Predictive value of a multi-biomarker disease activity score for clinical remission and radiographic progression in patients with early rheumatoid arthritis: a post-hoc study of the OPERA trial.

OBJECTIVES: Measurement of serum biomarkers at disease onset may improve prediction of disease course in patients with early rheumatoid arthritis (RA). We evaluated the multi-biomarker disease activity (MBDA) score and early changes in MBDA score for prediction of 28-joint Disease Activity Score based on C-reactive protein (DAS28-CRP) remission and radiographic progression in the double-blinded OPERA trial. METHOD: Treatment-naïve RA patients (N = 180) with moderate or high DAS28 were randomized to methotrexate (MTX) + adalimumab (n = 89) or MTX + placebo (n = 91) in combination with glucocorticoid injection into swollen joints. X-rays of hands and feet were evaluated at months 0 and 12 (n = 164) by the total Sharp van der Heijde score (TSS). The smallest detectable change (1.8 TSS units) defined radiographic progression (∆TSS ≥ 2). Clinical remission (DAS28-CRP < 2.6) was assessed at baseline and 6 months. MBDA score was determined at 0 and 3 months and tested in a multivariable logistic regression model for predicting DAS28 remission at 6 months and radiographic progression at 1 year. RESULTS: Baseline MBDA score was independently associated with radiographic progression at 1 year [odds ratio (OR) = 1.03/unit, 95% confidence interval (CI) = 1.01-1.06], and changes in MBDA score from baseline to 3 months with clinical remission at 6 months [OR = 0.98/unit, 95% CI 0.96-1.00). In anti-cyclic citrullinated peptide antibody (anti-CCP)-positive patients, 35 of 89 with high MBDA score (> 44) showed radiographic progression (PPV = 39%), compared with 0 of 15 patients (NPV = 100%) with low/moderate MBDA score (≤ 44) (p = 0.003). CONCLUSION: Early changes in MBDA score were associated with clinical remission based on DAS28-CRP at 6 months. In anti-CCP-positive patients, a non-high baseline MBDA score (≤ 44) had a clinical value by predicting very low risk of radiographic progression at 12 months.

Ultra-high mass multimer analysis of protein-1a capping domains by a silicon nanomembrane detector.

Conventional time of flight ion detectors are based on secondary electron multipliers encountering a significant loss in detection efficiency, sensitivity and resolution with protein mass above 50kDa. In this work we employ a silicon nanomembrane detector in a Matrix-Assisted Laser Desorption/Ionization coupled to time of flight (MALDI-TOF) mass spectrometer. The operating principle relies on phonon-assisted field emission with excellent performance in the high mass range from 0.001-2MDa. In addition to the analysis of standard proteins the nanomembrane detector (NMD) has the potential for the detection and structural investigation of complex macromolecular assemblies through non-covalent interactions. In order to investigate this hypothesis, the N-terminal capping/methyltransferase domain (CAP) of the Brome Mosaic Virus (BMV) 1a replication protein by MALDI-TOF-NMD is analyzed. The signals detected at the high m/z-ratios of 912.6/982.7 (×103) and 1333.3 (×103) could be modified species of CAP-tricta/tetractamer and the octadecamer. For the first time, the NMD is applied to detect biologically complex macromolecular protein assemblies. Hence, this technology overcomes the limitations of conventional TOF-detectors and increases the analytical range of MALDI-TOF. This technology will be a future alternative for the structural analysis of intact virus capsids that will complement other MS-based techniques such as native mass spectrometry.

Hand bone loss in early rheumatoid arthritis during a methotrexate-based treat-to-target strategy with or without adalimumab-a substudy of the optimized treatment algorithm in early RA (OPERA) trial.

