Nidovirales: evolving the largest RNA virus genome

This review focuses on the monophyletic group of animal RNA viruses united in the order Nidovirales. The order includes the distantly related coronaviruses, toroviruses, and roniviruses, which possess the largest known RNA genomes (from 26 to 32kb) and will therefore be called "large" nidoviruses in this review. They are compared with their arterivirus cousins, which also belong to the Nidovirales despite having a much smaller genome (13-16kb). Common and unique features that have been identified for either large or all nidoviruses are outlined. These include the nidovirus genetic plan and genome diversity, the composition of the replicase machinery and virus particles, virus-specific accessory genes, the mechanisms of RNA and protein synthesis, and the origin and evolution of nidoviruses with small and large genomes. Nidoviruses employ single-stranded, polycistronic RNA genomes of positive polarity that direct the synthesis of the subunits of the replicative complex, including the RNA-dependent RNA polymerase and helicase. Replicase gene expression is under the principal control of a ribosomal frameshifting signal and a chymotrypsin-like protease, which is assisted by one or more papain-like proteases. A nested set of subgenomic RNAs is synthesized to express the 3'-proximal ORFs that encode most conserved structural proteins and, in some large nidoviruses, also diverse accessory proteins that may promote virus adaptation to specific hosts. The replicase machinery includes a set of RNA-processing enzymes some of which are unique for either all or large nidoviruses. The acquisition of these enzymes may have improved the low fidelity of RNA replication to allow genome expansion and give rise to the ancestors of small and, subsequently, large nidoviruses.     

Transfer of specific immunity with RNA.

The term "immune RNA" (iRNA), as presently used, refers not to a characterized species of RNA but to phenol extracts of lymphocytes from immunized animals, which have the capacity to transfer a variety of immune products or activities. These include synthesis of specific antibody, development of specific cellular (delayed) hypersensitivity, and production of immunoglobulin allotypic or idiotypic specificities. Recent evidence that iRNA might be used as a specific therapeutic agent to induce immunity to tumors has stimulated an increased interest in this area of research. The purpose of this article is to review the current status of studies on iRNA.     

Differential gene expression between young and senescent, quiescent WI-38 cells.

To investigate age-related changes in gene expression in WI-38 cells, we isolated RNA from young and senescent, quiescent cultures and made subtracted cDNA libraries. Density-arrested cells were incubated in serum-free MCDB-104 for 3 days. RNA was then isolated and subtracted cDNA libraries were made in the phagemid vector pCDM8. Both by picking clones at random from these subtracted libraries and by differential hybridization screening with subtracted cDNA probes from young and senescent cells, we have identified a total of 11 genes for which RNA is expressed differentially in these quiescent young and senescent WI-38 cultures. Two genes, EPC-1 and EPC-A2, with elevated RNA levels in young cells, have sequences which have not previously been identified. Two of the genes with elevated RNA expression in the senescent cells are the mitochondria-coded genes for NADH dehydrogenase subunit 4 and for cytochrome b. We also identified seven other genes with elevated RNA levels in senescent cells. Three of these, LPC-1, LPC-14 and LPC-24, have been partially sequenced and have not previously been identified. These studies show that density-arrested, serum-deprived, quiescent young and senescent cells express a number of genes differentially. These differences are not growth-dependent, but are age-dependent. Our studies also show that the methods employed here, which include careful regulation of the cell cultures and subtraction of the libraries, result in libraries from which differentially expressed genes can be identified, either by random selection or by differential hybridization screening with subtracted probes.     

Chloroplast RNA polymerase genes of Chlamydomonas reinhardtii exhibit an unusual structure and arrangement

Summary Nucleotide sequence analysis of a 17043 basepair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5-and 3-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5 half of rpoC (termed rpoC1 in other species) is not present at the 5 end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.     

Formation of RNA and protein in cells infected with standard and defective Sindbis virus

Our studies on the formation of Sindbis virus proteins have established that: 1. one of the two envelope proteins (E2) accumulates in infected cells as a higher molecular weight precursor that is slowly converted to the virion protein; 2. the large protein (mol. wt 130000 daltons), that accumulates in cells infected with a temperature-sensitive mutant of Sindbis virus contains sequences of the three virion proteins; and 3. the protein (mol. wt. 100000 daltons) isolated from BHK cells infected with Sindbis virus is related in sequence to the two envelope proteins.We have investigated the formation of defective-interfering (DI) particles of Sindbis virus and their ability to inhibit the replication of standard virus. BHK cells infected with passages of Sindbis virus containing DI particles accumulate a species of RNA (20S) that is about half the molecular weight of the 26S RNA. We have demonstrated by competitive hybridization experiments that 20S RNA contains half the sequences of 26S RNA. We also present evidence that in contrast to 26S RNA, 20S does not bind to polysomesin vivo and is not translatedin vitro.Presented on the Meeting on Studies on Virus Replication of the Commission of the European Communities in Brüssel, May 1974.     

