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Results for “RNA Replication”

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At least 199 records · Page 11Linked to original sources

PubMed1996

Inhibition of VSV genome RNA replication but not transcription by monoclonal antibodies specific for the viral P protein.

The growth of vesicular stomatitis virus requires two distinct RNA synthetic events: transcription of messenger RNA molecules and replication of the viral genome RNA. We report the use of a panel of monoclonal antibodies directed against the viral phosphoprotein P in an attempt to assess the role of this protein in RNA synthesis. Using extracts derived from virus-infected cells, we show that several anti-P monoclonal antibodies can have an inhibitory effect on genome RNA replication by binding to a soluble form of the P protein. We also show that the P protein to which one of these antibodies (6D11) is directed is not complexed with the N protein and that the amount of soluble P protein that binds to the 6D11 antibody in immunoprecipitation reactions can be increased by treating extracts with alkaline phosphatase. In addition, phosphatase treatment of infected cell extracts results in an increased level of genome RNA replication. These results suggest that a soluble subspecies of the P protein that functions in genome RNA replication exists in infected cells and that this species of the P protein is not required for transcription.

Alkaline Phosphatase↗

PubMed2006

Host deadenylation-dependent mRNA decapping factors are required for a key step in brome mosaic virus RNA replication.

The genomes of positive-strand RNA [+RNA] viruses perform two mutually exclusive functions: they act as mRNAs for the translation of viral proteins and as templates for viral replication. A universal key step in the replication of +RNA viruses is the coordinated transition of the RNA genome from the cellular translation machinery to the viral replication complex. While host factors are involved in this step, their nature is largely unknown. By using the ability of the higher eukaryotic +RNA virus brome mosaic virus (BMV) to replicate in yeast, we previously showed that the host Lsm1p protein is required for efficient recruitment of BMV RNA from translation to replication. Here we show that in addition to Lsm1p, all tested components of the Lsm1p-7p/Pat1p/Dhh1p decapping activator complex, which functions in deadenylation-dependent decapping of cellular mRNAs, are required for BMV RNA recruitment for RNA replication. In contrast, other proteins of the decapping machinery, such as Edc1p and Edc2p from the deadenylation-dependent decapping pathway and Upf1p, Upf2p, and Upf3p from the deadenylation-independent decapping pathway, had no significant effects. The dependence of BMV RNA recruitment on the Lsm1p-7p/Pat1p/Dhh1p complex was linked exclusively to the 3' noncoding region of the BMV RNA. Collectively, our results suggest that the Lsm1p-7p/Pat1p/Dhh1p complex that transfers cellular mRNAs from translation to degradation might act as a key regulator in the switch from BMV RNA translation to replication.

Adenosine Monophosphate↗

PubMed2004

Reactive oxygen species suppress hepatitis C virus RNA replication in human hepatoma cells.

Hepatitis C virus (HCV) is a positive-stranded RNA virus that causes severe liver diseases, such as cirrhosis and hepatocellular carcinoma. HCV uses an RNA-dependent RNA polymerase to replicate its genome and an internal ribosomal entry site to translate its proteins. HCV infection is characterized by an increase in the concentrations of reactive oxygen species (ROS), the effect of which on HCV replication has yet to be determined. In this report, we investigated the effect of ROS on HCV replication, using a bicistronic subgenomic RNA replicon and a genomic RNA that can replicate in human hepatoma cells. The treatment with peroxide at concentrations that did not deplete intracellular glutathione or induce cell death resulted in significant decreases in the HCV RNA level in the cells. This response could be partially reversed by the antioxidant N-acetylcysteine. Further studies indicated that such a suppressive response to ROS was not due to the suppression of HCV protein synthesis or the destabilization of HCV RNA. Rather, it occurred rapidly at the level of RNA replication. ROS appeared to disrupt active HCV replication complexes, as they reduced the amount of NS3 and NS5A in the subcellular fraction where active HCV RNA replication complexes were found. In conclusion, our results show that ROS can rapidly inhibit HCV RNA replication in human hepatoma cells. The increased ROS levels in hepatitis C patients may therefore play an important role in the suppression of HCV replication.