This study aims to investigate 1-year hand bone loss (HBL1-year) in early rheumatoid arthritis (RA) patients treated with a methotrexate (MTX) and intra-articular triamcinolone treat-to-target strategy +/- adalimumab and to determine if HBL6months is associated with radiographic progression after 2 years. In a clinical trial (OPERA) of 180 treatment-naive early RA patients, bone mineral density (BMD) was estimated from hand radiographs with digital X-ray radiogrammetry (DXR) at baseline, after 6 (n = 90) and 12 months (n = 70) of follow-up. Baseline and 2-year radiographs were scored according to the Sharp/van der Heijde method. Baseline characteristics and HBL6months (0-6 months changes in DXR-BMD) were investigated as predictors of structural damage by univariate linear (∆ total Sharp/van der Heijde score (TSS) as dependent variable) and logistic (+/-radiographic progression (∆TSS >0) as dependent variable) regression analyses. Variables with p < 0.10 were included in multivariable models. In 70 patients with available HBL1-year data, HBL1-year was median (interquartile range (IQR)) -1.9 (-3.3; -0.26 mg/cm2) in the MTX + placebo group and -1.8 (-3.6; 0.06) mg/cm2 in the MTX + adalimumab group, p = 0.98, Wilcoxon signed-rank. Increased HBL (compared to general population reference values) was found in 26/37 and 23/33 patients in the MTX + placebo and MTX + adalimumab groups, chi-squared = 0.99. In 90 patients with HBL6months data and 2-year radiographic data, HBL6months was independently associated with ∆TSS after 2 years (ß = -0.086 (95% confidence interval = -0.15; -0.025) TSS unit/mg/cm2 increase, p = 0.006) but not with presence of radiographic progression (∆TSS >0) (OR 0.96 (0.92-1.0), p = 0.10). In early RA patients treated with a methotrexate-based treat-to-target strategy, the majority of patients had increased HBL1-year, irrespective of adalimumab; HBL6months was independently associated with ∆TSS after 2 years.

Prediction of treatment response to adalimumab: a double-blind placebo-controlled study of circulating microRNA in patients with early rheumatoid arthritis.

At least 30% of patients with rheumatoid arthritis (RA) do not respond to biologic agents, which emphasizes the need of predictive biomarkers. We aimed to identify microRNAs (miRNAs) predictive of response to adalimumab in 180 treatment-naïve RA patients enrolled in the OPtimized treatment algorithm for patients with early RA (OPERA) Study, an investigator-initiated, prospective, double-blind placebo-controlled study. Patients were randomized to adalimumab 40 mg (n=89) or placebo-adalimumab (n=91) subcutaneously in combination with methotrexate. Expressions of 377 miRNAs were determined using TaqMan Human MicroRNA LDA, A Card v2.0 (Applied Biosystems). Associations between miRNAs and treatment response were tested using interaction analyses. MiRNAs with a P-value <0.05 using three different normalizations were included in a multivariate model. After backwards elimination, the combination of low expression of miR-22 and high expression of miR-886.3p was associated with EULAR good response. Future studies to assess the utility of these miRNAs as predictive biomarkers are needed.

Clinical and radiographic outcome of a treat-to-target strategy using methotrexate and intra-articular glucocorticoids with or without adalimumab induction: a 2-year investigator-initiated, double-blinded, randomised, controlled trial (OPERA).

