Minus-strand RNA Research Articles

West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5′CS/3′CSI (conserved sequence) interaction, a 5′UAR/3′UAR ( upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5′UARs capable of forming the 5′/3′ viral RNA interaction were replicative. In contrast, WNV containing a 5′UAR mutation that abolished the 5′/3′ viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5′/3′ RNA interaction. The 5′UAR/3′UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5′UAR/3′UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3′UAR could alternate between pairing with the 5′UAR or with the 3′ end of the flaviviral genome. Therefore, the 5′UAR/3′UAR pairing may release the 3′ end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.

Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3′ of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

Expression of Plus- and Minus-strand Viral RNA in Coxsackievirus B3-Infected A/J Mice

In order to investigate the implication of viral replication in acute, subacute, and chronic infections of coxsackievirus B3 (CVB3), we examined the histopathological changes and plus- and minus-strand viral RNA dynamics in heart, pancreas, brain, and liver of CVB3-infected A/J mice. Mice were inoculated intraperitoneally with CVB3 and sacrificed on 1, 2, 3, 4, 7, 10, 14, 21, 30, 60, and 90 days post infection (p.i.). Plus- and minus-strand viral RNAs in the organs were quantitated and the organs were additionally evaluated histopathologically for inflammation. No inflammatory infiltrates were observed in the liver, brain, and heart. In contrast, massive lymphocyte infiltration and fat replacement were shown in the pancreas with loss of acinar cells. Both plus- and minus-strand viral RNA levels were detected by 21 days p.i. in heart, 90 days p.i. in pancreas, 4 days p.i. in liver, and 10 days p.i. in brain. The plus-strand RNA was found at least fifty fold higher than the minus-strand RNA by 4 days p.i. in heart and pancreas and by 3 days p.i. in liver. The plus- to minus-strand RNA ratio in brain was found less than 1:20. Our data indicate that viral replication was actively occurred in heart, pancreas, and liver during acute CVB3 infection, whereas viral replication was limited in brain. Furthermore, chronic persistent viral RNA was observed in pancreas. In conclusion, CVB3 at low dose of virus induces severe pancreatitis but marginal or no inflammatory changes in the heart, liver, and brain.

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Computer analysis of 158 hepatitis C virus (HCV) 5' untranslated region (5' UTR) sequences from the six genotypes showed that the 5' UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 5' UTR of the plus-strand RNA and the 3' end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome.

Rubella virus-induced superinfection exclusion studied in cells with persisting replicons

For the first time, homologous superinfection exclusion was documented for rubella virus (RUB) by using Vero cells harbouring persisting RUB replicons. Infection with wild-type RUB was reduced by tenfold, whereas Sindbis virus infection was unaffected. Replication following infection with packaged replicons and transfection with replicon transcripts was also restricted in these cells, indicating that restriction occurred after penetration and entry. Translation of such 'supertransfecting' replicon transcripts was not impaired, but no accumulation of supertransfecting replicon RNA could be detected. We tested the hypothesis favoured in the related alphaviruses that superinfection exclusion is mediated by cleavage of the incoming non-structural precursor by the pre-existing non-structural (NS) protease, resulting in an inhibition of minus-strand RNA synthesis. However, cleavage of a precursor translated from a supertransfecting replicon transcript with an NS protease catalytic-site mutation was not detected and the event in the replication cycle at which superinfection exclusion is executed remains to be elucidated.

Structural and functional analyses of stem–loop 1 of the Sindbis virus genome

Alphavirus genome function is controlled by elements at both the 5′ and 3′ ends. The 5′ 220 nt of the Sindbis virus genome is predicted to consist of four stem–loop structures the first of which has been demonstrated to be required for efficient minus-strand RNA synthesis. To understand the role of the structure of the first stem–loop (SL1) in regulating genome function, we performed enzymatic and chemical probing analyses. There were significant differences between the computer-predicted structures and our experimental data. In the 5′ terminus, two loop regions appear to be interacting in a complex and interdependent fashion with non-Watson–Crick interactions involving multiple adenosine residues playing a critical role in determining the overall structure. Some of the mutations that disrupted these interactions had significant affects, both positive and negative, on minus-strand synthesis, and translational efficiency was generally increased. In the context of full-length virus, these structural changes resulted in reduced virus growth kinetics particularly in mosquito cells suggesting host-specific effects of mutations in this region of the viral genome. Possible SL1 structures based on our experimental data are discussed.