Carcinoma, Hepatocellular↗

PubMed2004

Characterization of the inhibition of hepatitis C virus RNA replication by nonnucleosides.

The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.

Antiviral Agents↗

PubMed1998

Rescue of defective poliovirus RNA replication by 3AB-containing precursor polyproteins.

This study demonstrates the in vitro complementation of an RNA replication-defective lesion in poliovirus RNA by providing a replicase/polymerase precursor polypeptide [P3(wt) (wild type)] in trans. The replication-defective mutation was a phenylalanine-to-histidine change (F69H) in the hydrophobic domain of the membrane-associated viral protein 3AB. RNAs encoding wild-type forms of protein 3AB or the P3 precursor polypeptide were cotranslated with full-length poliovirus RNAs containing the F69H mutation in a HeLa cell-free translation/replication assay in an attempt to trans complement the RNA replication defect exhibited by the 3AB(F69H) lesion. Unexpectedly, generation of 3AB(wt) in trans was not able to efficiently complement the defective replication complex; however, cotranslation of the large P3(wt) precursor protein allowed rescue of RNA replication. Furthermore, P3 proteins harboring mutations that resulted in either an inactive polymerase or an inactive proteinase domain displayed differential abilities to trans complement the RNA replication defect. Our results indicate that replication proteins like 3AB may need to be delivered to the poliovirus replication complex in the form of a larger 3AB-containing protein precursor prior to complex assembly rather than as the mature viral cleavage product.

DNA-Directed RNA Polymerases↗

PubMed2005

Hairpin ribozymes in combination with siRNAs against highly conserved hepatitis C virus sequence inhibit RNA replication and protein translation from hepatitis C virus subgenomic replicons.

Chronic hepatitis C virus (HCV) infection is a clinically important liver disease with limited therapeutic options in a significant proportion of patients. Therefore, novel efficient therapeutic agents are needed. Because the 5'- and 3'-untranslated regions (UTRs) of HCV are highly conserved and functionally important for HCV replication, they are attractive targets for RNA-cleaving ribozymes or small interfering RNAs (siRNAs). In this study hairpin ribozymes (Rz) targeting HCV 5'- and 3'-UTR sequences were expressed from a retroviral vector transcript under control of two different RNA polIII promoters (tRNA(Val), U6). Ribozymes were evaluated in monocistronic, subgenomic I389/hyg-ubi/NS3-3'/5.1 HCV replicon cells as single agents or in combination with siRNAs against HCV 5'- or 3'-UTR recently demonstrated to inhibit HCV replicons. Additionally, ribozyme constructs were generated with the 3'-terminus of the ribozyme flanked by constitutive transport element (CTE) sequences, an RNA motif that has previously been shown to enhance cleavage activity of hammerhead ribozymes. In our study, tRNA(Val) as well as U6 promoter-driven Rzs markedly reduced HCV replicon RNA expression and HCV internal ribosome entry site (IRES)-mediated HCV NS5B protein translation from monocistronic subgenomic replicons. However, attachment of CTE sequences to the 3'-terminus did not significantly enhance activity of Rzs tested in this study. Interestingly, we detected additive HCV inhibitory effects for combinations of tRNA(Val)-driven Rzs and U6-derived siRNAs both directed against highly conserved 5'- and 3'-UTR sequence, suggesting that a dual strategy of ribozymes and siRNAs might become a powerful molecular tool to specifically silence HCV RNA replication.

Blotting, Western↗

PubMed1995

Persistent hepatitis C virus RNA replication in haemophiliacs: role of co-infection with human immunodeficiency virus.

