18(7): e1010616. https://doi.org/10.1371/journal.ppat.1010616 Editor: Rachel Fearns, Boston University, UNITED STATES Published: July 28, 2022 Copyright: © 2022 Dolnik, Becker. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by the Deutsche Forschungsgemeinschaft (DFG) through the Sonderforschungsbereich SFB 1021 project A05 to SB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

1. Introduction Ebolavirus (EBOV) and Marburgvirus (MARV) represent 2 genera of the family Filoviridae, which includes important zoonotic pathogens. EBOV and MARV cause fatal outbreaks in humans with case fatality rates of up to 90% [1]. Because of their nonsegmented single-stranded RNA genome that has negative polarity, filoviruses belong to the order Mononegavirales. Filovirus particles have a characteristic filamentous shape that is approximately 1 μm in length and a diameter of approximately 90 nm. The viral genome is encapsidated by the nucleoprotein NP and is associated with 4 additional viral proteins (VP): VP35 (the analog of the P protein in other mononegaviruses), the polymerase L, the transcription factor VP30 and VP24, forming the nucleocapsid (NC). The matrix protein VP40 surrounds the NC, forming a regular layer beneath the viral envelope. The glycoprotein GP is incorporated into the viral envelope [2]. The entry of viral particles into cells is initiated by the recognition of target cells by GP, and entry is mediated by macropinocytosis (Fig 1) [3,4]. During endocytosis, macropinocytic vesicles mature into late endosomes/lysosomes, where GP is cleaved by cellular proteases. This process exposes the receptor-binding domain of GP to the endosomal receptor NPC1, and receptor binding initiates fusion of the viral and lysosomal membranes, which results in the release of NCs into the cytosol [5]. Here, the NC-associated polymerase complex, which consists of L, the polymerase cofactor VP35, and the transcription initiation factor VP30, initiates the primary transcription of viral mRNAs. Except for GP, which is translated at ER-bound ribosomes and transported through the classical secretory pathway to the plasma membrane, all the other viral proteins are translated at free ribosomes in the cytosol. Increasing amounts of viral proteins in the cytosol lead to the formation of inclusion bodies (IBs), which are a hallmark of filovirus infection [6,7]. IBs represent sites of secondary transcription, genome replication, and de novo NC formation of transport-competent NCs that are transported to budding sites at the plasma membrane for envelopment (Fig 1) [8–12]. In the literature, the term NC is often used equivalently to the term ribonucleoprotein complex (RNP). In this review, we consider RNPs as functional complexes composed of viral RNA, NP, VP35, L, and VP30 that are active in transcription or replication [13]. Therefore, RNPs can have different protein compositions consisting of the viral RNA, NP, VP35, and L with or without the transcription initiation factor VP30. In comparison, NCs are defined as discrete condensed structures that are also detected inside the viral particles composed of the genomic RNA, NP, VP35, L, VP30, and VP24 (Fig 1). The complete replication cycle of filoviruses was reviewed by Kolesnikova and colleagues [14]. Here, we review the current knowledge on the structure, assembly, and transport of filovirus NCs from their assembly site in IBs to the budding sites at the plasma membrane, where they are packaged into filamentous infectious virions. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Filovirus replication cycle. Viral entry into target cells is initiated after attachment to the plasma membrane and is accomplished by macropinocytosis (1). During maturation of macropinocytic vesicles into LE/Lys, the viral envelope fuses with the LE/Lys membrane, and the NC is released into the cytosol (2), where it serves as a template for the primary transcription of capped viral mRNAs (3). Translation of viral proteins (4) occurs at free ribosomes, with the exception of GP, which is translated at the rough endoplasmatic reticulum (ER/RER). GP is transported through the classical secretory pathway via the Golgi apparatus to the plasma membrane. The matrix protein VP40 associates with membrane vesicles and is transported to the plasma membrane to form the viral envelope together with GP and cellular lipids (10). Expression of NC proteins (NP, VP35, VP30, VP24, and L) leads to the formation of IBs (5) where secondary transcription (6), genome replication (7), and NC assembly are organized (8). NCs are transported directionally from IBs to budding sites through actin polymerization-driven transport mediated by actin tail formation at one end (9). Envelopment of NCs by membranes containing VP40 and GP occurs at the plasma membrane where budding of filoviruses take place (11). Figure was created with BioRender.com. IB, inclusion body; LE/Lys, late endosomes/lysosomes; RER, rough endoplasmatic reticulum; RNP, ribonucleoprotein complex. https://doi.org/10.1371/journal.ppat.1010616.g001