Association between toll-like receptor 8 expression and adverse clinical outcomes in patients with enterovirus-associated dilated cardiomyopathy

In recent reports, human toll-like receptor (TLR) 8 mediates the antiviral response by recognizing single-stranded RNA. The inflammatory response against enteroviral (EV) RNA replication may play an important role in dilated cardiomyopathy (DCM). The purpose of this study was to determine whether TLR8 was expressed with EV replication in patients with enterovirus-associated DCM. Reverse transcriptase-polymerase chain reaction analysis was performed to screen the detection of myocardial EV RNA in 198 consecutive patients with DCM. Seventy-two EV RNA-positive patients with DCM and 20 control samples constituted the study population of the present study. Levels of TLR8 and myeloid differentiation factor (MyD) 88 adaptor protein mRNA and EV RNA (plus- and minus-strand RNAs) were measured by real-time RT-PCR. Immunohistochemistry was performed to identify the cellular source of these molecules. Toll-like receptor 8 and MyD88 mRNA levels were higher in patients with DCM than in controls (P < .001). Immunostainings of TLR8, MyD88, and EV protein showed localization of these proteins in cardiac myocytes in patients with DCM. After a mean follow-up of 426 days, clinical outcomes (development of heart failure n = 11, cardiac death n = 3) were associated with increased levels of TLR8 and MyD88 (P < .05). Multivariate analysis showed that TLR8 (relative risk 3.2, 95% CI 1.6-6.2) was a strong predictor of heart failure and cardiac death after adjustment for baseline characteristics. Toll-like receptor 8 and MyD88 expressions may be involved in the immune response to EV replication in enterovirus-associated DCM. In addition, TLR8 may provide important prognostic information in patients with enterovirus-associated DCM.

Interaction between the Cellular Protein eEF1A and the 3′-Terminal Stem-Loop of West Nile Virus Genomic RNA Facilitates Viral Minus-Strand RNA Synthesis

RNase footprinting and nitrocellulose filter binding assays were previously used to map one major and two minor binding sites for the cell protein eEF1A on the 3'(+) stem-loop (SL) RNA of West Nile virus (WNV) (3). Base substitutions in the major eEF1A binding site or adjacent areas of the 3'(+) SL were engineered into a WNV infectious clone. Mutations that decreased, as well as ones that increased, eEF1A binding in in vitro assays had a negative effect on viral growth. None of these mutations affected the efficiency of translation of the viral polyprotein from the genomic RNA, but all of the mutations that decreased in vitro eEF1A binding to the 3' SL RNA also decreased viral minus-strand RNA synthesis in transfected cells. Also, a mutation that increased the efficiency of eEF1A binding to the 3' SL RNA increased minus-strand RNA synthesis in transfected cells, which resulted in decreased synthesis of genomic RNA. These results strongly suggest that the interaction between eEF1A and the WNV 3' SL facilitates viral minus-strand synthesis. eEF1A colocalized with viral replication complexes (RC) in infected cells and antibody to eEF1A coimmunoprecipitated viral RC proteins, suggesting that eEF1A facilitates an interaction between the 3' end of the genome and the RC. eEF1A bound with similar efficiencies to the 3'-terminal SL RNAs of four divergent flaviviruses, including a tick-borne flavivirus, and colocalized with dengue virus RC in infected cells. These results suggest that eEF1A plays a similar role in RNA replication for all flaviviruses.

An RNA pseudoknot in the 3' end of the arterivirus genome has a critical role in regulating viral RNA synthesis.

In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3' terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3' end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.