In order to evaluate the evolution of transfusional hepatitis C in haemophiliacs, we performed a retrospective study of ALT levels and HCV viraemia with a RNA PCR assay in 57 patients. We found that the vast majority of HCV-infected patients remained viraemic (43/57 = 75%) and higher ALT levels correlated with HCV viraemia. Although indicators of the transfusional viral load (age, severity of haemophilia) and HBV co-infection did not correlate with HCV RNA replication, HIV seropositivity was strongly associated with persistence of HCV viraemia (23/25 = 92% in HIV-positive versus 20/32 = 62% in HIV-negative patients), without any correlation with CD4 counts. Genotyping of HCV in the 43 viraemic patients shows more frequent genotype 1 in the HIV-seropositive group (14/23) than in the seronegative group (6/20). Our data emphasize that besides the role of the immunodeficiency status, the genotypes of HCV might be involved in the differences observed in terms of HCV RNA replication between the HIV-seropositive and seronegative haemophiliacs.

AIDS-Related Opportunistic Infections↗

PubMed2001

Biochemical and genetic studies of the initiation of human rhinovirus 2 RNA replication: purification and enzymatic analysis of the RNA-dependent RNA polymerase 3D(pol).

The replication of human rhinovirus 2 (HRV2), a positive-stranded RNA virus belonging to the Picornaviridae, requires a virus-encoded RNA polymerase. We have expressed in Escherichia coli and purified both a glutathione S-transferase fusion polypeptide and an untagged form of the HRV2 RNA polymerase 3D(pol). Using in vitro assay systems previously described for poliovirus RNA polymerase 3D(pol) (J. B. Flanegan and D. Baltimore, Proc. Natl. Acad. Sci. USA 74:3677-3680, 1977; A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998), we have analyzed the biochemical properties of the two different enzyme preparations. HRV2 3D(pol) is both template and primer dependent, and it catalyzes two types of synthetic reactions in the presence of UTP, Mn(2+), and a poly(A) template. The first consists of an elongation reaction of an oligo(dT)(15) primer into poly(U). The second is a protein-priming reaction in which the enzyme covalently links UMP to the hydroxyl group of tyrosine in the terminal protein VPg, yielding VPgpU. This precursor is elongated first into VPgpUpU and then into VPg-linked poly(U), which is identical to the 5' end of picornavirus minus strands. The two forms of the enzyme are about equally active both in the oligonucleotide elongation and in the VPg-primed reaction. Various synthetic mutant VPgs were tested as substrates in the VPg uridylylation reaction.

Amino Acid Sequence↗

PubMed2004

Subgenomic RNA as a riboregulator: negative regulation of RNA replication by Barley yellow dwarf virus subgenomic RNA 2.

Barley yellow dwarf virus (BYDV) generates three 3'-coterminal subgenomic RNAs (sgRNAs) in infected cells. Translation of BYDV genomic RNA (gRNA) and sgRNA1 is mediated by the BYDV cap-independent translation element (BTE) in the 3' untranslated region. sgRNAs 2 and 3 are unlikely to be mRNAs. We proposed that accumulation of sgRNA2, which contains the BTE in its 5' UTR, regulates BYDV replication by trans-inhibiting translation of the viral polymerase from genomic RNA (gRNA). Here, we tested this hypothesis and found that: (i) co-inoculation of the BTE or sgRNA2 with BYDV RNA inhibits BYDV RNA accumulation in protoplasts; (ii) Brome mosaic virus (BMV), engineered to contain the BTE, trans-inhibits BYDV replication; and (iii) sgRNA2 generated during BYDV infection trans-inhibits both GFP expression from BMV RNA and translation of a non-viral reporter mRNA. We conclude that sgRNA2, via its BTE, functions as a riboregulator to inhibit translation of gRNA. This may make gRNA available as a replicase template and for encapsidation. Thus, BYDV sgRNA2 joins a growing list of trans-acting regulatory RNAs.