RNA-transfected dendritic cells

Based on their unique ability to stimulate primary immune responses, dendritic cells are the most potent antigen-presenting cells known. This ability stems from the fact that they are very efficient at the uptake and processing of antigen and they express high levels of major histocompatibility complex class I and class II, as well as costimulatory molecules, which are required to prime naive cytotoxic T-cells. Many groups of investigators have tried to take advantage of these features by developing dendritic cell-based vaccines against tumors and infectious diseases. While the basic principle in these studies is the same--dendritic cells pulsed with antigen are used to elicit cytotoxic T-cell responses--the methods used are varied. This is particularly true with respect to the nature of the antigen used and the method of antigen delivery. In this article, we will focus on the use of RNA as a form of antigen with which to load dendritic cells. We will discuss the rationale behind using RNA as an antigen source and will review recent studies in both murine and human settings that use RNA-pulsed dendritic cells as vaccines.     

Interaction of poliovirus-encoded 2C/2BC polypeptides with the 3' terminus negative-strand cloverleaf requires an intact stem-loop b

The poliovirus-encoded, membrane-associated polypeptide 2C and its precursor, 2BC, is believed to be required for initiation and elongation of viral RNA synthesis. Previous studies have shown that the 2C polypeptide specifically interacts with the 3'-terminal sequence of poliovirus negative-strand RNA. This interaction is facilitated by the presence of the conserved sequence UGUUUU in the "stem a" within the 3'-terminal cloverleaf structure. We show that the 2C precursor, 2BC, also interacts with the 3'-terminal cloverleaf of the negative-strand RNA. We also demonstrate here that interaction of 2C/2BC with the negative strand 3'-terminal sequence not only depends on an intact "stem a" containing the UGUUUU sequence but is equally influenced by the presence of an intact "stem b" within the negative-strand cloverleaf. The results presented here suggest that the spatial configuration of stem a UGUUUU sequence with respect to an intact stem-b is crucial for 2C/2BC interaction with the 3'-terminal negative-strand cloverleaf structure.     

A "mitochondrial cascade hypothesis" for sporadic Alzheimer's disease

Alzheimer's disease (AD) includes etiologically heterogeneous disorders characterized by senile or presenile dementia, extracellular amyloid protein aggregations containing an insoluble amyloid precursor protein derivative, and intracytoplasmic tau protein aggregations. Recent studies also show excess neuronal aneuploidy, programmed cell death (PCD), and mitochondrial dysfunction. The leading AD molecular paradigm, the "amyloid cascade hypothesis", is based on studies of rare autosomal dominant variants and does not specify what initiates the common late-onset, sporadic form. We propose for late-onset, sporadic AD a "mitochondrial cascade hypothesis" that comprehensively reconciles seemingly disparate histopathologic and pathophysiologic features. In our model, the inherited, gene-determined make-up of an individual's electron transport chain sets basal rates of reactive oxygen species (ROS) production, which determines the pace at which acquired mitochondrial damage accumulates. Oxidative mitochondrial DNA, RNA, lipid, and protein damage amplifies ROS production and triggers three events: (1) a reset response in which cells respond to elevated ROS by generating the beta-sheet protein, beta amyloid, which further perturbs mitochondrial function, (2) a removal response in which compromised cells are purged via PCD mechanisms, and (3) a replace response in which neuronal progenitors unsuccessfully attempt to re-enter the cell cycle, with resultant aneuploidy, tau phosphorylation, and neurofibrillary tangle formation. In addition to defining a role for aging in AD pathogenesis, the mitochondrial cascade hypothesis also allows and accounts for histopathologic overlap between the sporadic, late-onset and autosomal dominant, early onset forms of the disease.     

Membrane-bound virions of coxsackievirus B4: Cellular localization,analysis of the genomic RNA,genome-linked protein,and effect on host macromolecular synthesis

Summary Hela cells infected with several group B coxsackieviruses contain, in addition to standard virions, a population of virus-specific ribonucleoprotein particles which we (5) designated membrane-bound virions (MBV). MBVs differ from standard virions in buoyant density, yield, appearance, protein composition and infectivity. Here we present several new features of MBVs of coxsackievirus B4. The MBVs are lighter ( about 1.30) and are localized in rough membranes, intermixed with virions. They contain 35S virion RNA covalently linked with a small protein, VPg. The VPg contain two proteins of different charge. MBV VPg is considerably smaller than the 5300-dalton virion VPg. MBV RNA is homologous to the base sequence present in B4 virus double-stranded RNA. The T₁ oligonucleotide fingerprint of MBV RNA is distinguishable from that of virion RNA by one oligonucleotide. Several oligonucleotides of virion RNA appear to occur in submolar quantities in MBV RNA. MBVs are 75 to >200 times less infective; they inhibit host cell macromolecular synthesis less efficiently than virions. In coinfected cells, the extent of inhibition of host synthesis is less severe than in cells infected with virions alone, which suggest interference by MBV particles.With 6 Figures     

Structure of avian orthoreovirus virion by electron cryomicroscopy and image reconstruction

