The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.     

Role of host reticulon proteins in rearranging membranes for positive-strand RNA virus replication

Positive-strand RNA [(+)RNA] viruses are responsible for numerous human, animal, and plant diseases. Because of the limiting coding capacity of (+)RNA viruses, their replication requires a complex orchestration of interactions between the viral genome, viral proteins and exploited host factors. To replicate their genomic RNAs, (+)RNA viruses induce membrane rearrangements that create membrane-linked RNA replication compartments. Along with substantial advances on the ultrastructure of the membrane-bound RNA replication compartments, recent results have shed light into the role that host factors play in rearranging these membranes. This review focuses on recent insights that have driven a new understanding of the role that the membrane-shaping host reticulon homology domain proteins (RHPs) play in facilitating the replication of various (+)RNA viruses.     

The dependence of viral RNA replication on co-opted host factors

Positive-sense RNA ((+)RNA) viruses such as hepatitis C virus exploit host cells by subverting host proteins, remodelling subcellular membranes, co-opting and modulating protein and ribonucleoprotein complexes, and altering cellular metabolic pathways during infection. To facilitate RNA replication, (+)RNA viruses interact with numerous host molecules through protein-protein, RNA-protein and protein-lipid interactions. These interactions lead to the formation of viral replication complexes, which produce new viral RNA progeny in host cells. This Review presents the recent progress that has been made in understanding the role of co-opted host proteins and membranes during (+)RNA virus replication, and discusses common themes employed by different viruses.     

Chloroplast: the Trojan horse in plant–virus interaction

The chloroplast is one of the most dynamic organelles of a plant cell. It carries out photosynthesis, synthesizes major phytohormones, plays an active part in the defence response and is crucial for interorganelle signalling. Viruses, on the other hand, are extremely strategic in manipulating the internal environment of the host cell. The chloroplast, a prime target for viruses, undergoes enormous structural and functional damage during viral infection. Indeed, large proportions of affected gene products in a virus‐infected plant are closely associated with the chloroplast and the process of photosynthesis. Although the chloroplast is deficient in gene silencing machinery, it elicits the effector‐triggered immune response against viral pathogens. Virus infection induces the organelle to produce an extensive network of stromules which are involved in both viral propagation and antiviral defence. From studies over the last few decades, the involvement of the chloroplast in the regulation of plant–virus interaction has become increasingly evident. This review presents an exhaustive account of these facts, with their implications for pathogenicity. We have attempted to highlight the intricacies of chloroplast–virus interactions and to explain the existing gaps in our current knowledge, which will enable virologists to utilize chloroplast genome‐based antiviral resistance in economically important crops.     

Barley stripe mosaic virus γb protein disrupts chloroplast antioxidant defenses to optimize viral replication

The plant antioxidant system plays important roles in response to diverse abiotic and biotic stresses. However, the effects of virus infection on host redox homeostasis and how antioxidant defense pathway is manipulated by viruses remain poorly understood. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein is recruited to the chloroplast by the viral αa replicase to enhance viral replication. Here, we show that BSMV infection induces chloroplast oxidative stress. The versatile γb protein interacts directly with NADPH‐dependent thioredoxin reductase C (NTRC), a core component of chloroplast antioxidant systems. Overexpression of NbNTRC significantly impairs BSMV replication in Nicotiana benthamiana plants, whereas disruption of NbNTRC expression leads to increased viral accumulation and infection severity. To counter NTRC‐mediated defenses, BSMV employs the γb protein to competitively interfere with NbNTRC binding to 2‐Cys Prx. Altogether, this study indicates that beyond acting as a helicase enhancer, γb also subverts NTRC‐mediated chloroplast antioxidant defenses to create an oxidative microenvironment conducive to viral replication.     

Gene amplification and expression by RNA viruses and potential for further application to plant gene transfer

The great majority of plant viruses encapsidate messenger-sense ssRNA and have no natural DNA phase in their life cycle. Despite their RNA nature, essentially any desired change can be introduced into such genomes by using recombinant DNA techniques with suitably constructed, expressible viral cDNA clones. For some viruses such as brome mosaic virus, these methods have been used to define the sequences controlling RNA-directed genomic RNA replication and the expression of internal genes via subgenomic mRNAs. The results suggest a surprising degree of genetic flexibility, which appears to be reflected in the varied gene complements and genetic organizations of presumably related plant and animal RNA viruses sharing conserved replication genes. Foreign genes inserted in such RNA virus genomes can be amplified and expressed to a high level in transfected plant cells. In addition to the potential use of such viruses as episomal expression vectors, it should be possible to couple the viral pathways of RNA-dependent RNA synthesis to amplify and to further regulate the expression of genes transformed into plant chromosomes.     

Chloroplast Proteome of <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> Infected by Tomato Blistering Mosaic Virus

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase β subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein–protein interaction studies to confirm their roles in virus replication and disease development.     

