The cellular chaperone heat shock protein 90 facilitates Flock House virus RNA replication in Drosophila cells

The assembly of viral RNA replication complexes on intracellular membranes represents a critical step in the life cycle of positive-strand RNA viruses. We investigated the role of the cellular chaperone heat shock protein 90 (Hsp90) in viral RNA replication complex assembly and function using Flock House virus (FHV), an alphanodavirus whose RNA-dependent RNA polymerase, protein A, is essential for viral RNA replication complex assembly on mitochondrial outer membranes. The Hsp90 chaperone complex transports cellular mitochondrial proteins to the outer mitochondrial membrane import receptors, and thus we hypothesized that Hsp90 may also facilitate FHV RNA replication complex assembly or function. Treatment of FHV-infected Drosophila S2 cells with the Hsp90-specific inhibitor geldanamycin or radicicol potently suppressed the production of infectious virions and the accumulation of protein A and genomic, subgenomic, and template viral RNA. In contrast, geldanamycin did not inhibit the activity of preformed FHV RNA replication complexes. Hsp90 inhibitors also suppressed viral RNA and protein A accumulation in S2 cells expressing an FHV RNA replicon. Furthermore, Hsp90 inhibition with either geldanamycin or RNAi-mediated chaperone downregulation suppressed protein A accumulation in the absence of viral RNA replication. These results identify Hsp90 as a host factor involved in FHV RNA replication and suggest that FHV uses established cellular chaperone pathways to assemble its RNA replication complexes on intracellular membranes.     

A positive-strand RNA virus replication complex parallels form and function of retrovirus capsids

We show that brome mosaic virus (BMV) RNA replication protein 1a, 2a polymerase, and a cis-acting replication signal recapitulate the functions of Gag, Pol, and RNA packaging signals in conventional retrovirus and foamy virus cores. Prior to RNA replication, 1a forms spherules budding into the endoplasmic reticulum membrane, sequestering viral positive-strand RNA templates in a nuclease-resistant, detergent-susceptible state. When expressed, 2a polymerase colocalizes in these spherules, which become the sites of viral RNA synthesis and retain negative-strand templates for positive-strand RNA synthesis. These results explain many features of replication by numerous positive strand RNA viruses and reveal that these viruses, reverse transcribing viruses, and dsRNA viruses share fundamental similarities in replication and may have common evolutionary origins.     

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.     

Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle

Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.     

A replication silencer element in a plus-strand RNA virus

Replication represents a key step in the infectious cycles of RNA viruses. Here we describe a regulatory RNA element, termed replication silencer, that can down-regulate complementary RNA synthesis of a positive-strand RNA virus via an RNA-RNA interaction. This interaction occurs between the 5-nucleotide-long, internally positioned replication silencer and the extreme 3'-terminus of the viral RNA comprising part of the minimal minus-strand initiation promoter. Analysis of RNA synthesis in vitro, using model defective interfering (DI) RNA templates of tomato bushy stunt virus and a partially purified, RNA-dependent RNA polymerase preparation from tombusvirus-infected plants, revealed that this interaction inhibits minus-strand synthesis 7-fold. This functional interaction was supported further by: (i) RNA structure probing; (ii) phylogenetic analysis; (iii) inhibition of activity by short complementary DNAs; and (iv) compensatory mutational analysis. The silencer was found to be essential for accumulation of DI RNAs in protoplasts, indicating that it serves an important regulatory role(s) in vivo. Because similar silencer-promoter interactions are also predicted in other virus genera, this type of RNA-based regulatory mechanism may represent a widely utilized strategy for modulating replication.     

Requirements for assembly of poliovirus replication complexes and negative-strand RNA synthesis

HeLa cells were transfected with several plasmids that encoded all poliovirus (PV) nonstructural proteins. Viral RNAs were transcribed by T7 RNA polymerase expressed from recombinant vaccinia virus. All plasmids produced similar amounts of viral proteins that were processed identically; however, RNAs were designed either to serve as templates for replication or to contain mutations predicted to prevent RNA replication. The mutations included substitution of the entire PV 5' noncoding region (NCR) with the encephalomyocarditis virus (EMCV) internal ribosomal entry site, thereby deleting the 5'-terminal cloverleaf-like structure, or insertion of three nucleotides in the 3Dpol coding sequence. Production of viral proteins was sufficient to induce the characteristic reorganization of intracellular membranes into heterogeneous-sized vesicles, independent of RNA replication. The vesicles were stably associated with viral RNA only when RNA replication could occur. Nonreplicating RNAs localized to distinct, nonoverlapping regions in the cell, excluded from the viral protein-membrane complexes. The absence of accumulation of positive-strand RNA from both mutated RNAs in transfected cells was documented. In addition, no minus-strand RNA was produced from the EMCV chimeric template RNA in vitro. These data show that the 5'-terminal sequences of PV RNA are essential for initiation of minus-strand RNA synthesis at its 3' end.     