OBJECTIVES: To study clinical and radiographic outcomes after withdrawing 1 year's adalimumab induction therapy for early rheumatoid arthritis (eRA) added to a methotrexate and intra-articular triamcinolone hexacetonide treat-to-target strategy (NCT00660647). METHODS: Disease-modifying antirheumatic drug (DMARD)-naive patients with eRA started methotrexate (20 mg/week) and intra-articular triamcinolone (20 mg/ml) for 2 years. In addition, they were randomised to receive placebo adalimumab (DMARD group, n=91) or adalimumab (40 mg/every other week) (DMARD+adalimumab group, n=89) during the first year. Sulfasalazine and hydroxychloroquine were added if disease activity persisted after 3 months. During year 2, synthetic DMARDs continued. Adalimumab was (re)initiated if active disease reoccurred. Clinical response, remission, disability, quality of life and radiographic changes were assessed. RESULTS: One year after adalimumab withdrawal, treatment profiles and clinical responses did not differ between groups. In the DMARD/DMARD+adalimumab groups, the median 2-year methotrexate dose was 20/20 mg/week (p=0.45), triple DMARD therapy had been initiated in 33/27 patients (p=0.49), adalimumab was (re)initiated in 12/12 patients and cumulative triamcinolone dose was 160/120 mg (p=0.15). The treatment target (disease activity score, 4 variables, C-reactive protein (DAS28CRP) ≤3.2 or DAS28>3.2 without swollen joints) was achieved at all visits in ≥85% of patients in year 2; remission rates were DAS28CRP<2.6:69%/66%; Clinical Disease Activity Index ≤2.8:55%/57%; Simplified Disease Activity Index <3.3:54%/49%; American College of Rheumatology/European League against Rheumatism (28 joints):44%/45% (p=0.66-1.00). Radiographic progression (Δtotal Sharp score/year) was similar 1.31/0.53 (p=0.12). Erosive progression (Δerosion score (ES)/year) was year 1:0.57/0.06 (p=0.02); year 2:0.38/0.05 (p=0.005). Proportion of patients without erosive progression (ΔES≤0) was year 1: 59%/76% (p=0.03); year 2:64%/79% (p=0.04). CONCLUSIONS: An aggressive triamcinolone and synthetic DMARD treat-to-target strategy in eRA provided excellent 2-year clinical and radiographic disease control independent of adalimumab induction therapy. ES progression was slightly less during and following adalimumab induction therapy. TRIAL REGISTRATION NUMBER: NCT00660647.

DNA-directed expression of an animal virus RNA for replication-dependent colony formation in Saccharomyces cerevisiae.

To date, the insect nodavirus flock house virus (FHV) is the only virus of a higher eukaryote that has been shown to undergo a full replicative cycle and produce infectious progeny in the yeast Saccharomyces cerevisiae. The genome of FHV is composed of two positive-sense RNA segments: RNA1, encoding the RNA replicase, and RNA2, encoding the capsid protein precursor. When yeast cells expressing FHV RNA replicase were transfected with a chimeric RNA composed of a selectable gene flanked by the termini of RNA2, the chimeric RNA was replicated and transmitted to daughter cells indefinitely. In the work reported here, we developed a system in which a selectable chimeric RNA replicon was transcribed from an inducible RNA polymerase II (polII) promoter in vivo in yeast. To render marker gene expression absolutely dependent on RNA replication, the primary polII transcript was made negative in sense and contained an intron that blocked the translation of cryptic transcripts from the opposite DNA strand. The RNA products of DNA-templated transcription, processing, and RNA replication were characterized by Northern blot hybridization and primer extension analysis. Marker gene expression and colony growth under selective conditions depended strictly on FHV RNA replication, with background colonies arising at a frequency of fewer than 1 in 10(8) plated cells. The utility of the system was demonstrated by introducing a second chimeric replicon and showing that at least two different selectable markers could be simultaneously expressed by means of RNA replication. This is the first example of FHV RNA1-dependent selectable marker expression initiated in vivo and will greatly facilitate the identification and characterization of the requirements and inhibitors of RNA replication.

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.

Identification of sequences in Brome mosaic virus replicase protein 1a that mediate association with endoplasmic reticulum membranes.

