Specific Encapsidation of Nodavirus RNAs Is Mediated through the C Terminus of Capsid Precursor Protein Alpha

Flock house virus (FHV) is a small icosahedral insect virus with a bipartite, messenger-sense RNA genome. Its T=3 icosahedral capsid is initially assembled from 180 subunits of a single type of coat protein, capsid precursor protein alpha (407 amino acids). Following assembly, the precursor particles undergo a maturation step in which the alpha subunits autocatalytically cleave between Asn363 and Ala364. This cleavage generates mature coat proteins beta (363 residues) and gamma (44 residues) and is required for acquisition of virion infectivity. The X-ray structure of mature FHV shows that gamma peptides located at the fivefold axes of the virion form a pentameric helical bundle, and it has been suggested that this bundle plays a role in release of viral RNA during FHV uncoating. To provide experimental support for this hypothesis, we generated mutant coat proteins that carried deletions in the gamma region of precursor protein alpha. Surprisingly, we found that these mutations interfered with specific recognition and packaging of viral RNA during assembly. The resulting particles contained large amounts of cellular RNAs and varying amounts of the viral RNAs. Single-site amino acid substitution mutants showed that three phenylalanines located at positions 402, 405, and 407 of coat precursor protein alpha were critically important for specific recognition of the FHV genome. Thus, in addition to its hypothesized role in uncoating and RNA delivery, the C-terminal region of coat protein alpha plays a significant role in recognition of FHV RNA during assembly. A possible link between these two functions is discussed.     

Maturation cleavage required for infectivity of a nodavirus.

Nodaviral morphogenesis involves formation of labile precursor particles, called provirions, which mature by autocatalytic cleavage of the 407-residue coat precursor protein between asparagine residue 363 and alanine residue 364. It has previously been demonstrated that maturation results in increased physicochemical stability of the virion. We show here that cleavage of coat protein in purified provirions of Flock House virus was accompanied by a five- to eightfold increase in specific infectivity. Cleavage-negative provirions, produced by site-directed mutagenesis of asparagine residue 363 to aspartate, threonine, or alanine, displayed no infectivity above revertant frequencies as measured by plaque assay. All viable revertants (nine of nine) restored asparagine to the mutated position, suggesting high specificity for asparagine at the cleavage site.     

Reconstitution of Flock House provirions: a model system for studying structure and assembly.

Assembly of Flock House virus in infected Drosophila cells proceeds through an intermediate, the provirion, which lacks infectivity until the coat precursor protein, alpha, undergoes a spontaneous "maturation" cleavage (A. Schneemann, W. Zhong, T. M. Gallagher, and R. R. Rueckert, J. Virol 6:6728, 1992). We describe here methods for purifying provirions in a state which permitted dissociation and reassembly. Dissociation, to monomeric alpha protein and free RNA, was accomplished by freezing at pH 9.0 in the presence of 0.5 M salt and 0.1 M urea. When dialyzed at low ionic strength and pH 6.5, the dissociation products reassembled spontaneously to form homogeneous provirions with a normal complement of RNA as judged by cosedimentation with authentic virions and by ability to undergo maturation cleavage with acquisition of substantial, though subnormal, infectivity. Reconstitution experiments, i.e., remixing components after separating RNA from capsid protein, generated abnormal particles, suggesting the presence in the unfractionated dissociation products of an unidentified "nucleating" component.     

Assembly-dependent maturation cleavage in provirions of a small icosahedral insect ribovirus.

Extracts from nodavirus-infected Drosophila cells contained detergent-labile 140S "young" particles much richer than mature virions in their content of protein alpha, a precursor of coat proteins beta and gamma. Incorporation studies in infected cells showed that most newly synthesized alpha protein was assembled into young particles within a few minutes. Incubation of the particles, either in cytoplasmic extracts or after purification, resulted in spontaneous first-order cleavage of alpha protein to form beta-plus-gamma chains. Alpha protein that was not associated with particles failed to cleave. Cleavage was accompanied by a marked increase in detergent stability of the particles and was unaffected by a broad spectrum of protease inhibitors or by coating with precipitating antibody. We conclude (i) that alpha chains are cleaved only after assembly into provirions, (ii) that cleavage occurs internally and is likely therefore autocatalytic, and (iii) that cleavage stabilizes the mature virus particles.     