Plus- and minus-stranded foot-and-mouth disease virus RNA quantified simultaneously using a novel real-time RT-PCR

Even though tagged RT-PCR and rTth RT-PCR have been developed to improve strand-specific detection of RNA virus, these assays are not quantitative. In this study, a novel real-time RT-PCR assay, which combines the benefits of both the tagged and rTth RT-PCR has been developed to quantify the strand-specific RNA of foot-and-mouth disease virus (FMDV). The tagged-primers plus a TaqMan probe located within the highly conserved viral 3D region were used. The in vitro synthesized minus-and plus-stranded RNA templates were used as a dual control along with the copy number of viral RNA molecules, which is more reliable than reported RT-PCR employing a DNA-based standard. This assay was used to quantify FMDV RNA from 10(9) to 10(1) copies with a maximum sensitivity of between ten and five copies and was shown to be highly reproducible with low intra-and inter-assay variation. Coefficients of variation (CV) values were 0.70-1.39% and 0.98-2.1%, respectively. Importantly, the method was applied to simultaneously quantify both plus-stranded and minus-stranded FMDV RNA using tagged-RT and tagged-FP primer during a high-temperature reverse transcription. Highly sensitive and strand-specific real-time RT-PCR assay has been established. We tested the ratio of viral plus-stranded to minus-stranded RNA in acutely infected and persistently infected BHK-21 cells, for which the values ranged from 22/1 to 143/1 and from 287/1 to 334/1, respectively, suggesting different replication patterns of plus-and minus-stranded RNA in acutely infected and persistently infected cells. This value ranged from 83/1 to 93/1 in enriched FMDV virions, indicating that FMDV encapsidation is highly specific for plus-stranded RNAs. In addition, the method was applied to surveille the FMDV replication at animal level.

Molecular biology of potexviruses: recent advances

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5' non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3' NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr-R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

Genetic Adaptation to Untranslated Region-Mediated Enterovirus Growth Deficits by Mutations in the Nonstructural Proteins 3AB and 3CD

Both untranslated regions (UTRs) of plus-strand RNA virus genomes jointly control translation and replication of viral genomes. In the case of the Enterovirus genus of the Picornaviridae family, the 5'UTR consists of a cloverleaf-like terminus preceding the internal ribosomal entry site (IRES) and the 3' terminus is composed of a structured 3'UTR and poly(A). The IRES and poly(A) have been implicated in translation control, and all UTR structures, in addition to cis-acting genetic elements mapping to the open reading frame, have been assigned roles in RNA replication. Viral UTRs are recognized by viral and host cell RNA-binding proteins that may co-determine genome stability, translation, plus- and minus-strand RNA replication, and scaffolding of viral replication complexes within host cell substructures. In this report, we describe experiments with coxsackie B viruses with a cell type-specific propagation deficit in Sk-N-Mc neuroblastoma cells conferred by the combination of a heterologous IRES and altered 3'UTR. Serial passage of these constructs in Sk-N-Mc cells yielded genetic adaptation by mutations within the viral nonstructural proteins 3A and 3C. Our data implicate 3A and/or 3C or their precursors 3AB and/or 3CD in a functional complex with the IRES and 3'UTR that drives viral propagation. Adaptation to neuroblastoma cells suggests an involvement of cell type-specific host factors or the host cell cytoplasmic milieu in this phenomenon.

Detection of viral genome in the myocardium: Lack of prognostic and functional relevance in patients with acute dilated cardiomyopathy

The presence of viral genome in the myocardium of patients with dilated cardiomyopathy (DCM) has been suggested as causative for the underlying cardiac disease. Nevertheless, the results of present studies are conflicting regarding the natural course of heart diseases associated with detection of viral genome. This study was undertaken to determine if the detection of viral genome in the myocardium of patients with DCM is of functional and prognostic relevance under modern treatment strategies of heart insufficiency. In 197 patients with DCM, left ventricular endomyocardial biopsies were performed. Analysis for genome of adenovirus, enterovirus (EV), and parvovirus B19 as well as enteroviral replication and immunohistology was performed. The increase in ejection fraction (EF) was 14.5 +/- 12.4% in the EV-positive group compared with 11.1 +/- 14.2 in the EV-negative group (P = not significant [NS]) after a mean follow-up (FU) of 19.5 and 17.6 months. The increase in EF in the virus-positive group (positive for EV, adenovirus, or parvovirus B19) was 15.3 +/- 13.3% compared with 12.3 +/- 11.9% in the virus-negative group (P = NS) after a mean FU of 17.6 and 11.5 months. There was no significant difference in the change of EF between the EV-positive and virus-negative groups. Detection of enteroviral RNA replication (detection of EV minus-strand RNA) did not result in a deterioration of left ventricular function compared with the virus-negative group (P = NS) after mean FU of 11.2 and 12.0 months. The transplantation-free survival of the patients was not influenced by detection of viral genome. Our results favor the view that the presence of viral genome in the myocardium of patients with DCM is of no functional and prognostic relevance.