RNA replication of all positive-strand RNA viruses is closely associated with intracellular membranes. Brome mosaic virus (BMV) RNA replication occurs on the perinuclear region of the endoplasmic reticulum (ER), both in its natural plant host and in the yeast Saccharomyces cerevisiae. The only viral component in the BMV RNA replication complex that localizes independently to the ER is 1a, a multifunctional protein with an N-terminal RNA capping domain and a C-terminal helicase-like domain. The other viral replication components, the RNA polymerase-like protein 2a and the RNA template, depend on 1a for recruitment to the ER. We show here that, in membrane extracts, 1a is fully susceptible to proteolytic digestion in the absence of detergent and thus, a finding consistent with its roles in RNA replication, is wholly or predominantly on the cytoplasmic face of the ER with no detectable lumenal protrusions. Nevertheless, 1a association with membranes is resistant to high-salt and high-pH treatments that release most peripheral membrane proteins. Membrane flotation gradient analysis of 1a deletion variants and 1a segments fused to green fluorescent protein (GFP) showed that sequences in the N-terminal RNA capping module of 1a mediate membrane association. In particular, a region C-terminal to the core methyltransferase homology was sufficient for high-affinity ER membrane association. Confocal immunofluorescence microscopy showed that even though these determinants mediate ER localization, they fail to localize GFP to the narrow region of the perinuclear ER, where full-length 1a normally resides. Instead, they mediate a more globular or convoluted distribution of ER markers. Thus, additional sequences in 1a that are distinct from the primary membrane association determinants contribute to 1a's normal subcellular distribution, possibly through effects on 1a conformation, orientation, or multimerization on the membrane.

The brome mosaic virus RNA3 intergenic replication enhancer folds to mimic a tRNA TpsiC-stem loop and is modified in vivo.

The genome of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, consists of three capped, messenger-sense RNAs. RNA1 and RNA2 encode viral replication proteins 1a and 2a, respectively. RNA3 encodes the 3a movement protein and the coat protein, which are essential for systemic infection in plants but dispensable for RNA3 replication in plants and yeast. A subset of the 250-base intergenic region (IGR), the replication enhancer (RE), contains all cis-acting signals necessary for a crucial, early template selection step, the 1a-dependent recruitment of RNA3 into replication. One of these signals is a motif matching the conserved box B sequence of RNA polymerase III transcripts. Using chemical modification with CMCT, kethoxal, DMS, DEPC, and lead, we probed the structure of the IGR in short, defined transcripts and in full-length RNA3 in vitro, in yeast extracts, and in whole yeast cells. Our results reveal a stable, unbranched secondary structure that is not dependent on the surrounding ORF sequences or on host factors within the cell. Functional 5' and 3' deletions that defined the minimal RE in earlier deletion studies map to the end of a common helical segment. The box B motif is presented as a hairpin loop of 7 nt closed by G:C base pairs in perfect analogy to the TpsiC-stem loop in tRNA(Asp). An adjacent U-rich internal loop, a short helix, and another pyrimidine-rich loop were significantly protected from base modifications. This same arrangement is conserved between BMV and cucumoviruses CMV, TAV, and PSV. In the BMV box B loop sequence, uridines corresponding to tRNA positions T54 and psi55 were found to be modified in yeast and plants to 5mU and pseudouridine. Together with the aminoacylated viral 3'-end, this is thus the second RNA replication signal within BMV where the virus has evolved a tRNA structural mimicry to a degree that renders it a substrate for classical tRNA modification reactions in vivo.

Brome mosaic virus Protein 1a recruits viral RNA2 to RNA replication through a 5' proximal RNA2 signal.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes two RNA replication factors. Membrane-associated 1a protein contains a helicase-like domain and RNA capping functions. 2a, which is targeted to membranes by 1a, contains a central polymerase-like domain. In the absence of 2a and RNA replication, 1a acts through an intergenic replication signal in BMV genomic RNA3 to stabilize RNA3 and induce RNA3 to associate with cellular membrane. Multiple results imply that 1a-induced RNA3 stabilization reflects interactions involved in recruiting RNA3 templates into replication. To determine if 1a had similar effects on another BMV RNA replication template, we constructed a plasmid expressing BMV genomic RNA2 in vivo. In vivo-expressed RNA2 templates were replicated upon expression of 1a and 2a. In the absence of 2a, 1a stabilized RNA2 and induced RNA2 to associate with membrane. Deletion analysis demonstrated that 1a-induced membrane association of RNA2 was mediated by sequences in the 5'-proximal third of RNA2. The RNA2 5' untranslated region was sufficient to confer 1a-induced membrane association on a nonviral RNA. However, sequences in the N-terminal region of the 2a open reading frame enhanced 1a responsiveness of RNA2 and a chimeric RNA. A 5'-terminal RNA2 stem-loop important for RNA2 replication was essential for 1a-induced membrane association of RNA2 and, like the 1a-responsive RNA3 intergenic region, contained a required box B motif corresponding to the TPsiC stem-loop of host tRNAs. The level of 1a-induced membrane association of various RNA2 mutants correlated well with their abilities to serve as replication templates. These results support and expand the conclusion that 1a-induced BMV RNA stabilization and membrane association reflect early, 1a-mediated steps in viral RNA replication.

Mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus RNA replication between template recognition and RNA synthesis.

All positive-strand RNA viruses assemble their RNA replication complexes on intracellular membranes. Brome mosaic virus (BMV) replicates its RNA in endoplasmic reticulum (ER)-associated complexes in plant cells and the yeast Saccharomyces cerevisiae. BMV encodes RNA replication factors 1a, with domains implicated in RNA capping and helicase functions, and 2a, with a central polymerase-like domain. Factor 1a interacts independently with the ER membrane, viral RNA templates, and factor 2a to form RNA replication complexes on the perinuclear ER. We show that BMV RNA replication is severely inhibited by a mutation in OLE1, an essential yeast chromosomal gene encoding delta9 fatty acid desaturase, an integral ER membrane protein and the first enzyme in unsaturated fatty acid synthesis. OLE1 deletion and medium supplementation show that BMV RNA replication requires unsaturated fatty acids, not the Ole1 protein, and that viral RNA replication is much more sensitive than yeast growth to reduced unsaturated fatty acid levels. In ole1 mutant yeast, 1a still becomes membrane associated, recruits 2a to the membrane, and recognizes and stabilizes viral RNA templates normally. However, RNA replication is blocked prior to initiation of negative-strand RNA synthesis. The results show that viral RNA synthesis is highly sensitive to lipid composition and suggest that proper membrane fluidity or plasticity is essential for an early step in RNA replication. The strong unsaturated fatty acid dependence also demonstrates that modulating fatty acid balance can be an effective antiviral strategy.

DNA-Directed expression of functional flock house virus RNA1 derivatives in Saccharomyces cerevisiae, heterologous gene expression, and selective effects on subgenomic mRNA synthesis.

Flock house virus (FHV), a positive-strand RNA animal virus, is the only higher eukaryotic virus shown to undergo complete replication in yeast, culminating in production of infectious virions. To facilitate studies of viral and host functions in FHV replication in Saccharomyces cerevisiae, yeast DNA plasmids were constructed to inducibly express wild-type FHV RNA1 in vivo. Subsequent translation of FHV replicase protein A initiated robust RNA1 replication, amplifying RNA1 to levels approaching those of rRNA, as in FHV-infected animal cells. The RNA1-derived subgenomic mRNA, RNA3, accumulated to even higher levels of >100,000 copies per yeast cell, compared to 10 copies or less per cell for 95% of yeast mRNAs. The time course of RNA1 replication and RNA3 synthesis in induced yeast paralleled that in yeast transfected with natural FHV virion RNA. As in animal cells, RNA1 replication and RNA3 synthesis depended on FHV RNA replicase protein A and 3'-terminal RNA1 sequences but not viral protein B2. Additional plasmids were engineered to inducibly express RNA1 derivatives with insertions of the green fluorescent protein (GFP) gene in subgenomic RNA3. These RNA1 derivatives were replicated, synthesized RNA3, and expressed GFP when provided FHV polymerase in either cis or trans, providing the first demonstration of reporter gene expression from FHV subgenomic RNA. Unexpectedly, fusing GFP to the protein A C terminus selectively inhibited production of positive- and negative-strand subgenomic RNA3 but not genomic RNA1 replication. Moreover, changing the first nucleotide of the subgenomic mRNA from G to T selectively inhibited production of positive-strand but not negative-strand RNA3, suggesting that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3.

