Flock House Virus RNA Polymerase Initiates RNA Synthesis De Novo and Possesses a Terminal Nucleotidyl Transferase Activity

Flock House virus (FHV) is a positive-stranded RNA virus with a bipartite genome of RNAs, RNA1 and RNA2, and belongs to the family Nodaviridae. As the most extensively studied nodavirus, FHV has become a well-recognized model for studying various aspects of RNA virology, particularly viral RNA replication and antiviral innate immunity. FHV RNA1 encodes protein A, which is an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for RNA replication. Although the RNA replication of FHV has been studied in considerable detail, the mechanism employed by FHV protein A to initiate RNA synthesis has not been determined. In this study, we characterized the RdRP activity of FHV protein A in detail and revealed that it can initiate RNA synthesis via a de novo (primer-independent) mechanism. Moreover, we found that FHV protein A also possesses a terminal nucleotidyl transferase (TNTase) activity, which was able to restore the nucleotide loss at the 3′-end initiation site of RNA template to rescue RNA synthesis initiation in vitro, and may function as a rescue and protection mechanism to protect the 3′ initiation site, and ensure the efficiency and accuracy of viral RNA synthesis. Altogether, our study establishes the de novo initiation mechanism of RdRP and the terminal rescue mechanism of TNTase for FHV protein A, and represents an important advance toward understanding FHV RNA replication.     

DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

Coordinate replication of alfalfa mosaic virus RNAs 1 and 2 involves cis- and trans-acting functions of the encoded helicase-like and polymerase-like domains

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and coat protein. Transient expression of wild-type (wt) and mutant viral RNAs and proteins by agroinfiltration of plant leaves was used to study cis- and trans-acting functions of the helicase-like domain in P1 and the polymerase-like domain in P2. Three mutations in conserved motifs of the helicase-like domain of P1 affected one or more steps leading to synthesis of minus-strand RNAs 1, 2, and 3. In leaves containing transiently expressed P1 and P2, replication of wt but not mutant RNA 1 was observed. Apparently, the transiently expressed P1 could not complement the defect in replication of the RNA 1 mutant. Moreover, the transiently expressed wt replicase supported replication of RNA 2, but this replication was blocked in trans by coexpression of mutant RNA 1. However, expression of mutant RNA 1 did not interfere with the replication of RNA 3 by the wt replicase. Similarly, a mutation in the GDD motif encoded by RNA 2 could not be complemented in trans and affected the replication of RNA 1 by a wt replicase, while replication of RNA 3 remained unaffected. In competition assays, the transient wt replicase preferentially replicated RNA 3 over RNAs 1 and 2. The results indicate that one or more functions of P1 and P2 act in cis and point to the existence of a mechanism that coordinates the replication of RNAs 1 and 2.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

Flock House Virus RNA Replicates on Outer Mitochondrial Membranes in Drosophila Cells

The identification and characterization of host cell membranes essential for positive-strand RNA virus replication should provide insight into the mechanisms of viral replication and potentially identify novel targets for broadly effective antiviral agents. The alphanodavirus flock house virus (FHV) is a positive-strand RNA virus with one of the smallest known genomes among animal RNA viruses, and it can replicate in insect, plant, mammalian, and yeast cells. To investigate the localization of FHV RNA replication, we generated polyclonal antisera against protein A, the FHV RNA-dependent RNA polymerase, which is the sole viral protein required for FHV RNA replication. We detected protein A within 4 h after infection of Drosophila DL-1 cells and, by differential and isopycnic gradient centrifugation, found that protein A was tightly membrane associated, similar to integral membrane replicase proteins from other positive-strand RNA viruses. Confocal immunofluorescence microscopy and virus-specific, actinomycin D-resistant bromo-UTP incorporation identified mitochondria as the intracellular site of protein A localization and viral RNA synthesis. Selective membrane permeabilization and immunoelectron microscopy further localized protein A to outer mitochondrial membranes. Electron microscopy revealed 40- to 60-nm membrane-bound spherical structures in the mitochondrial intermembrane space of FHV-infected cells, similar in ultrastructural appearance to tombusvirus- and togavirus-induced membrane structures. We concluded that FHV RNA replication occurs on outer mitochondrial membranes and shares fundamental biochemical and ultrastructural features with RNA replication of positive-strand RNA viruses from other families.     

Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus.