Crystal Structure of the Dengue Virus RNA-Dependent RNA Polymerase Catalytic Domain at 1.85-Angstrom Resolution

Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-A resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.

Long-distance RNA–RNA interactions between terminal elements and the same subset of internal elements on the potato virus X genome mediate minus- and plus-strand RNA synthesis

Potexvirus genomes contain conserved terminal elements that are complementary to multiple internal octanucleotide elements. Both local sequences and structures at the 5' terminus and long-distance interactions between this region and internal elements are important for accumulation of potato virus X (PVX) plus-strand RNA in vivo. In this study, the role of the conserved hexanucleotide motif within SL3 of the 3' NTR and internal conserved octanucleotide elements in minus-strand RNA synthesis was analyzed using both a template-dependent, PVX RNA-dependent RNA polymerase (RdRp) extract and a protoplast replication system. Template analyses in vitro indicated that 3' terminal templates of 850 nucleotides (nt), but not 200 nt, supported efficient, minus-strand RNA synthesis. Mutational analyses of the longer templates indicated that optimal transcription requires the hexanucleotide motif in SL3 within the 3' NTR and the complementary CP octanucleotide element 747 nt upstream. Additional experiments to disrupt interactions between one or more internal conserved elements and the 3' hexanucleotide element showed that long-distance interactions were necessary for minus-strand RNA synthesis both in vitro and in vivo. Additionally, multiple internal octanucleotide elements could serve as pairing partners with the hexanucleotide element in vivo. These cis-acting elements and interactions correlate in several ways to those previously observed for plus-strand RNA accumulation in vivo, suggesting that dynamic interactions between elements at both termini and the same subset of internal octanucleotide elements are required for both minus- and plus-strand RNA synthesis and potentially other aspects of PVX replication.

Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus

The tertiary structure in the 3′-untranslated region (3′-UTR) of Bamboo mosaic virus (BaMV) RNA is known to be involved in minus-strand RNA synthesis. Proteins found in the RNA-dependent RNA polymerase (RdRp) fraction of BaMV-infected leaves interact with the radio labeled 3′-UTR probe in electrophoretic mobility shift assays (EMSA). Results derived from the ultraviolet (UV) cross-linking competition assays suggested that two cellular factors, p43 and p51, interact specifically with the 3′-UTR of BaMV RNA. p43 and p51 associate with the poly(A) tail and the pseudoknot of the BaMV 3′-UTR, respectively. p51-containing extracts specifically down-regulated minus-strand RNA synthesis when added to in vitro RdRp assays. LC/MS/MS sequencing indicates that p43 is a chloroplast phosphoglycerate kinase (PGK). When the chloroplast PKG levels were knocked down in plants, using virus-induced gene silencing system, the accumulation level of BaMV coat protein was also reduced.

Helper virus-independent trans-replication of hepatitis C virus-derived minigenome

We have previously described a synthetic T7-driven cDNA minigenome containing the antisense sequence of luciferase gene and internal ribosome entry site of encephalomyocarditis virus flanked by 5′- and 3′-end sequences of hepatitis C virus (HCV) that contain cis-acting replication elements. Synthesis of minus-strand RNA from the artificial minigenome was determined by using Huh-7 cells harboring autonomously replicating HCV subgenome as a helper for provision of functional replication components. To further confirm and extend these studies, we investigated here whether the minigenome replication system could be reconstituted by transfection of naïve Huh-7 cells with plasmid expressing nonstructural (NS) proteins. Reporter assay and Northern blot analysis revealed that trans-expression of NS proteins from 3 to 5 resulted in high level of luciferase activity and synthesized minus-strand RNA. The analogous result was also obtained with the minigenome derived from HCV 2a, and both HCV 1b- and 2a-derived NS protein were able to support the chimeric minigenomes whose 5′- or 3′-end was replaced by the respective region of the heterologous virus. These results provide a basis for establishing the reverse genetic system that is helpful to study cis- and trans-acting factors involved in HCV RNA replication.