A mutant allele of essential, general translation initiation factor DED1 selectively inhibits translation of a viral mRNA.

Positive-strand RNA virus genomes are substrates for translation, RNA replication, and encapsidation. To identify host factors involved in these functions, we used the ability of brome mosaic virus (BMV) RNA to replicate in yeast. We report herein identification of a mutation in the essential yeast gene DED1 that inhibited BMV RNA replication but not yeast growth. DED1 encodes a DEAD (Asp-Glu-Ala-Asp)-box RNA helicase required for translation initiation of all yeast mRNAs. Inhibition of BMV RNA replication by the mutant DED1 allele (ded1-18) resulted from inhibited expression of viral polymerase-like protein 2a, encoded by BMV RNA2. Inhibition of RNA2 translation was selective, with no effect on general cellular translation or translation of BMV RNA1-encoded replication factor 1a, and was independent of p20, a cellular antagonist of DED1 function in translation. Inhibition of RNA2 translation in ded1-18 yeast required the RNA2 5' noncoding region (NCR), which also conferred a ded1-18-specific reduction in expression on a reporter gene mRNA. Comparison of the similar RNA1 and RNA2 5' NCRs identified a 31-nucleotide RNA2-specific region that was required for the ded1-18-specific RNA2 translation block and attenuated RNA2 translation in wild-type yeast. Further comparisons and RNA structure predictions suggest a modular arrangement of replication and translation signals in RNA1 and RNA2 5' NCRs that appears conserved among bromoviruses. The 5' attenuator and DED1 dependence of RNA2 suggest that, despite its divided genome, BMV regulates polymerase translation relative to other replication factors, just as many single-component RNA viruses use translational read-through and frameshift mechanisms to down-regulate polymerase. The results show that a DEAD-box helicase can selectively activate translation of a specific mRNA and may provide a paradigm for translational regulation by other members of the ubiquitous DEAD-box RNA helicase family.

Helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand RNA synthesis, and viral RNA capping.

Brome mosaic virus (BMV) encodes two RNA replication proteins: 1a, which contains RNA capping and helicase-like domains, and 2a, which is related to polymerases. BMV 1a and 2a can direct virus-specific RNA replication in the yeast Saccharomyces cerevisiae, which reproduces the known features of BMV replication in plant cells. We constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on BMV RNA3 replication in yeast. The helicase mutants showed no function in any assays used: they were strongly defective in template recruitment for RNA replication, as measured by 1a-induced stabilization of RNA3, and they synthesized no detectable negative-strand or subgenomic RNA. Capping domain mutants divided into two groups. The first exhibited increased template recruitment but nevertheless allowed only low levels of negative-strand and subgenomic mRNA synthesis. The second was strongly defective in template recruitment, made very low levels of negative strands, and made no detectable subgenomes. To distinguish between RNA synthesis and capping defects, we deleted chromosomal gene XRN1, encoding the major exonuclease that degrades uncapped mRNAs. XRN1 deletion suppressed the second but not the first group of capping mutants, allowing synthesis and accumulation of large amounts of uncapped subgenomic mRNAs, thus providing direct evidence for the importance of the viral RNA capping function. The helicase and capping enzyme mutants showed no complementation. Instead, at high levels of expression, a helicase mutant dominantly interfered with the function of the wild-type protein. These results are discussed in relation to the interconnected functions required for different steps of positive-strand RNA virus replication.

Identification and characterization of a host protein required for efficient template selection in viral RNA replication.

