RNA Synthesis Activity Research Articles

The structure of a birnavirus polymerase reveals a distinct active site topology

Single-subunit polymerases are universally encoded in both cellular organisms and viruses. Their three-dimensional structures have the shape of a right-hand with the active site located in the palm region, which has a topology similar to that of the RNA recognition motif (RRM) found in many RNA-binding proteins. Considering that polymerases have well conserved structures, it was surprising that the RNA-dependent RNA polymerases from birnaviruses, a group of dsRNA viruses, have their catalytic motifs arranged in a permuted order in sequence. Here we report the 2.5 A structure of a birnavirus VP1 in which the polymerase palm subdomain adopts a new active site topology that has not been previously observed in other polymerases. In addition, the polymerase motif C of VP1 has the sequence of -ADN-, a highly unusual feature for RNA-dependent polymerases. Through site-directed mutagenesis, we have shown that changing the VP1 motif C from -ADN- to -GDD- results in a mutant with an increased RNA synthesis activity. Our results indicate that the active site topology of VP1 may represent a newly developed branch in polymerase evolution, and that birnaviruses may have acquired the -ADN- mutation to control their growth rate.

A site on the influenza A virus NS1 protein mediates both inhibition of PKR activation and temporal regulation of viral RNA synthesis

It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123–127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2α is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123–127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR−/− cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.

Biochemical characterization of a recombinant Japanese encephalitis virus RNA-dependent RNA polymerase

BackgroundJapanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated.ResultsTo characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template.ConclusionAs a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.

Characterization of norovirus 3Dpol RNA-dependent RNA polymerase activity and initiation of RNA synthesis

Norovirus (NV) 3D(pol) is a non-structural protein predicted to play an essential role in the replication of the NV genome. In this study, the characteristics of NV 3D(pol) activity and initiation of RNA synthesis have been examined in vitro. Recombinant NV 3D(pol), as well as a 3D(pol) active-site mutant were expressed in Escherichia coli and purified. NV 3D(pol) was able to synthesize RNA in vitro and displayed flexibility with respect to the use of Mg(2+) or Mn(2+) as a cofactor. NV 3D(pol) yielded two different products when incubated with synthetic RNA in vitro: (i) a double-stranded RNA consisting of two single strands of opposite polarity or (ii) the single-stranded RNA template labelled at its 3' terminus by terminal transferase activity. Initiation of RNA synthesis occurred de novo rather than by back-priming, as evidenced by the fact that the two strands of the double-stranded RNA product could be separated, and by dissociation in time-course analysis of terminal transferase and RNA synthesis activities. In addition, RNA synthesis was not affected by blocking of the 3' terminus of the RNA template by a chain terminator, sustaining de novo initiation of RNA synthesis. NV 3D(pol) displays in vitro properties characteristic of RNA-dependent RNA polymerases, allowing the implementation of this in vitro enzymic assay for the development and validation of antiviral drugs against NV, a so far non-cultivated virus and an important human pathogen.

TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter

Activator-dependent recruitment of TFIID initiates formation of the transcriptional preinitiation complex. TFIID binds core promoter DNA elements and directs the assembly of other general transcription factors, leading to binding of RNA polymerase II and activation of RNA synthesis. How TATA box-binding protein (TBP) and the TBP-associated factors (TAFs) are assembled into a functional TFIID complex with promoter recognition and coactivator activities in vivo remains unknown. Here, we use RNAi to knock down specific TFIID subunits in Drosophila tissue culture cells to determine which subunits are most critical for maintaining stability of TFIID in vivo. Contrary to expectations, we find that TAF4 rather than TBP or TAF1 plays the most critical role in maintaining stability of the complex. Our analysis also indicates that TAF5, TAF6, TAF9, and TAF12 play key roles in stability of the complex, whereas TBP, TAF1, TAF2, and TAF11 contribute very little to complex stability. Based on our results, we propose that holo-TFIID comprises a stable core subcomplex containing TAF4, TAF5, TAF6, TAF9, and TAF12 decorated with peripheral subunits TAF1, TAF2, TAF11, and TBP. Our initial functional studies indicate a specific and significant role for TAF1 and TAF4 in mediating transcription from a TATA-less, downstream core promoter element (DPE)-containing promoter, whereas a TATA-containing, DPE-less promoter was far less dependent on these subunits. In contrast to both TAF1 and TAF4, RNAi knockdown of TAF5 had little effect on transcription from either class of promoter. These studies significantly alter previous models for the assembly, structure, and function of TFIID.

