New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

Abstract

Infections by negative strand RNA viruses (NSVs) induce the formation of viral inclusion bodies (IBs) in the host cell that segregate viral as well as cellular proteins to enable efficient viral replication. The induction of those membrane-less viral compartments leads inevitably to structural remodeling of the cellular architecture. Recent studies suggested that viral IBs have properties of biomolecular condensates (or liquid organelles), as have previously been shown for other membrane-less cellular compartments like stress granules or P-bodies. Biomolecular condensates are highly dynamic structures formed by liquid-liquid phase separation (LLPS). Key drivers for LLPS in cells are multivalent protein:protein and protein:RNA interactions leading to specialized areas in the cell that recruit molecules with similar properties, while other non-similar molecules are excluded. These typical features of cellular biomolecular condensates are also a common characteristic in the biogenesis of viral inclusion bodies. Viral IBs are predominantly induced by the expression of the viral nucleoprotein (N, NP) and phosphoprotein (P); both are characterized by a special protein architecture containing multiple disordered regions and RNA-binding domains that contribute to different protein functions. P keeps N soluble after expression to allow a concerted binding of N to the viral RNA. This results in the encapsidation of the viral genome by N, while P acts additionally as a cofactor for the viral polymerase, enabling viral transcription and replication. Here, we will review the formation and function of those viral inclusion bodies upon infection with NSVs with respect to their nature as biomolecular condensates.

Keywords: biomolecular condensates; liquid-liquid phase separation (LLPS); negative strand RNA viruses (NSV); nucleoprotein; phosphoprotein; viral inclusion bodies; viral replication.

Figure 1

Overview on viral replication organelles in cells upon infection with different RNA viruses. Viral replication compartments associated with membranes are depicted in green, membrane-less compartments are indicated in blue. Those with liquid phase properties are depicted as droplets. DMV, double membrane vesicles. ER, endoplasmatic reticulum. IAV, Influenza A virus. MTOC, microtubule organizing center, ERGIC, endoplasmtic-reticulum–Golgi intermediate compartment.

Figure 2

(A) Schematic diagram of a NSV particle. (B) General genome organization of a NSV. (C) Replication cycle of an NSV (based on a filoviral replication cycle). After entry into the cell (1) and release of the nucleocapsid into the cytoplasm (2), primary viral transcription (3) is initiated by the integrated viral polymerase complex. Viral mRNAs are translated by the host translation machinery (4). Synthesized viral proteins support new rounds of viral transcription (5), replication (6) and nucleocapsid assembly (7). Nucleocapsids are transported (8) to the cell periphery where they assemble to virions (9) and bud from the plasma membrane (10).

Figure 3

Summary of inclusion body (IB) formation upon infection with NSV. Small IBs indicate minimal required viral proteins for IB formation, while larger IBs represent mature IBs as biomolecular condensates formed by LLPS. * IB formation by LLPS suspected. Different steps of the viral life cycle taking place in IBs as indicated. In red cellular proteins that localize to IBs. RABV, Rabies virus [106]. VSV, vesicular stomatitis virus [103,107]. RSV, Respiratory syncytial virus [101,108]. MeV, Measles virus [109,110]. NiV, Nipah virus [111]. EBOV, Ebola virus [104,105]. MARV, Marburg virus [112,113]. IAV, Influenza A virus [114,115]. IBAG, IB associated granules. IBperi, perinuclear IB. IBpm, IB plasma membrane. NC, nucleocapsid. HSP70/90, heat shock protein 70/90. FAK, focal adhesion kinase. G3BP, Ras GTPase-activating protein-binding protein 1. TIA1, T-cell restricted intracellular antigen 1. TIAR, TIA1-related protein. PCBP2, Poly(RC) Binding Protein 2. p65, NF-κB subunit p65. p65 MAPK, p38 mitogen-activated protein kinase. OGT, O-linked N-acetylglucosamine transferase. MAVS, mitochondrial antiviral-signaling protein. MDA5, melanoma differentiation-associated protein 5. WDR5, WD repeat protein 5. Tsg101, tumor susceptibility gene 101. IQGAP1, Ras GTPase-activating-like protein 1. NXF1, Nuclear RNA export factor 1. CAD, carbamoyl-phosphate synthetase 2, aspar-tate transcarbamylase, and dihydroorotase. SRPK1, Serine-arginine protein kinase. PP2A-B56, protein phosphatase 2 B56 subunit. eIF4G, Eukaryotic translation initiation factor 4 G. eIF3, Eukaryotic initiation factor 3. PABP, Poly(A)-binding protein. Rab11, Ras-related protein Rab-11.