Biochemical studies suggest that positive-strand RNA virus replication involves host as well as viral functions. Brome mosaic virus (BMV) is a member of the alphavirus-like superfamily of animal and plant positive-strand RNA viruses. Yeast expressing the BMV RNA replication proteins 1a and 2a supports BMV RNA replication and mRNA synthesis. Using the ability of BMV to replicate in yeast, we show that efficient BMV RNA replication requires Lsm1p, a yeast protein related to core RNA splicing factors but shown herein to be cytoplasmic. Haploid yeast with an Lsm1p mutation was defective in an early template selection step in BMV RNA replication, involving the helicase-like replication protein 1a and an internal viral RNA element conserved with tRNAs. Lsm1p dependence of this interaction was suppressed by adding 3' poly(A) to the normally unpolyadenylated BMV RNA. Our results show Lsm1p involvement in a specific step of BMV RNA replication and connections between Lsm1p and poly(A) function, possibly through interaction with factors binding mRNA 5' ends.

Brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a.

Brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, encodes RNA replication proteins 1a and 2a. 1a contains a C-terminal helicase-like domain and an N-terminal domain implicated in viral RNA capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (ER)-associated replication complex that is the site of BMV-specific RNA-dependent RNA synthesis in plant and yeast cells. 1a also localizes to the ER in the absence of 2a or viral RNA replication templates. To investigate the determinants of 2a localization, we fused 2a to the green fluorescent protein (GFP), creating a functional GFP-2a fusion that supported BMV RNA replication and subgenomic mRNA transcription. In the absence of 1a, the GFP-2a fusion was found to be diffused throughout the cytoplasm and in punctate spots not associated with any cytoplasmic organelle so far tested. Formation of these spots was dependent on the C-terminal half of 2a and may represent aggregation of a fraction of 2a. When coexpressed with 1a, GFP-2a colocalized with 1a and ER-resident protein Kar2p in a partial or complete ring around the nucleus. Consistent with these results, cell fractionation showed that both the GFP-2a fusion and wild-type (wt) 2a remained soluble when expressed alone, while in cells coexpressing 1a, most of the GFP-2a fusion or wt 2a cofractionated with 1a in the rapidly sedimenting membrane fraction. Deletion analysis showed that the N-terminal 120-amino-acid segment of 2a, containing one of two 2a regions previously shown to interact with 1a, was necessary and sufficient for 1a-directed localization of GFP-2a derivatives to the ER. These results suggest that 1a, which also interacts independently with the ER and viral RNA, is a key organizer of RNA replication complex assembly.

Putative RNA capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a.

Brome mosaic virus (BMV) RNA replication is directed by two virus-encoded proteins, 1a and 2a. The amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsP1, which has been implicated in capping viral RNAs. In this study, we examined the enzymatic activities of BMV 1a expressed in yeast, where the protein is fully functional in RNA replication. 1a methylated GTP, dGTP, and the cap analogs GpppG and GpppA, using S-adenosylmethionine (AdoMet) as the methyl donor. Product analysis by nuclear magnetic resonance spectroscopy showed that 1a methylation was specific for guanine position 7. Additionally, 1a interacted with GTP to form a covalent 1a-m(7)GMP complex. This reaction was specific for GTP, required AdoMet, and was accompanied by transfer of (3)H-methyl from AdoMet to the covalent 1a-guanylate complex. The covalent complex could be immunoprecipitated by 1a antibodies. The 1a-m(7)GMP complex was inhibited in catalyzing further methyltransferase reactions. Mutation of conserved amino acids in the N-terminal half of 1a reduced both methyltransferase and covalent complex formation activities to very low or undetectable levels. Covalent 1a-guanylate complex formation took place in similar, AdoMet-dependent fashion in extracts of BMV-infected barley protoplasts. These results show that BMV 1a has activities similar to those of alphavirus nsP1, demonstrating conservation of these putative capping functions across a wide span of sequence divergence within the alphavirus-like superfamily. Conservation of this unusual combination of functions also supports the inference that the superfamily caps viral RNAs by an unusual pathway proceeding via a m(7)GMP intermediate.

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

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

RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein.

Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. The results presented here reveal a striking in vivo switch in CP assembly. Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers. Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid. This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions. Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly. Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate. The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures. The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids. RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.