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.     

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.     

Temperature-sensitive poliovirus mutant fails to cleave VP0 and accumulates provirions.

A temperature-sensitive mutant of poliovirus, VP2-103, was isolated and characterized. A single nucleotide change, resulting in the substitution of glutamine for arginine at amino acid 76 of the capsid protein VP2, prevented the maturation of virions at the nonpermissive temperature. Particles indistinguishable from the previously elusive provirions were observed; these particles have been proposed to be penultimate in virion morphogenesis. Cleavage of VP0 into VP2 and VP4, the products found in mature virions, was not observed in VP2-103-infected cells at the nonpermissive temperature. The cleavage of VP0 in wild-type poliovirus-infected cells is dependent on RNA packaging; this reaction has been postulated to be autocatalytic. The existence of RNA-containing provirionlike particles in VP2-103-infected cells shows that RNA packaging can be uncoupled from VP0 cleavage.     

Crystallization of viruslike particles assembled from flock house virus coat protein expressed in a baculovirus system.

Flock house virus coat protein expressed in a baculovirus system spontaneously assembles into viruslike particles, which undergo an autocatalytic postassembly cleavage equivalent to that of the native virus. Mutations of the asparagine at the Asn/Ala cleavage site result in assembly of provirion-like particles that are cleavage defective. Crystals of the mutant provirions have been grown, and they diffract X rays beyond 3.3-A (0.33-nm) resolution. The crystals are monoclinic space group P2(1) (a = 464.8 A [46.48 nm]; b = 333.9 A [33.39 nm]; c = 325.2 A [32.52 nm]; beta = 91.9 degrees) with two provirion-like particles per unit cell. Thus, it should be possible to determine the high-resolution structure of the provirion, which will be compared with the crystal structure of the mature authentic virion. This collation should provide mechanistic detail for understanding the cleavage event. Moreover, this demonstrates that the baculovirus expression system displays sufficient fidelity to permit crystallographic analysis of the assembly process of biological macromolecules.     

Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses.

In this work, we evaluate the stability, dynamics and protein-nucleic acid interaction in Flock House virus (FHV). FHV is an RNA insect virus, non-enveloped, member of the family Nodaviridae. It is composed of a bipartite single-stranded RNA genome packaged in an icosahedral capsid of 180 copies of an identical protein (alpha protein). A fundamental property of many animal viruses is the post-assembly maturation required for infectivity. FHV is constructed as a provirion, which matures to an infectious virion by cleavage of alpha protein into beta and gamma subunits. We used high pressure, temperature and chemical denaturing agents to promote perturbation of the viral capsid. These effects were monitored by spectroscopy measurements (fluorescence, light scattering and CD) and size-exclusion chromatography. The data showed that FHV was stable to pressures up to 310 MPa at room temperature. The fluorescence emission and light scattering values showed small changes that were reversible after decompression. When we combined pressure and sub-denaturing urea concentrations (1 M), the changes were more drastic, suggesting dissociation of the capsid. However, these changes were reversible after pressure release. The complete dissociation of FHV could be observed only under high urea concentrations (10 M). There were no significant changes in emission spectra up to 5 M urea. FHV also was stable when we used temperature treatments (high and low). We also compared the effects of urea and pressure on FHV wild type and cleavage-defective mutant VLPs (virus-like particles). The VLPs and authentic particles are distinguishable by protein-RNA interactions, since VLPs pack cellular RNA and native particles contain viral RNA. Our results demonstrated that native particles are more stable than VLPs to physical and chemical treatments. Our data point to the specificity of the interaction between the capsid protein and the viral RNA. This specificity is crucial to the stability of the particle, which makes this interaction an excellent target for drug development.     

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.     

Morphogenesis of hepatitis A virus: isolation and characterization of subviral particles.