3' Untranslated Regions↗

PubMed2004

Structure-activity relationship of heterobase-modified 2'-C-methyl ribonucleosides as inhibitors of hepatitis C virus RNA replication.

Hepatitis C virus infection constitutes a significant health problem in need of more effective therapies. We have recently identified 2'-C-methyladenosine and 2'-C-methylguanosine as potent nucleoside inhibitors of HCV RNA replication in vitro. However, both of these compounds suffered from significant limitations. 2'-C-Methyladenosine was found to be susceptible to enzymatic conversions by adenosine deaminase and purine nucleoside phosphorylase, and it displayed limited oral bioavailability in the rat. 2'-C-Methylguanosine, on the other hand, was neither efficiently taken up in cells nor phosphorylated well. As part of an attempt to address these limitations, we now report upon the synthesis and evaluation of a series of heterobase-modified 2'-C-methyl ribonucleosides. The structure-activity relationship within this series of nucleosides reveals 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine as potent and noncytotoxic inhibitors of HCV RNA replication. Both 4-amino-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine and 4-amino-5-fluoro-7-(2-C-methyl-beta-d-ribofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine display improved enzymatic stability profiles as compared to that of 2'-C-methyladenosine. Consistent with these observations, the most potent compound, 4-amino-5-fluoro-7H-pyrrolo[2,3-d]pyrimidine ribonucleoside, is orally bioavailable in the rat. Together, the potency of the 2'-C-methyl-4-amino-pyrrolo[2,3-d]pyrimidine ribonucleosides and their improved pharmacokinetic properties relative to that of 2'-C-methyladenosine suggests that this class of compounds may have clinical utility.

Adenosine Deaminase↗

PubMed2005

Phosphorylation of the p33 replication protein of Cucumber necrosis tombusvirus adjacent to the RNA binding site affects viral RNA replication.

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase C. The related p28 replication protein of Turnip crinkle virus was also found to be phosphorylated in vivo, suggesting that posttranslational modification of replication proteins is a general feature among members of the large Tombusviridae family. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Phosphorylation-mimicking aspartic acid mutations rendered p33 nonfunctional in plant protoplasts and in yeast, a model host. Comparable mutations within the prereadthrough portion of p92 did not abolish replication. The nonphosphorylation-mimicking alanine mutants of CNV were able to replicate in plant protoplasts and in yeast, albeit with reduced efficiency when compared to the wild type. These alanine mutants also showed altered subgenomic RNA synthesis and a reduction in the ratio between plus- and minus-strand RNAs produced during CNV infection. These findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

Amino Acid Sequence↗

PubMed1999

Identification of a new element for RNA replication within the internal ribosome entry site of poliovirus RNA.

Several mutants of the Mahoney strain of poliovirus type 1 have been generated by introducing mutations into the stem-loop II (SLII) structure within the internal ribosomal entry site (IRES). Four of these mutants (SLII-1, -4, -5 and -6 mutants) have been characterized previously and are host-range mutants that replicate well in human HeLa cells but not in mouse cells. Two deletion mutants, SLII-2 and SLII-3, were non-viable, even in HeLa cells. It is now reported that SLII-2 was defective in genome RNA synthesis and viral protein synthesis, while SLII-3 was defective only in viral protein synthesis. These results indicate that the SLII region contains a cis-element for RNA replication as well as for IRES-dependent translation and that these two functions lie at the same sites within the SLII region. The host cellular factors that interacted with wild-type SLII and mutant SLII-2 and SLII-3 RNAs were different, suggesting that different host-factor binding regulates expression of mutant phenotypes.

HeLa Cells↗

PubMed1991

Rescue of Sindbis virus-specific RNA replication and transcription by using a vaccinia virus recombinant.

