Influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and Rab11-dependent vesicles.

Transport of neo-synthesized influenza A virus (IAV) viral ribonucleoproteins (vRNPs) from the nucleus to the plasma membrane involves Rab 11 but the precise mechanism remains poorly understood. We used metal-tagging and immunolabeling to visualize viral proteins and cellular endomembrane markers by electron microscopy of IAV-infected cells. Unexpectedly, we provide evidence that the vRNP components and the Rab11 protein are present at the membrane of a modified, tubulated endoplasmic reticulum (ER) that extends all throughout the cell, and on irregularly coated vesicles (ICVs). Some ICVs are found very close to the ER and to the plasma membrane. ICV formation is observed only in infected cells and requires an active Rab11 GTPase. Against the currently accepted model in which vRNPs are carried onto Rab11-positive recycling endosomes across the cytoplasm, our findings reveal that the endomembrane organelle that is primarily involved in the transport of vRNPs is the ER.

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.

Screening a yeast library of temperature-sensitive mutants reveals a role for actin in tombusvirus RNA recombination.

Genetic recombination in RNA viruses drives the evolutionary arms race with host's antiviral strategies and recombination also facilitates adaptation of viruses to new hosts. In this paper, the authors used tombusvirus and a temperature-sensitive (ts) mutant library of yeast to identify 40 host proteins affecting viral recombination in yeast model host. Subsequent detailed analysis with two identified actin-related proteins, Act1p and Arp3p, has revealed that the wt actin network helps TBSV to maintain low level viral recombination. Pharmacological inhibition of actin in plant protoplasts confirmed the role of the actin network in tombusvirus recombination. An in vitro approach revealed the altered activity of the tombusvirus replicase in the presence of mutated Act1p. The authors show more efficient recruitment of a cellular DEAD-box helicase, which enhances tombusvirus recombination, into the membrane-bound replicase in Act1p mutant yeast. Overall, this work shows that the actin network affects tombusvirus recombination in yeast and plant cells.

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase.

UNLABELLED: Plus-stranded RNA viruses induce membrane deformations in infected cells in order to build viral replication complexes (VRCs). Tomato bushy stunt virus (TBSV) co-opts cellular ESCRT (endosomal sorting complexes required for transport) proteins to induce the formation of vesicle (spherule)-like structures in the peroxisomal membrane with tight openings toward the cytosol. In this study, using a yeast (Saccharomyces cerevisiae) vps23Δ bro1Δ double-deletion mutant, we showed that the Vps23p ESCRT-I protein (Tsg101 in mammals) and Bro1p (ALIX) ESCRT-associated protein, both of which bind to the viral p33 replication protein, play partially complementary roles in TBSV replication in cells and in cell extracts. Dual expression of dominant-negative versions of Arabidopsis homologs of Vps23p and Bro1p inhibited tombusvirus replication to greater extent than individual expression in Nicotiana benthamiana leaves. We also demonstrated the critical role of Snf7p (CHMP4), Vps20p, and Vps24p ESCRT-III proteins in tombusvirus replication in yeast and in vitro. Electron microscopic imaging of vps23Δ yeast revealed the lack of tombusvirus-induced spherule-like structures, while crescent-like structures are formed in ESCRT-III deletion yeasts replicating TBSV RNA. In addition, we also showed that the length of the viral RNA affects the sizes of spherules formed in N. benthamiana cells. The 4.8-kb genomic RNA is needed for the formation of spherules 66 nm in diameter, while spherules formed during the replication of the ∼600-nucleotide (nt)-long defective interfering RNA in the presence of p33 and p92 replication proteins are 42 nm. We propose that the viral RNA serves as a "measuring string" during VRC assembly and spherule formation. IMPORTANCE: Plant positive-strand RNA viruses, similarly to animal positive-strand RNA viruses, replicate in membrane-bound viral replicase complexes in the cytoplasm of infected cells. Identification of cellular and viral factors affecting the formation of the membrane-bound viral replication complex is a major frontier in current virology research. In this study, we dissected the functions of co-opted cellular ESCRT-I (endosomal sorting complexes required for transport I) and ESCRT-III proteins and the viral RNA in tombusvirus replicase complex formation using in vitro, yeast-based, and plant-based approaches. Electron microscopic imaging revealed the lack of tombusvirus-induced spherule-like structures in ESCRT-I or ESCRT-III deletion yeasts replicating TBSV RNA, demonstrating the requirement for these co-opted cellular factors in tombusvirus replicase formation. The work could be of broad interest in virology and beyond.

Co-opted oxysterol-binding ORP and VAP proteins channel sterols to RNA virus replication sites via membrane contact sites.

Viruses recruit cellular membranes and subvert cellular proteins involved in lipid biosynthesis to build viral replicase complexes and replication organelles. Among the lipids, sterols are important components of membranes, affecting the shape and curvature of membranes. In this paper, the tombusvirus replication protein is shown to co-opt cellular Oxysterol-binding protein related proteins (ORPs), whose deletion in yeast model host leads to decreased tombusvirus replication. In addition, tombusviruses also subvert Scs2p VAP protein to facilitate the formation of membrane contact sites (MCSs), where membranes are juxtaposed, likely channeling lipids to the replication sites. In all, these events result in redistribution and enrichment of sterols at the sites of viral replication in yeast and plant cells. Using in vitro viral replication assay with artificial vesicles, we show stimulation of tombusvirus replication by sterols. Thus, co-opting cellular ORP and VAP proteins to form MCSs serves the virus need to generate abundant sterol-rich membrane surfaces for tombusvirus replication.