The morphogenesis of hepatitis A virus (HAV) in BS-C-1 cells was examined by immunoblotting with antisera to capsid proteins and labeling of virus-specific proteins with L-[35S]methionine. Antiserum to VP2 detected two virus-specific proteins with apparent molecular masses of 30.6 and 30 kDa, representing VP0 and VP2, while antiserum to VP1 detected proteins with molecular masses of 33 and 40 kDa, representing VP1 and a virus-specific protein which we designated PX, respectively. Sedimentation of cell lysates revealed the presence of virions, procapsids, and pentamers, but particles analogous to the protomers of other picornaviruses were not detected. Although provirions and virions were not found as discrete species in our gradient system, it was evident that the rate of sedimentation was proportional to the relative amounts of VP0 and VP2 in particles, with slower-sedimenting particles (provirions) containing predominantly VP0 rather than VP2. Procapsids contained VP0 in addition to VP1 and VP3. Pentamers also contained VP0, but PX was present rather than VP1. These results suggest that PX is a precursor to VP1 and is most likely 1D2A. Primary cleavage of the viral polyprotein also occurs at the 2A-2B junction in cardioviruses and aphthoviruses, but assembly of pentamers containing 1D2A has not been reported for those viruses. The absence of detectable levels of protomers suggests a high efficiency of pentamer formation, which may be related to the high efficiency of viral RNA encapsidation for HAV (D.A. Anderson, B.C. Ross, and S.A. Locarnini, J. Virol. 62:4201-4206, 1988). The results of this study reveal further unusual aspects of the HAV replicative cycle which distinguish it from other picornaviruses and may contribute to its restricted replication in cell culture.     

Large-scale,pH-dependent,quaternary structure changes in an RNA virus capsid are reversible in the absence of subunit autoproteolysis

The assembly and maturation of the coat protein of a T=4, nonenveloped, single-stranded RNA virus, Nudaurelia capensis omega virus (N omega V), was examined by using a recombinant baculovirus expression system. At pH 7.6, the coat protein assembles into a stable particle called the procapsid, which is 450 A in diameter and porous. Lowering the pH to 5.0 leads to a concerted reorganization of the subunits into a 410-A-diameter particle called the capsid, which has no obvious pores. This conformational change is rapid but reversible until slow, autoproteolytic cleavage occurs in at least 15% of the subunits at the lower pH. In this report, we show that expression of subunits with replacement of Asn-570, which is at the cleavage site, with Thr results in assembly of particles with expected morphology but that are cleavage defective. The conformational change from procapsid to capsid is reversible in N570T mutant virus-like particles, in contrast to wild-type particles, which are locked into the capsid conformation after cleavage of the coat protein. The reexpanded procapsids display slightly different properties than the original procapsid, suggesting hysteretic effects. Because of the stability of the procapsid under near-neutral conditions and the reversible properties of the cleavage-defective mutant, N omega V provides an excellent model for the study of pH-induced conformational changes in macromolecular assemblies. Here, we identify the relationship between cleavage and the conformational change and propose a pH-dependent helix-coil transition that may be responsible for the structural rearrangement in N omega V.     

Rescue of maturation-defective flock house virus infectivity with noninfectious, mature, viruslike particles

The infectivity of flock house virus (FHV) requires autocatalytic maturation cleavage of the capsid protein at residue 363, liberating the C-terminal 44-residue γ peptides, which remain associated with the particle. In vitro studies previously demonstrated that the amphipathic, helical portion (amino acids 364 to 385) of γ is membrane active, suggesting a role for γ in RNA membrane translocation during infection. Here we show that the infectivity of a maturation-defective mutant of FHV can be restored by viruslike particles that lack the genome but undergo maturation cleavage. We propose that the colocalization of the two defective particle types in an entry compartment allows the restoration of infectivity by γ.     

Amino acid substitutions in the poliovirus maturation cleavage site affect assembly and result in accumulation of provirions.