Nuclear localization of flavivirus RNA synthesis in infected cells.

Flaviviral replication is believed to be exclusively cytoplasmic, occurring within virus-induced membrane-bound replication complexes in the host cytoplasm. Here we show that a significant proportion (20%) of the total RNA-dependent RNA polymerase (RdRp) activity from cells infected with West Nile virus, Japanese encephalitis virus (JEV), and dengue virus is resident within the nucleus. Consistent with this, the major replicase proteins NS3 and NS5 of JEV also localized within the nucleus. NS5 was found distributed throughout the nucleoplasm, but NS3 was present at sites of active flaviviral RNA synthesis, colocalizing with NS5, and visible as distinct foci along the inner periphery of the nucleus by confocal and immunoelectron microscopy. Both these viral replicase proteins were also present in the nuclear matrix, colocalizing with the peripheral lamina, and revealed a well-entrenched nuclear location for the viral replication complex. In keeping with this observation, antibodies to either NS3 or NS5 coimmunoprecipitated the other protein from isolated nuclei along with newly synthesized viral RNA. Taken together these data suggest an absolute requirement for both of the replicase proteins for nucleus-localized synthesis of flavivirus RNA. Thus, we conclusively demonstrate for the first time that the host cell nucleus functions as an additional site for the presence of functionally active flaviviral replicase complex.

The Novel Nucleoside Analog R1479 (4′-Azidocytidine) Is a Potent Inhibitor of NS5B-dependent RNA Synthesis and Hepatitis C Virus Replication in Cell Culture

Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479.

Association of functional influenza viral proteins and RNAs with nuclear chromatin and sub-chromatin structure

Transcription and replication of the influenza virus genome occur in the nucleus. However, the intra-nuclear localization of viral RNP complexes and the function of nuclear domains involved in viral transcription and replication, if any, are not well known. In the present study, we determined the intra-nuclear localization of viral proteins and viral RNAs and the in vitro RNA synthesis activity of viral RNP complexes associated with distinct nuclear fractions prepared from infected nuclei. A majority of viral RNA polymerases and M1 were recovered in DNase-sensitive fractions, whereas some portion of RNA polymerases and approximately 25% of NP were tightly associated with so-called nuclear matrix fractions. The amount of vRNA associated with the nuclear matrix was significantly more than that of cRNA. The in vitro viral RNA synthesis activity was detected in DNase-insensitive fractions, including the nuclear matrix. In contrast, newly synthesized viral RNAs were recovered in the DNase-sensitive fraction. These observations suggest that vRNP complexes are, at least partially, associated with densely packed chromatin, where viral transcription and replication occur, and the newly synthesized vRNP complexes to be transported into the cytoplasm are released into the nucleoplasm.

Haemophilus parasuis invades porcine brain microvascular endothelial cells

Haemophilus parasuis, an important swine pathogen, is the aetiological agent of Glässer's disease. It is responsible for cases of polyserositis, meningitis and pneumonia in young pigs. To date, 15 serotypes have been described, although several non-typable isolates are frequently recovered from diseased animals. The pathogenesis of H. parasuis infection is poorly understood. To cause meningitis, H. parasuis would have to cross the blood-brain barrier (BBB), composed of brain microvascular endothelial cells (BMEC). The objective of this study was to investigate the ability of H. parasuis to interact with porcine brain microvascular endothelial cells (PBMEC). It was demonstrated that the serotype 5 reference strain of H. parasuis, Nagasaki (originally recovered from a case of meningitis), was able to adhere at very high levels to and, most importantly, invade PBMEC. These capacities were confirmed by electron microscopy. Actinobacillus pleuropnemoniae serotype 7 (strain WF 83), used as negative control, was not able to adhere to or invade PBMEC. Comparisons of the levels of adhesion and invasion by several H. parasuis field strains from different serotypes isolated from cases of either meningitis or pneumonia showed that isolates of serotypes 4 and 5 had a higher invasion capacity than isolates belonging to other serotypes. Inhibition studies demonstrated that PBMEC invasion by H. parasuis required rearrangement of actin microfilaments and microtubular cytoskeletal elements but not active bacterial DNA, RNA or protein synthesis. Characterization studies demonstrated that proteinaceous invasin(s) does not seem to play a major role in entry of H. parasuis into PBMEC. Intracellular viable H. parasuis were found in PBMEC up to 6 h after antibiotic treatment. Even at high bacterial doses, H. parasuis was not toxic to PBMEC. In swine, the invasion of endothelial cells of the BBB may play an important role in the pathogenesis of meningitis caused by H. parasuis.