Host-dependent restriction of mengovirus replication. II. Effect of host restriction on late viral RNA synthesis and viral maturation.

Restricted mengovirus replication in Mandin-Darby bovine kidney (MDBK) cells is characterized by a 400-fold reduction in infectious virus yield and a 40-fold increase in the production of noninfectious virus. Using conditions which insure that all MDBK cells are infected, virus-specific RNA and protein synthesis were measured in the restrictive host and in a permissive host for mengovirus, HeLa cells. Labeling kinetics and sucrose gradient analysis of mengovirus-specific RNA from MDBK cells show a reduction of 10-fold in virion RNA, 5-fold in double-stranded RNA, and 12.5-fold in single-stranded RNA. The viral RNA biosynthetic processes which occur late in the replicative cycle and result in the production of 90% of the single-stranded viral RNA that is packaged into capsid proteins in the permissive host are absent in restrictive MDBK cells. Viral protein synthesis as measured by labeled viral-specific polysome is decreased, and there is an accumulation of 80S subviral particles in the restricted host. It is suggested that restriction may act at a number of stages of viral replication and maturation.     

Brome mosaic virus RNA replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum

The universal membrane association of positive-strand RNA virus RNA replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. Brome mosaic virus (BMV) encodes two mutually interacting RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and the polymerase-like 2a protein. In cells from the natural plant hosts of BMV, 1a and 2a colocalize on the endoplasmic reticulum (ER). 1a and 2a also direct BMV RNA replication and subgenomic mRNA synthesis in the yeast Saccharomyces cerevisiae, but whether the distribution of 1a, 2a, and active replication complexes in yeast duplicates that in plant cells has not been determined. For yeast expressing 1a and 2a and replicating BMV genomic RNA3, we used double-label confocal immunofluorescence to define the localization of 1a, 2a, and viral RNA and to explore the determinants of replication complex targeting. As in plant cells, 1a and 2a colocalized on and were retained on the yeast ER, with no detectable accumulation in the Golgi apparatus. 1a and 2a were distributed over most of the ER surface, with strongest accumulation on the perinuclear ER. In vivo labeling with bromo-UTP showed that the sites of 1a and 2a accumulation were the sites of nascent viral RNA synthesis. In situ hybridization showed that completed viral RNA products accumulated predominantly in the immediate vicinity of replication complexes but that some, possibly more mature cells also accumulated substantial viral RNA in the surrounding cytoplasm distal to replication complexes. Additionally, we find that 1a localizes to the ER when expressed in the absence of other viral factors. These results show that BMV RNA replication in yeast duplicates the normal localization of replication complexes, reveal the intracellular distribution of RNA replication products, and show that 1a is at least partly responsible for the ER localization and retention of the RNA replication complex.     

Aichi virus leader protein is involved in viral RNA replication and encapsidation

Aichi virus, a member of the family Picornaviridae, encodes a leader (L) protein of 170 amino acids (aa). The Aichi virus L protein exhibits no significant sequence homology to those of other picornaviruses. In this study, we investigated the function of the Aichi virus L protein in virus growth. In vitro translation and cleavage assays indicated that the L protein has no autocatalytic activity and is not involved in polyprotein cleavage. The L-VP0 junction was cleaved by 3C proteinase. Immunoblot analysis showed that the L protein is stably present in infected cells. Characterization of various L mutants derived from an infectious cDNA clone revealed that deletion of 93 aa of the center part (aa 43 to 135), 50 aa of the N-terminal part (aa 4 to 53), or 90 aa of the C-terminal part (aa 74 to 163) abolished viral RNA replication. A mutant (Delta114-163) in which 50 aa of the C-terminal part (aa 114 to 163) were deleted exhibited efficient RNA replication and translation abilities, but the virus yield was 4 log orders lower than that of the wild type. Sedimentation analysis of viral particles generated in mutant Delta114-163 RNA-transfected cells showed that the mutant has a severe defect in the formation of mature virions, but not in that of empty capsids. Thus, the data obtained in this study indicate that the Aichi virus L protein is involved in both viral RNA replication and encapsidation.     

Complete replication of poliovirus in vitro: preinitiation RNA replication complexes require soluble cellular factors for the synthesis of VPg-linked RNA.