The assembly of infectious poliovirus virions requires a proteolytic cleavage between an asparagine-serine amino acid pair (the maturation cleavage site) in VP0 after encapsidation of the genomic RNA. In this study, we have investigated the effects that mutations in the maturation cleavage site have on P1 polyprotein processing, assembly of subviral intermediates, and encapsidation of the viral genomic RNA. We have made mutations in the maturation cleavage site which change the asparagine-serine amino acid pair to either glutamine-glycine or threonine-serine. The mutations were created by site-directed mutagenesis of P1 cDNAs which were recombined into wild-type vaccinia virus to generate recombinant vaccinia viruses. The P1 polyproteins expressed from the recombinant vaccinia viruses were analyzed for proteolytic processing and assembly defects in cells coinfected with a recombinant vaccinia virus (VV-P3) that expresses the poliovirus 3CD protease. A trans complementation system using a defective poliovirus genome was utilized to assess the capacity of the mutant P1 proteins to encapsidate genomic RNA (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993). The mutant P1 proteins containing the glutamine-glycine amino acid pair (VP4-QG) and the threonine-serine pair (VP4-TS) were processed by 3CD provided in trans from VV-P3. The processed capsid proteins VP0, VP3, and VP1 derived from the mutant precursor VP4-QG were unstable and failed to assemble into subviral structures in cells coinfected with VV-P3. However, the capsid proteins derived from VP4-QG did assemble into empty-capsid-like structures in the presence of the defective poliovirus genome. In contrast, the capsid proteins derived from processing of the VP4-TS mutant assembled into subviral intermediates both in the presence and in the absence of the defective genome RNA. By a sedimentation analysis, we determined that the capsid proteins derived from the VP4-TS precursor encapsidated the defective genome RNA. However, the cleavage of VP0 to VP4 and VP2 was delayed, resulting in the accumulation of provirions. The maturation cleavage of the VP0 protein containing the VP4-TS mutation was accelerated by incubation of the provirions at 37 degrees C. The results of these studies demonstrate that mutations in the maturation cleavage site have profound effects on the subsequent capability of the capsid proteins to assemble and provide evidence for the existence of the provirion as an assembly intermediate.     

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.     

Isolation and characterization of cDNA clones encoding a 18.8 kDa polypeptide,the product of the gene psaL,associated with photosystem I reaction center from spinach

Several cDNA clones encoding subunit XI of photosystem I reaction center (PSI-L) have been isolated from two gt11 expression libraries based on polyadenylated RNA of spinach seedlings illuminated for 4 and 16 h, respectively. The precursor polypeptide made from these recombinant DNAs in vitro can be efficiently imported into isolated spinach chloroplasts. It is correctly processed to the size of the authentic polypeptide and integrates into the photosystem I assembly. The 834 nucleotide sequence of the longest cDNA insert encodes a precursor polypeptide of 24 kDa (216 residues) and a mature protein of probably 18.8 kDa (169 residues). Hydropathy analysis suggests that the polypeptide contains two transmembrane segments. The protein appears to originate in a single-copy gene in spinach and to be decoded from RNA species of ca. 900 bases.     

Synthesis of Black Beetle Virus Proteins in Cultured Drosophila Cells: Differential Expression of RNAs 1 and 2

Black beetle virus is an insect virus with a split genome consisting of two single-stranded, messenger-active RNA molecules with molecular weights of 1.0 x 10(6) (RNA 1) and 0.5 x 10(6) (RNA 2), respectively. Virions contained two proteins, beta with a molecular weight of 43,000 (43K) and gamma (5K), and traces of a third protein, alpha (47K). When translated in cell-free extracts of rabbit reticulocytes, RNA 1 directed the synthesis of protein A (104K), whereas RNA 2 synthesized protein alpha. The in vitro translation efficiency of the two RNAs was roughly equal. Infection of cultured Drosophila cells induced the synthesis of five new proteins: A, alpha, beta, gamma, and B (10K), detected by autoradiography of polyacrylamide gels after electrophoresis of extracts from [(35)S]methionine-labeled cultures. All but protein gamma could also be detected by staining with Coomassie brilliant blue, indicating vigorous synthesis of viral proteins. Pulse-chase experiments in infected cells revealed the disappearance of protein alpha and the coordinate appearance of proteins beta and gamma, supporting an earlier proposal that coat protein of mature virions is made by cleavage of precursor alpha. Proteins A and B were stable in such pulse-chase experiments. The three classes of virus-induced proteins, represented by A, B, and alpha, were synthesized in markedly different amounts and with different kinetics. Synthesis of proteins A and B peaked early in infection and then declined, whereas synthesis of coat protein precursor alpha peaked much later. These results suggest that RNA 1 controls early replication functions via protein A (and also possibly protein B), whereas RNA 2 controls synthesis of coat protein required later for virion assembly.