Spatiotemporal Analysis of Hepatitis C Virus Infection

Hepatitis C virus (HCV) entry, translation, replication, and assembly occur with defined kinetics in distinct subcellular compartments. It is unclear how HCV spatially and temporally regulates these events within the host cell to coordinate its infection. We have developed a single molecule RNA detection assay that facilitates the simultaneous visualization of HCV (+) and (−) RNA strands at the single cell level using high-resolution confocal microscopy. We detect (+) strand RNAs as early as 2 hours post-infection and (−) strand RNAs as early as 4 hours post-infection. Single cell levels of (+) and (−) RNA vary considerably with an average (+):(−) RNA ratio of 10 and a range from 1–35. We next developed microscopic assays to identify HCV (+) and (−) RNAs associated with actively translating ribosomes, replication, virion assembly and intracellular virions. (+) RNAs display a defined temporal kinetics, with the majority of (+) RNAs associated with actively translating ribosomes at early times of infection, followed by a shift to replication and then virion assembly. (−) RNAs have a strong colocalization with NS5A, but not NS3, at early time points that correlate with replication compartment formation. At later times, only ~30% of the replication complexes appear to be active at a given time, as defined by (−) strand colocalization with either (+) RNA, NS3, or NS5A. While both (+) and (−) RNAs colocalize with the viral proteins NS3 and NS5A, only the plus strand preferentially colocalizes with the viral envelope E2 protein. These results suggest a defined spatiotemporal regulation of HCV infection with highly varied replication efficiencies at the single cell level. This approach can be applicable to all plus strand RNA viruses and enables unprecedented sensitivity for studying early events in the viral life cycle.     

Nodavirus-induced membrane rearrangement in replication complex assembly requires replicase protein a, RNA templates, and polymerase activity

Positive-strand RNA [(+)RNA] viruses invariably replicate their RNA genomes on modified intracellular membranes. In infected Drosophila cells, Flock House nodavirus (FHV) RNA replication complexes form on outer mitochondrial membranes inside ～50-nm, virus-induced spherular invaginations similar to RNA replication-linked spherules induced by many (+)RNA viruses at various membranes. To better understand replication complex assembly, we studied the mechanisms of FHV spherule formation. FHV has two genomic RNAs; RNA1 encodes multifunctional RNA replication protein A and RNA interference suppressor protein B2, while RNA2 encodes the capsid proteins. Expressing genomic RNA1 without RNA2 induced mitochondrial spherules indistinguishable from those in FHV infection. RNA1 mutation showed that protein B2 was dispensable and that protein A was the only FHV protein required for spherule formation. However, expressing protein A alone only "zippered" together the surfaces of adjacent mitochondria, without inducing spherules. Thus, protein A is necessary but not sufficient for spherule formation. Coexpressing protein A plus a replication-competent FHV RNA template induced RNA replication in trans and membrane spherules. Moreover, spherules were not formed when replicatable FHV RNA templates were expressed with protein A bearing a single, polymerase-inactivating amino acid change or when wild-type protein A was expressed with a nonreplicatable FHV RNA template. Thus, unlike many (+)RNA viruses, the membrane-bounded compartments in which FHV RNA replication occurs are not induced solely by viral protein(s) but require viral RNA synthesis. In addition to replication complex assembly, the results have implications for nodavirus interaction with cell RNA silencing pathways and other aspects of virus control.     

Viral evolution and insects as a possible virologic turning table

Summary Three lines of observation demonstrate the role of arthropods in transmission and evolution of viruses. a) Recent outbreaks of viruses from their niches took place and insects have played a major role in propagating the viruses. b) Examination of the list of viral families and their hosts shows that many infect invertebrates (I) and vertebrates (V) or (I) and plants (P) or all kingdoms (VIPs). This notion holds true irrespective of the genome type. At first glance the argument seems to be weak in the case of enveloped and non-enveloped RNA viruses with single-stranded (ss) segmented or non-segmented genomes of positive (+) or negative polarity. Here, there are several families infecting V or P only; no systematic relation to arthropods is found. c) In the non-enveloped plant viruses with ss RNA genomes there is a strong tendency for segmentation and individual packaging of the genome pieces. This is in contrast to ss+ RNA animal viruses and can only be explained by massive transmission by seed or insects or both, because individual packaging necessitates a multihit infection. Comparisons demonstrate relationships in the nonstructural proteins of double-stranded and ss+ RNA viruses irrespective of host range, segmentation, and envelope. Similar conclusions apply for the negative-stranded RNA viruses. Thus, viral supergroups can be created that infect V or P and exploit arthropods for infection or transmission or both. Examples of such relationships and explanations for viral evolution are reviewed and the arthropod orders important for cell culture are given.     

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate.     

Subviral molecules of RNA associated with plant ss(+)RNA viruses

Plant ss(+)RNA viruses besides their genome RNAs often are associated with additional subviral RNA molecules which occur naturally or are generated de novo during infection. There are such molecules like: satellite, defective, defective interfering and chimeric RNAs. Subviral RNAs can not replicate and encapsidate by oneself. Helper viruses supply the protein complexes that are necessary to these processes. The subviral molecules are characterized by small size. Recombination, deletion and accumulation of mutation are the main ways of arising subviral elements, although the origin of satRNAs is unknown. The unique feature of subviral RNAs is their ability to modify of infection progress caused by helper virus. They can attenuate or enhance the intensity of disease symptoms. The overall influence on disease development depends on three-component complex consisting of: plant host-virus' strain--subviral RNA. This article is a synthetic review of information concerning subviral RNA molecules of plant viruses, their structure, functions and origin.     