4. Transport of nucleocapsids from inclusion bodies to budding sites The ectopic expression of fluorescently labeled VP30 (VP30-GFP) in EBOV- or MARV-infected cells results in fluorescently labeled NCs, whose movement can be monitored by live-cell imaging. Using this approach, the ejection of single NCs from IBs could be observed, and their intracellular transport to the plasma membrane could be tracked and characterized in living cells [9,10]. Since the long filovirus NC cannot reach the budding site by diffusion alone, interactions with host cell factors are required to mediate its transport. The application of cytoskeleton-depolymerizing drugs revealed that the transport of filovirus NCs is driven by an actin-dependent mechanism [9,10,79,80]. Other intracellular pathogens, such as Listeria monocytogenes or baculovirus, express proteins that directly interact with actin and the actin polymerization complex Arp2/3 to promote the polymerization of branched actin filaments [81]. Such nucleation-promoting factors are required for the activation of the Arp2/3 complex to mediate actin polymerization [81,82]. Direct interactions between filovirus NC proteins and actin or the Arp2/3 complex have not yet been described. To promote actin polymerization for their transport, filovirus NCs may therefore recruit cellular nucleation-promoting factors, which provide a link to actin and the Arp2/3 complex (Fig 4). PPT PowerPoint slide

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TIFF original image Download: Fig 4. Transport of nucleocapsids from inclusion bodies to budding sites. Transport-competent NCs composed of all the NC proteins are formed inside IBs. The involvement of different host cell factors is required for the actin-dependent transport of NCs (gray box). Outside the IBs, actin tails are formed at one end of the NC in the cytosol, which drive their transport. After reaching the plasma membrane, budding of filoviruses occurs mainly at filopodia, in which myosin 10 may support the transport of NCs along parallel actin filaments. Figure was created with BioRender.com. IB, inclusion body; NC, nucleocapsid. https://doi.org/10.1371/journal.ppat.1010616.g004 Using specific inhibitors and silencing host cell factor expression by siRNA, it was demonstrated that the Arp2/3 complex, WAVE1 and Rac1 play important roles in the actin-dependent transport of NCLS and authentic viral NCs [9,10,83]. Moving filoviral NCLS and NCs display actin comet tails at one end, suggesting a transport mechanism previously described for the intracellular transport of vaccinia virus and baculovirus capsids that is based on the polymerization of branched actin filaments [81,84]. The NP of MARV was shown to recruit cellular factors into the IBs, such as Tsg101 and IQGAP1, a regulator of actin dynamics and filopodia formation, which is also involved in regulating the transport of NCs. Down-regulation of Tsg101 or IQGAP1 expression affected the transport of NCs, resulting in their accumulation close to the plasma membrane and reduced budding at filopodia suggesting different transport steps of NCs from the IBs at first to the planar plasma membrane and then further into filopodia [85]. Filopodia that are enriched with VP40 are the preferred budding sites of filoviruses [9,10,85,86]. This is consistent with data showing that EBOV VP40 strongly enhances the recruitment of NCLS into filopodia [79]. Filopodia are long, thin cellular protrusions containing characteristic parallel actin filaments that are cross-linked by fascin [87]. Unconventional myosin 10 mediates the transport of cargo along actin filaments within filopodia. Myosin 10 was shown to transport VP40, and it is hypothesized that NCs make use of this mechanism for transport inside filopodia (Fig 4) [9,10,86,88]. The exact molecular interactions between actin polymerization complexes that drive the actin-dependent transport of the NC and the viral NC proteins remain to be determined. Using a minimalistic live-cell imaging system based on fluorophore-tagged NC proteins, it was shown that NP, VP35, and VP24 are essential and sufficient for forming electron-dense and transport-competent helical NCLS [16,18–20,36,71,79,80]. Mutation of the YxxL motif of VP24 in EBOV resulted in NCLS that exhibited significantly impaired transport by an unknown mechanism [89]. Taken together, the current literature demonstrates that both NP and VP24 seem to regulate the actin-dependent transport of NCLS via different mechanisms. Further experiments with the minimalistic live-cell imaging system and application of advanced NC-tracking software are needed to define the molecular mechanism underlying the formation of the actin tail at one end of NCLS that drives their transport. Different transport kinetics were described for filovirus NCs depending on their intracellular