Identification of the Bunyamwera bunyavirus transcription termination signal

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae, which comprises segmented RNA viruses. Each of the BUNV negative-strand segments, small (S), medium (M) and large (L), serves as template for two distinct RNA-synthesis activities: (i) replication to generate antigenomes that are in turn replicated to yield further genomes; and (ii) transcription to generate a single species of mRNA. BUNV mRNAs are truncated at their 3' ends relative to the genome template, presumably because the BUNV transcriptase terminates transcription before reaching the 5' terminus of the genomic template. Here, identification of the transcription termination signal responsible for 3'-end truncation of BUNV S-segment mRNA was carried out. It was shown that efficient transcription termination was signalled by a 33 nt sequence within the 5' non-translated region (NTR) of the S segment. A 6 nt region (3'-GUCGAC-5') within this sequence was found to play a major role in termination signalling, with other nucleotides possessing individually minor, but collectively significant, signalling ability. By abrogating the signalling ability of these 33 nt, we identified a second, functionally independent termination signal located 32 nt downstream. This downstream signal was 9 nt in length and contained a pentanucleotide sequence, 3'-UGUCG-5', that overlapped the 6 nt major signalling component of the upstream signal. The pentanucleotide sequence was also found within the 5' NTR of the BUNV L segment and in several other members of the genus Orthobunyavirus, suggesting that the mechanism responsible for BUNV transcription termination may be common to other orthobunyaviruses.

Dipyridamole Reversibly Inhibits Mengovirus RNA Replication

Dipyridamole is an effective inhibitor of cardiovirus growth in cell culture. The effects of dipyridamole on mengovirus replication in vivo and in vitro were examined in the hope the drug could be used as an experimental analog of the poliovirus inhibitor guanidine. Guanidine selectively inhibits poliovirus RNA synthesis but not RNA translation, and as such, has been a valuable research tool. Although guanidine does not inhibit cardiovirus infection, a compound with similar discriminatory characteristics would be experimentally useful for parallel work with these viruses. We found that mengovirus plaque formation in HeLa or L cells was inhibited nearly 100% by the presence of 80 muM dipyridamole. The inhibitory effect was reversible and targeted an early step in the replication cycle. Studies with luciferase-expressing mengovirus replicons showed that viral protein synthesis was unaffected by dipyridamole, and rather, RNA synthesis was the step targeted by the drug. This assessment was confirmed by direct analyses of viral translation and RNA synthesis activities in a Krebs-2-derived in vitro system that supported complete, infectious cardiovirus replication. In Krebs extracts, dipyridamole specifically inhibited viral RNA synthesis to more than 95%, with no concomitant effect on viral protein translation or polyprotein processing. The observed inhibition reversibly affected an early step in both minus-strand and plus-strand RNA synthesis, although inhibition of plus-strand synthesis was more profound than that of minus-strand synthesis. We conclude that dipyridamole is a potent experimental tool that readily distinguishes between cardiovirus translation and RNA replication functions.

Inhibiting gene expression at transcription start sites in chromosomal DNA with antigene RNAs

Transcription start sites are critical switches for converting recognition of chromosomal DNA into active synthesis of RNA. Their functional importance suggests that they may be ideal targets for regulating gene expression. Here, we report potent inhibition of gene expression by antigene RNAs (agRNAs) complementary to transcription start sites within human chromosomal DNA. Silencing does not require methylation of DNA and differs from all known mechanisms for inhibiting transcription. agRNAs overlap DNA sequences within the open complex formed by RNA polymerase, and silencing is acutely sensitive to single base shifts. agRNAs effectively silence both TATA-less and TATA-box-containing promoters. Transcription start sites occur within every gene, providing predictable targets for agRNAs. Potent inhibition of multiple genes suggests that agRNAs may represent a natural mechanism for controlling transcription, may complement siRNAs and miRNAs that target mRNA, and will be valuable agents for silencing gene expression.

Role of the conserved nucleotide mismatch within 3'- and 5'-terminal regions of Bunyamwera virus in signaling transcription.