Mathematical Modeling of Subgenomic Hepatitis C Virus Replication in Huh-7 Cells

Cell-based hepatitis C virus (HCV) replicon systems have provided a means for understanding HCV replication mechanisms and for testing new antiviral agents. We describe here a mathematical model of HCV replication that assumes that the translation of the HCV polyprotein occurs in the cytoplasm, that HCV RNA synthesis occurs in vesicular-membrane structures, and that the strategy of replication involves a double-stranded RNA intermediate. Our results shed light on the intracellular dynamics of subgenomic HCV RNA replication from transfection to steady state within Huh-7 cells. We predict the following: (i) about 6 x 10(3) ribosomes are involved in generating millions of HCV NS5B-polymerase molecules in a Huh-7 cell, (ii) the observed 10:1 asymmetry of plus- to minus-strand RNA levels can be explained by a higher-affinity (200-fold) interaction of HCV NS5B polymerase-containing replication complexes with HCV minus-strand RNA over HCV plus-strand RNA in order to initiate synthesis, (iii) the latter higher affinity can also account for the observed approximately 6:1 plus-strand/minus-strand ratio in vesicular-membrane structures, and (iv) the introduction of higher numbers of HCV plus-strand RNA by transfection leads to faster attainment of steady-state but does not change the steady-state HCV RNA level. Fully permissive HCV replication systems have been developed, and the model presented here is a first step toward building a comprehensive model for complete HCV replication. Moreover, the model can serve as an important tool in understanding HCV replication mechanisms and should prove useful in designing and evaluating new antivirals against HCV.

Tracking and Elucidating Alphavirus-Host Protein Interactions

Viral infections cause profound alterations in host cells. Here, we explore the interactions between proteins of the Alphavirus Sindbis and host factors during the course of mammalian cell infection. Using a mutant virus expressing the viral nsP3 protein tagged with green fluorescent protein (GFP) we directly observed nsP3 localization and isolated nsP3-interacting proteins at various times after infection. These results revealed that host factor recruitment to nsP3-containing complexes was time dependent, with a specific early and persistent recruitment of G3BP and a later recruitment of 14-3-3 proteins. Expression of GFP-tagged G3BP allowed reciprocal isolation of nsP3 in Sindbis infected cells, as well as the identification of novel G3BP-interacting proteins in both uninfected and infected cells. Note-worthy interactions include nuclear pore complex components whose interactions with G3BP were reduced upon Sindbis infection. This suggests that G3BP is a nuclear transport factor, as hypothesized previously, and that viral infection may alter RNA transport. Immunoelectron microscopy showed that a portion of Sindbis nsP3 is localized at the nuclear envelope, suggesting a possible site of G3BP recruitment to nsP3-containing complexes. Our results demonstrate the utility of using a standard GFP tag to both track viral protein localization and elucidate specific viral-host interactions over time in infected mammalian cells.

Template requirements for recognition and copying by Sindbis virus RNA-dependent RNA polymerase

The Sindbis virus (SIN) nonstructural protein nsP4 possesses the RNA-dependent RNA polymerase activity required for the replication of the SIN genome and transcription of a subgenomic mRNA during infection. Isolation of this protein from other viral components of the RNA synthetic complex allowed the characterization of template requirements for nsP4-mediated genome replication. The major findings of this study are: (i) in the absence of other viral proteins nsP4 is capable of copying SIN plus- and minus-strand templates, but does not transcribe subgenomic RNA; (ii) mutations in the 3′ conserved sequence element and poly(A) tail of the plus-strand template prevent nsP4-mediated de novo initiation of minus-strand RNA synthesis; (iii) nsP4-dependent terminal addition of nucleotides occurs on template RNA possessing certain mutations in the 3′CSE and polyadenylate tail ; (iv) nsP4 is capable of minus-strand synthesis independent of the sequence at the 5′ end of the template; (v) an A–U rich sequence in the 3′CSE represents a binding site for a replicase component, probably nsP4; (vi) plus-strand genomic RNA synthesis is dependent on the 3′ end of the minus-strand template. These studies begin to define the specific interactions with the viral RNA templates mediated by individual components of the viral replication complex and suggest a model for ternary complex formation during the initiation of minus-strand RNA synthesis.