Translation of poliovirion RNA in HeLa S10 extracts resulted in the formation of RNA replication complexes which catalyzed the asymmetric replication of poliovirus RNA. Synthesis of poliovirus RNA was detected in unfractionated HeLa S10 translation reactions and in RNA replication complexes isolated from HeLa S10 translation reactions by pulse-labeling with [32P]CTP. The RNA replication complexes formed in vitro contained replicative-intermediate RNA and were enriched in viral protein 3CD and the membrane-associated viral proteins 2C, 2BC, and 3AB. Genome-length poliovirus RNA covalently linked to VPg was synthesized in large amounts by the replication complexes. RNA replication was highly asymmetric, with predominantly positive-polarity RNA products. Both anti-VPg antibody and guanidine HCl inhibited RNA replication and virus formation in the HeLa S10 translation reactions without affecting viral protein synthesis. The inhibition of RNA synthesis by guanidine was reversible. The reversible nature of guanidine inhibition was used to demonstrate the formation of preinitiation RNA replication complexes in reaction mixes containing 2 mM guanidine HCl. Preinitiation complexes sedimented upon centrifugation at 15,000 x g and initiated RNA replication upon their resuspension in reaction mixes lacking guanidine. Initiation of RNA synthesis by preinitiation complexes did not require active protein synthesis or the addition of soluble viral proteins. Initiation of RNA synthesis by preinitiation complexes, however, was absolutely dependent on soluble HeLa cytoplasmic factors. Preinitiation complexes also catalyzed the formation of infectious virus in reaction mixes containing exogenously added capsid proteins. The titer of infectious virus produced in such trans-encapsidation reactions reached 4 x 10(7) PFU/ml. The HeLa S10 translation-RNA replication reactions represent an efficient in vitro system for authentic poliovirus replication, including protein synthesis, polyprotein processing, RNA replication, and virus assembly.     

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.     

Parallels among positive-strand RNA viruses, reverse-transcribing viruses and double-stranded RNA viruses

Viruses are divided into seven classes on the basis of differing strategies for storing and replicating their genomes through RNA and/or DNA intermediates. Despite major differences among these classes, recent results reveal that the non-virion, intracellular RNA-replication complexes of some positive-strand RNA viruses share parallels with the structure, assembly and function of the replicative cores of extracellular virions of reverse-transcribing viruses and double-stranded RNA viruses. Therefore, at least four of seven principal virus classes share several underlying features in genome replication and might have emerged from common ancestors. This has implications for virus function, evolution and control.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

Assembly of alphavirus replication complexes from RNA and protein components in a novel trans-replication system in mammalian cells

For positive-strand RNA viruses, the viral genomic RNA also acts as an mRNA directing the translation of the replicase proteins of the virus. Replication takes place in association with cytoplasmic membranes, which are heavily modified to create specific replication compartments. Here we have expressed by plasmid DNA transfection the large replicase polyprotein of Semliki Forest virus (SFV) in mammalian cells from a nonreplicating mRNA and provided a separate RNA containing the replication signals. The replicase proteins were able to efficiently and specifically replicate the template in trans, leading to accumulation of RNA and marker gene products expressed from the template RNA. The replicase proteins and double-stranded RNA replication intermediates localized to structures similar to those seen in SFV-infected cells. Using correlative light electron microscopy (CLEM) with fluorescent marker proteins to relocate those transfected cells, in which active replication was ongoing, abundant membrane modifications, representing the replication complex spherules, were observed both at the plasma membrane and in intracellular endolysosomes. Thus, replication complexes are faithfully assembled and localized in the trans-replication system. We demonstrated, using CLEM, that the replication proteins alone or a polymerase-negative polyprotein mutant together with the template did not give rise to spherule formation. Thus, the trans-replication system is suitable for cell biological dissection and examination in a mammalian cell environment, and similar systems may be possible for other positive-strand RNA viruses.     

Mutational analysis of the rubella virus nonstructural polyprotein and its cleavage products in virus replication and RNA synthesis

Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.     

Role of RNA helicases in HIV-1 replication

Viruses are replication competent genomes which are relatively gene-poor. Even the largest viruses (i.e. Herpesviruses) encode only slightly >200 open reading frames (ORFs). However, because viruses replicate obligatorily inside cells, and considering that evolution may be driven by a principle of economy of scale, it is reasonable to surmise that many viruses have evolved the ability to co-opt cell-encoded proteins to provide needed surrogate functions. An in silico survey of viral sequence databases reveals that most positive-strand and double-stranded RNA viruses have ORFs for RNA helicases. On the other hand, the genomes of retroviruses are devoid of virally-encoded helicase. Here, we review in brief the notion that the human immunodeficiency virus (HIV-1) has adopted the ability to use one or more cellular RNA helicases for its replicative life cycle.