Bunyamwera virus (BUNV) is the prototype of the Bunyaviridae family of tri-partite negative-sense RNA viruses. The three BUNV segments possess 3' and 5' nontranslated regions (NTRs) that signal two RNA synthesis activities: (i) transcription to generate mRNAs and (ii) replication to generate antigenomes that are replicated to yield further genomes. While the genome acts as a template for synthesis of both transcription and replication products, the antigenome allows synthesis of only replication products, with mRNAs being undetectable. Here, we investigate the basis for the fundamentally different signaling abilities of genomic and antigenomic strands. We show that the identity of only nucleotide position 9 within the genomic 3' NTR is critical for the different RNA synthesis characteristics of genomic and antigenomic strands, thus identifying this nucleotide as an essential component of the transcription promoter. This nucleotide is distinctive, as it interrupts an unbroken run of conserved complementary nucleotides within the 3' and 5' NTRs of all three segments. Our results show that the conserved mismatched arrangement of this nucleotide plays no detectable role in signaling transcription. Instead, we show that the transcription-signaling ability of this position is entirely dependent on its nucleotide identity. We further show that while a U residue at 3' position 9 is strongly preferred for transcription activity in the context of the genomic promoter, it does not signal transcription in the context of the antigenomic promoter. Therefore, our results show that the identity of 3' position 9 is crucial for signaling BUNV transcription; however, it is not the sole determinant.

Inhibition of native hepatitis C virus replicase by nucleotide and non-nucleoside inhibitors

A number of nucleotide and non-nucleoside inhibitors of HCV polymerase are currently under investigation as potential antiviral agents to treat HCV-infected patients. HCV polymerase is part of a replicase complex including the polymerase subunit NS5B together with other viral and host proteins and viral RNA. The RNA synthesis activity of the native replicase complex was inhibited by 3′-deoxy-CTP, a chain-terminating nucleotide analog, but not inhibited by non-nucleoside NS5B polymerase inhibitors of three different structural classes. The HCV replicase was also resistant to heparin, a broad-spectrum, RNA-competitive polymerase inhibitor. Prebinding of the recombinant NS5B protein with a RNA template rendered the polymerase largely resistant to the inhibition by heparin and the non-nucleoside inhibitors, but did not affect the inhibitory potency of 3′-deoxy-CTP. Therefore, the HCV replicase showed a similar pattern of inhibitor sensitivity as compared to RNA-bound NS5B. These results suggest that the native HCV replicase complex represents a stable and productive polymerase–RNA complex. The allosteric non-nucleoside NS5B polymerase inhibitors are inactive against established HCV replicase but may function antagonistically with the formation of a productive enzyme–template complex.

Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. The xeroderma pigmentosum group B (XPB) helicase subunit of TFIIH functions in NER and transcription. The serine 751 (S751) residue of XPB was found to be phosphorylated in vivo. This phosphorylation inhibits NER and the microinjection of a phosphomimicking XPB-S751E mutant is unable to correct the NER defect of XP-B cells. Conversely, XPB-S751 dephosphorylation or its substitution with alanine (S751A) restores NER both in vivo and in vitro. Surprisingly, phospho/dephosphorylation of S751 spares TFIIH-dependent transcription. Finally, the phosphorylation of XPB-S751 does not impair the TFIIH unwinding of the DNA around the lesion, but rather prevents the 5' incision triggered by the ERCC1-XPF endonuclease. These data support an additional role for XPB in promoting the incision of the damaged fragment and reveal a point of NER regulation on TFIIH without interference in its transcription activity.

Cytokinin-binding protein from Arabidopsis thaliana leaves participating in transcription regulation

The 67 kDa cytokinin-binding protein (CBP) has been isolated from old rosette leaves of 9-week-oldArabidopsis thaliana plants. The procedure of CBP isolation included the protein puri?cation by SephadexG-50, hydrophobic chromatography on phenyl-Sepharose and a?nity chromatography on zeatin riboside-Sepharose. Nuclear localization in leaves of 67 kDa CBP was demonstrated. The recognition of a naturalcytokinin trans-zeatin by 67 kDa CBP from Arabidopsis leaves was detected by a very speci?c and sensitiveassay based on trans-zeatin competition in ELISA with anti-idiotype antibodies (Aba-i) for complex formationwith CBP. Aba-i were raised against antibodies against zeatin. trans-Zeatin competed with Aba-i in adose-dependent manner, demonstrating a high a?nity for the protein. In combination with trans-zeatin,67 kDa protein activated RNA synthesis in vitro in the transcription elongation systems containingchromatin-bound RNA polymerase I from barley leaves (heterologous system) or from Arabidopsis leaves(homologous system). These data permit a suggestion that the 67 kDa CBPs from monocots and dicotsful?ll similar functions. A comparison of functional activity of 67 kDa CBP from young, growing rosetteleaves of 3-week-old Arabidopsis plants, from mature leaves of 7-week-old plants and from senescent leavesof 9-week-old plants demonstrated age-dependent changes of the protein properties. The data are discussedin context of recent advances in the study of transcription regulation in eukaryotic cells.