Identification of Yeast Factors Involved in the Replication of Mungbean Yellow Mosaic India Virus Using Yeast Temperature-Sensitive Mutants

正Dear Editor,The geminiviruses are small single-stranded plant DNA viruses belonging to the family Geminiviridae, which cause serious diseases in many economically important     

Protein composition of 6K2‐induced membrane structures formed during Potato virus A infection

The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein‐induced membranous structures from Potato virus A (PVA)‐infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N‐terminal Twin‐Strep‐tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non‐tagged Cerulean‐6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep‐tag‐based affinity chromatography was developed. Both (+)‐ and (–)‐strand PVA RNA and viral protein VPg were co‐purified specifically with the affinity tagged PVA‐SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA‐SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2‐induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co‐purified with PVA‐derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.     

Reverse genetic analysis of Ourmiaviruses reveals the nucleolar localization of the coat protein in Nicotiana benthamiana and unusual requirements for virion formation

Ourmia melon virus (OuMV) is the type member of the genus Ourmiavirus. These viruses have a trisegmented genome, each part of which encodes a single protein. Ourmiaviruses share a distant similarity with other plant viruses only in their movement proteins (MP), whereas their RNA-dependent RNA polymerase (RdRP) shares features only with fungal viruses of the family Narnaviridae. Thus, ourmiaviruses are in a unique phylogenetic position among existing plant viruses. Here, we developed an agroinoculation system to launch infection in Nicotiana benthamiana plants. Using different combinations of the three segments, we demonstrated that RNA1 is necessary and sufficient for cis-acting replication in the agroinfiltrated area. RNA2 and RNA3, encoding the putative movement protein and the coat protein (CP), respectively, are both necessary for successful systemic infection of N. benthamiana. The CP is dispensable for long-distance transport of the virus through vascular tissues, but its absence prevents efficient systemic infection at the exit sites. Virion formation occurred only when the CP was translated from replication-derived RNA3. Transient expression of a green fluorescent protein-MP (GFP-MP) fusion via agroinfiltration showed that the MP is present in cytoplasmic connections across plant cell walls; in protoplasts the GFP-MP fusion stimulates the formation of tubular protrusions. Expression through agroinfiltration of a GFP-CP fusion displays most of the fluorescence inside the nucleus and within the nucleolus in particular. Nuclear localization of the CP was also confirmed through Western blot analysis of purified nuclei. The significance of several unusual properties of OuMV for replication, virion assembly, and movement is discussed in relation to other positive-strand RNA viruses.     

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.

Authors Summary

Cellular proteins and cellular membranes are usurped by positive-stranded RNA viruses to assemble viral replicase complexes required for their replication. Tombusviruses, which are small RNA viruses of plants, depend on sterol-rich membranes for replication. The authors show that the tombusviral replication protein binds to cellular oxysterol-binding ORP proteins. Moreover, the endoplasmic reticulum resident cellular VAP proteins also co-localize with viral replication proteins. These protein interactions likely facilitate the formation of membrane contact sites that are visible in cells replicating tombusvirus RNA. The authors also show that sterols are recruited and enriched to the sites of viral replication. In vitro replication assay was used to show that sterols indeed stimulate tombusvirus replication. In summary, tombusviruses use subverted cellular proteins to build sterol-rich membrane microdomain to promote the assembly of the viral replicase complex. The paper connects efficient virus replication with cellular lipid transport and membrane structures.     

RNA‐Directed rna polymerases of plants

The report in 1971 by Comuet and Astier‐Manifacier that Chinese cabbage contains an active RNA‐dependent RNA polymerase has been extended to all plants studied. This has met with much opposition because the central dogma of molecular biology requires no replication mechanism for RNA. Only upon RNA virus infection are such enzymes needed, and it was generally believed that these were always and only virus‐coded. The purification and characterization of several of these plant viruses will be reviewed, with particular reference to the fact that while their amount in plant tissue is variably increased by various RNA virus infections their nature is unaffected by the viral genome and is strictly host‐specific. It will be noted, however, that in a specific instance viral infection has been shown to affect an important property of the enzyme. Also, it has become evident that certain plant viruses resemble animal picorna viruses (e.g., polio virus) and that these viruses carry an RNA polymerase gene. The same may be true, but has not been proven, for a small group of plant viruses that shows resemblances to the prokaryotic RNA phages in which a viral gene product together with host proteins form the RNA polymerase. An important question that remains to be solved in future work is the role of RNA polymerases in normal plant cell biology. Also, the mechanism by which viral infection causes the enzyme to become largely membrane or organelle bound and possibly conformationally changed in the process remains to be elucidated.