A heterologous system expressing functional Sindbis virus nonstructural proteins (nsPs) has several possible uses for studying Sindbis virus-specific RNA replication and transcription in vivo and in vitro. Of the many possible approaches, vaccinia virus offers an attractive transient expression system given that Sindbis virus replication can occur in cells which have been previously infected by vaccinia virus. In this report, a vaccinia virus recombinant (called vSINNS), which contains the cDNA encoding the Sindbis virus nsPs under the control of either the vaccinia virus 7.5K promoter or the bacteriophage T7 promoter, has been constructed and characterized. Upon infection of several cell types with vSINNS, Sindbis virus nsP precursors and processed forms, including nsP1, nsP2, and both phosphorylated and nonphosphorylated forms of nsP3, were synthesized. Proteins containing the putative RNA-dependent RNA polymerase domain (nsP4 and nsP34), which are normally produced in small amounts by readthrough of an opal termination codon, were not detected in vSINNS-infected cells. However, all nsP functions necessary for Sindbis virus-specific RNA synthesis must have been expressed, since both replication and subgenomic mRNA transcription of an engineered Sindbis virus defective interfering RNA in cells infected with vSINNS was observed. Furthermore, vSINNS could be used as a helper virus to amplify, to relatively high titers, a replication-defective Sindbis virus mutant containing an in-frame deletion in the conserved N-terminal domain of nsP3. These data, as well as the observation that normal yields of parental Sindbis virus are produced in cells which have been previously infected with vSINNS, indicate that expression of Sindbis virus nsPs, in the absence of Sindbis virus-specific RNA replication, is not sufficient to block the formation of active RNA replication complexes by superinfecting Sindbis virus.

Animals↗

PubMed2005

Hepatitis C virus RNA replication is regulated by host geranylgeranylation and fatty acids.

Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Our laboratory has previously demonstrated that high-level HCV replication during acute infection of chimpanzees is associated with the modulation of multiple genes involved in lipid metabolism, and that drugs that regulate cholesterol and fatty acid biosynthesis regulate the replication of the subgenomic HCV replicon in Huh-7 cells. In this article, we demonstrate that Huh-7 cells harboring replicating, full-length HCV RNAs express elevated levels of ATP citrate lyase and acetyl-CoA synthetase genes, both of which are involved in cholesterol and fatty acid biosynthesis. Further, we confirm that the cholesterol-biosynthetic pathway controls HCV RNA replication by regulating the cellular levels of geranylgeranyl pyrophosphate, we demonstrate that the impact of geranylgeranylation depends on the fatty acid content of the cell, and we show that fatty acids can either stimulate or inhibit HCV replication, depending on their degree of saturation. These results illustrate a complex cellular-regulatory network that controls HCV RNA replication, presumably by modulating the trafficking and association of cellular and/or viral proteins with cellular membranes, suggesting that pharmacologic manipulation of these pathways may have a therapeutic effect in chronic HCV infection.

ATP Citrate (pro-S)-Lyase↗

PubMed2002

Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique beta-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and beta-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the beta-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMTR(345)), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416-8428).

Amino Acid Motifs↗

PubMed2005

Stimulation of poliovirus synthesis in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro.

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3C(pro) or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.

3C Viral Proteases↗

PubMed1995

Enzymatic RNA replication in self-reproducing vesicles: an approach to a minimal cell.

The replication of a RNA template catalyzed by Q beta replicase was obtained in oleic acid/oleate vesicles simultaneously with the self-reproduction of the vesicles themselves. This was accomplished by entrapping the enzyme Q beta replicase, the RNA template, and the ribonucleotides ATP, CTP, GTP, and UTP inside the vesicles. The water-insoluble oleic anhydride was then added externally. It binds to the vesicle bilayer where it is catalytically hydrolyzed yielding the carboxylate surfactant in situ, which then brings about growth and reproduction of the vesicles themselves. This experiment is presented as a first approach to a synthetic minimal cell, in which the reproduction of the membrane and the replication of the internalized RNA molecules proceed simultaneously.

Catalysis↗

PubMed2001

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.

Animals↗