Invasion of Porcine Brain Microvascular Endothelial Cells by Streptococcus suis Serotype 2

Streptococcus suis is an important swine pathogen that mainly causes meningitis and occasionally causes other infections, such as endocarditis, arthritis, and pneumonia. The pathogenesis of S. suis infection has not been completely defined. However, in order to cause meningitis, S. suis has to cross the blood-brain barrier (BBB) made up of brain microvascular endothelial cells. The objective of this work was to study the interactions of S. suis serotype 2 with porcine brain microvascular endothelial cells (PBMEC). The ability of North American and European S. suis serotype 2 strains to adhere to PBMEC and, most importantly, to invade PBMEC was demonstrated by using an antibiotic protection assay and was confirmed by electron microscopy. The polysaccharide capsule of S. suis seemed to partially interfere with the adhesion and invasion abilities of the bacterium. Our results showed that intracellular viable S. suis could be found in PBMEC up to 7 h after antibiotic treatment. Inhibition studies demonstrated that invasion of PBMEC by S. suis required actin microfilaments but not microtubular cytoskeletal elements or active bacterial RNA or protein synthesis. At high bacterial doses, suilysin-positive strains were toxic for PBMEC. The role of suilysin in cytotoxicity was confirmed by using purified suilysin, electron microscopy, and the lack of toxicity of a suilysin-negative mutant. In swine, the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by S. suis.

Hepatitis C Virus Non-structural Proteins in the Probable Membranous Compartment Function in Viral Genome Replication

The molecular mechanism of hepatitis C virus(HCV) RNA replication is still unknown. Recently, a cell culture system in which the HCV subgenomic replicon is efficiently replicated and maintained for a long period in Huh-7 cells has been established. Taking advantage of this replicon system, we detected the activity to synthesize the subgenomic RNA in the digitonin-permeabilized replicon cells. To elucidate how and where this viral RNA replicates in the cells, we monitored the activity for HCV RNA synthesis in the permeabilized replicon cells under several conditions. We obtained results suggesting that HCV replication complexes functioning to synthesize the replicon RNA are protected from access of nuclease and proteinase by possible cellular lipid membranes. We also found that a large part of the replicon RNA, including newly synthesized RNA, was present in such a membranous structure but a large part of each NS protein was not. A small part of each NS protein that was resistant to the proteinase action was shown to contribute sufficiently to the synthesis of HCV subgenomic RNA in the permeabilized replicon cells. These results suggested that a major subcellular site of HCV genome replication is probably compartmentalized by lipid membranes and that only a part of each NS protein forms the active replication complex in the replicon cells.

Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane.

Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

Enzymatic activities of the GB virus-B RNA-dependent RNA polymerase

The GB virus-B (GBV-B) nonstructural protein 5B (NS5B) encodes an RNA-dependent RNA polymerase (RdRp) with greater than 50% sequence similarity to the hepatitis C virus (HCV) NS5B. Recombinant GBV-B NS5B was reported to possess RdRp activity (W. Zhong et al., 2000, J. Viral Hepat. 7, 335–342). In this study, the GBV-B RdRp was examined more thoroughly for different RNA synthesis activities, including primer-extension, de novo initiation, template switch, terminal nucleotide addition, and template specificity. The results can be compared with previous characterizations of the HCV RdRp. The two RdRps share similarities in terms of metal ion and template preference, the abilities to add nontemplated nucleotides, perform both de novo initiation and extension from a primer, and switch templates. However, several differences in RNA synthesis between the GBV-B and HCV RdRps were observed, including (i) optimal temperatures for activity, (ii) ranges of Mn 2+ concentration tolerated for activity, and (iii) cation requirements for de novo RNA synthesis and terminal transferase activity. To assess whether the recombinant GBV-B RdRp may represent a relevant surrogate system for testing HCV antiviral agents, two compounds demonstrated to be active at nanomolar concentrations against HCV NS5B were tested on the GBV RdRp. A chain terminating nucleotide analog could prevent RNA synthesis, while a nonnucleoside HCV inhibitor was unable to affect RNA synthesis by the GBV RdRp.