Flock house virus (FHV) is a small icosahedral insect virus of the family Nodaviridae. Its genome consists of two messenger-sense RNA molecules, both of which are encapsidated in the same particle. RNA1 (3.1 kb) encodes proteins required for viral RNA replication; RNA2 (1.4 kb) encodes protein alpha (43 kDa), the precursor of the coat protein. When Spodoptera frugiperda cells were infected with a recombinant baculovirus containing a cDNA copy of RNA2, coat protein alpha assembled into viruslike precursor particles (provirions) that matured normally by autocatalytic cleavage of protein alpha into polypeptide chains beta (38 kDa) and gamma (5 kDa). The particles were morphologically indistinguishable from authentic FHV and contained RNA derived from the coat protein message. These results showed that RNA1 was required neither for virion assembly nor for maturation of provirions. Expression of mutants in which Asn-363 at the beta-gamma cleavage site of protein alpha was replaced by either aspartate, threonine, or alanine resulted in assembly of particles that were cleavage defective. For two of the mutants, unusual structural features were observed after preparation for electron microscopy. Particles containing Asp at position 363 were labile and showed a strong tendency to break into half-shells. Particles in which Asn-363 was replaced by Ala displayed a distinct hole in an otherwise complete shell. The third mutant, containing Thr at position 363, was indistinguishable in morphology from authentic FHV.     

Cytoplasmic Granule Formation and Translational Inhibition of Nodaviral RNAs in the Absence of the Double-Stranded RNA Binding Protein B2

Flock House virus (FHV) is a positive-sense RNA insect virus with a bipartite genome. RNA1 encodes the RNA-dependent RNA polymerase, and RNA2 encodes the capsid protein. A third protein, B2, is translated from a subgenomic RNA3 derived from the 3′ end of RNA1. B2 is a double-stranded RNA (dsRNA) binding protein that inhibits RNA silencing, a major antiviral defense pathway in insects. FHV is conveniently propagated in Drosophila melanogaster cells but can also be grown in mammalian cells. It was previously reported that B2 is dispensable for FHV RNA replication in BHK21 cells; therefore, we chose this cell line to generate a viral mutant that lacked the ability to produce B2. Consistent with published results, we found that RNA replication was indeed vigorous but the yield of progeny virus was negligible. Closer inspection revealed that infected cells contained very small amounts of coat protein despite an abundance of RNA2. B2 mutants that had reduced affinity for dsRNA produced analogous results, suggesting that the dsRNA binding capacity of B2 somehow played a role in coat protein synthesis. Using fluorescence in situ hybridization of FHV RNAs, we discovered that RNA2 is recruited into large cytoplasmic granules in the absence of B2, whereas the distribution of RNA1 remains largely unaffected. We conclude that B2, by binding to double-stranded regions in progeny RNA2, prevents recruitment of RNA2 into cellular structures, where it is translationally silenced. This represents a novel function of B2 that further contributes to successful completion of the nodaviral life cycle.     

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.     

Nodamura virus RNA replication in Saccharomyces cerevisiae: heterologous gene expression allows replication-dependent colony formation

Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 10(4)-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]     

Assembly of two independent populations of flock house virus particles with distinct RNA packaging characteristics in the same cell

Flock House virus (FHV; Nodaviridae) is a positive-strand RNA virus that encapsidates a bipartite genome consisting of RNA1 and RNA2. We recently showed that specific recognition of these RNAs for packaging into progeny particles requires coat protein translated from replicating viral RNA. In the present study, we investigated whether the entire assembly pathway, i.e., the formation of the initial nucleating complex and the subsequent completion of the capsid, is restricted to the same pool of coat protein subunits. To test this, coat proteins carrying either FLAG or hemagglutinin epitopes were synthesized from replicating or nonreplicating RNA in the same cell, and the resulting particle population and its RNA packaging phenotype were analyzed. Results from immunoprecipitation analysis and ion-exchange chromatography showed that the differentially tagged proteins segregated into two distinct populations of virus particles with distinct RNA packaging phenotypes. Particles assembled from coat protein that was translated from replicating RNA contained the FHV genome, whereas particles assembled from coat protein that was translated from nonreplicating mRNA contained random cellular RNA. These data demonstrate that only coat proteins synthesized from replicating RNA partake in the assembly of virions that package the viral genome and that RNA replication, coat protein translation, and virion assembly are processes that are tightly coupled during the life cycle of FHV.     

High-frequency leader sequence switching during coronavirus defective interfering RNA replication.

A system was developed that exploited defective interfering (DI) RNAs of coronavirus to study the role of free leader RNA in RNA replication. A cDNA copy of mouse hepatitis virus DI RNA was placed downstream of the T7 RNA polymerase promoter to generate DI RNAs capable of extremely efficient replication in the presence of a helper virus. We demonstrated that, in the DI RNA-transfected cells, the leader sequence of these DI RNAs was switched to that of the helper virus during one round of replication. This high-frequency leader sequence exchange was not observed if a nine-nucleotide stretch of sequence (UUUAUAAAC) at the junction between the leader and the remaining DI sequence was deleted. This observation suggests that a free leader RNA generated from the genomic RNA of mouse hepatitis virus may participate in the replication of DI RNA.