location. The velocities range from 100 nm/s in the filopodia to 400 nm/s in the cytosol, and these differences suggest the involvement of different actin-based transport machineries during transport from IBs to budding sites [9,10,79,80]. The molecular details of the observed differences in the transport velocities of NCs are still unclear. In addition to actin tail-driven transport, NCs were observed to move along actin filaments, suggesting motor protein-dependent and motor protein-independent transport mechanisms [10]. For measles virus, the transport of RNPs from IBs to the plasma membrane is dependent on actin filaments, whereas their incorporation into new virions requires actin dynamics at the cell periphery [90]. Transport kinetics and cytoskeleton inhibitor studies of NCs of other mononegaviruses, such as vesicular stomatitis virus, revealed the involvement of microtubule- and actin-dependent transport mechanisms [91]. Most likely, microtubule-dependent transport does not play a major role in the transport of filovirus NC [9,10,79,80]. The formation of an actin tail at one end of the filovirus NC resulted in directional transport and budding of the particles with the pointed end of the NC in front. It is unknown how the binding of the actin polymerization machinery to one end of the NC is regulated. For example, it was shown that positioning the baculoviral proteins p78/83 or VP80 at one end of the NC influenced the directionality of transport [92–94]. Thus, the structural polarity of the filovirus NC may be the result of the recruitment of the cellular actin polymerization machinery to the barbed end, since budding was shown to occur with the pointed end first (Fig 4) [28,40]. It is tempting to hypothesize that the position of the viral polymerase complex in the NC at the 3′ end of the genome, where it can initiate primary transcription as shown for other mononegaviruses, may influence the recruitment of transport machinery [95,96]. For vesicular stomatitis virus, however, super-resolution imaging revealed that the viral polymerase is located at the blunt end of the bullet-shaped particles where the 5′ end of the genome is located [97]. The viral determinants responsible for recruitment of the actin polymerization machinery at one end of the NC remain to be identified using super-resolution techniques and live-cell imaging. Interactions of the matrix protein VP40 with NP in the IBs inhibit viral transcription and replication possibly by partial NC condensation and on the other hand enable NC envelopment at the plasma membrane and budding [27,38,73,98,99]. Live-cell imaging studies of MARV-infected cells revealed that outside of IBs association of VP40 with NCs was detected only at the plasma membrane, arguing against a contribution of VP40 to the transport of NCs from IBs to the plasma membrane [10]. The incorporation of NCLSs into infectious virus-like particles (also referred to as transcription and replication competent virus-like particles, trVLPs) at the plasma membrane was dependent on tyrosine phosphorylation of VP40, which occurs at cellular membranes, indicating that docking of NCLS to the plasma membrane and envelopment are regulated by the posttranslational modification of VP40 [98].

5. Conclusions and perspectives Current data suggest that NP is a highly dynamic protein that organizes protein–protein and protein–RNA interactions to promote RNP and NC formation. The resulting helical structure with boomerang-shaped protrusions has a pointed and a barbed end, generating structural asymmetry. The complete 3D structure showing the location of all NC proteins remains to be solved, which will require high-resolution Cryo-EM and Cryo-ET studies and structure reconstruction methods based on authentic viruses and trVLPs. The assembly of NCs occurs in NP-induced IBs, which are likely liquid-like organelles. Posttranslational modifications of viral NC proteins, such as phosphorylation and interactions with host cell factors, seem to regulate their formation. Techniques used in liquid organelle biology have to be applied to investigate the liquid–liquid and liquid–solid phase transitions in IBs formation and to further dissect the role of the different viral and cellular factors involved. NP, VP35, and VP24 are sufficient and essential for forming mature and transport-competent NCs, which require the recruitment of actin polymerization factors for transport from the IBs to budding sites. RNA labeling techniques, multicolor live-cell imaging, and super-resolution imaging and techniques such as correlative light electron microscopy can help to identify the molecular determinants of the viral and host factors that are involved in the formation and transport of NCs.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010616

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