Among members of the genus Orthoreovirus, family Reoviridae, a group of non-enveloped viruses with genomes comprising ten segments of double-stranded RNA, only the "non-fusogenic" mammalian orthoreoviruses (MRVs) have been studied to date by electron cryomicroscopy and three-dimensional image reconstruction. In addition to MRVs, this genus comprises other species that induce syncytium formation in cultured cells, a property shared with members of the related genus Aquareovirus. To augment studies of these "fusogenic" orthoreoviruses, we used electron cryomicroscopy and image reconstruction to analyze the virions of a fusogenic avian orthoreovirus (ARV). The structure of the ARV virion, determined from data at an effective resolution of 14.6 A, showed strong similarities to that of MRVs. Of particular note, the ARV virion has its pentameric lambda-class core turret protein in a closed conformation as in MRVs, not in a more open conformation as reported for aquareovirus. Similarly, the ARV virion contains 150 copies of its monomeric sigma-class core-nodule protein as in MRVs, not 120 copies as reported for aquareovirus. On the other hand, unlike that of MRVs, the ARV virion lacks "hub-and-spokes" complexes within the solvent channels at sites of local sixfold symmetry in the incomplete T=13l outer capsid. In MRVs, these complexes are formed by C-terminal sequences in the trimeric mu-class outer-capsid protein, sequences that are genetically missing from the homologous protein of ARVs. The channel structures and C-terminal sequences of the homologous outer-capsid protein are also genetically missing from aquareoviruses. Overall, the results place ARVs between MRVs and aquareoviruses with respect to the highlighted features.     

A novel species-specific RNA related to alternatively spliced amyloid precursor protein mRNAs.

Using an S1 nuclease protection assay, we have identified a novel "variant" Amyloid Precursor Protein (APP) RNA in human brain which is 3-6-fold more abundant than APP-770, but less abundant than APP-751 or APP-695. This variant, referred to as amyloid precursor-related protein 365 (APRP-365), is not detected in mouse and rat brain RNAs. A 1.6 kilo-basepair cDNA clone corresponding to this variant APP RNA predicts the existence of a 365 amino acid protein that is similar to the amino-terminal end of APP-770 but lacks the beta-amyloid peptide and any hydrophobic transmembrane spanning domains. In a modified polymerase chain reaction (PCR), we used amplification of reverse transcribed mRNA to confirm and extend our S1 observations. Together, the features of APRP-365 suggest that the human variant is a soluble protein containing a Kunitz protease inhibitor domain.     

Multiple small RNA species and the viroid hypothesis for the sunblotch disease of avocado

Multiple RNA species (ASV(1-5)) ranging in size from about 61,000 to 185,000 daltons have been detected in association with sunblotch disease of avocado. The predominant species, ASV(5), with a molecular weight of 61,000 accumulates to concentration approaching 5 S RNA. All RNA species are detected in extracts from symptomatic tissue of avocado var. Fuerte but not from asymptomatic tissue of the same leaves from which the disease agent can be transmitted to avocado. The probability that the predominant ASV(5) species is either the putative sunblotch "viroid" or a noninfectious "viroid-related" molecule is discussed.     

Japanese encephalitis virus replication: A procedure for the selective isolation and characterization of viral RNA species

Summary The viral RNA species synthesized in a porcine kidney cell line, PS(Y-15), by Japanese encephalitis virus (JEV) are described. Virus titers on these cells ranged between 10⁶ to 10⁷ p.f.u./ml at the end of 2 to 3 days incubation at 35° C. Actinomycin D (AD) could not be used to unmask JEV RNA synthesis since it inhibited virus replication at concentrations necessary to substantially reduce host cell RNA synthesis. Treatment of cells with 1 g AD/ml and removal prior to infection permitted good JEV replication, and at the same time strongly suppressed synthesis of 28 S and 18 S cellular ribosomal RNA. The problem of separating viral RNA from non-ribosomal RNA that was still being synthesized by AD pretreated cells was resolved by the isolation of the cytoplasmic membrane fraction of infected cells. RNA extracted from the membranes of infected AD pretreated cells and analyzed for sedimentation characteristics on sucrose gradients has four RNA species not found in uninfected cells.They are: (1) 45 S single stranded RNA believed to be the infectious RNA found in the virion; (2) a 27 S RNA single stranded RNA; (3) a 20 S ribonuclease resistant RNA believed to be double stranded and (4) an 8 S RNA species. The RNA species found in JEV infected cells, except for the 8 S form, have been found in group A arboviruses. The procedure described utilizing AD pretreatment of host cells and the separation of the cellular cytoplasmic fraction may well have value for the study of the biosynthetic events involved in the replication of other animal viruses that are inhibited by AD.This work was supported by the Bureau of Medicine and Surgery, Department of the Navy, Project MR 041.05.01-0006B3GJ.The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large.Postdoctoral Research Associate, National Research Council, supported by the Bureau of Medicine and Surgery, Department of the Navy.     

Hansa: an automated method for discriminating disease and neutral human nsSNPs

Variations are mostly due to nonsynonymous single nucleotide polymorphisms (nsSNPs), some of which are associated with certain diseases. Phenotypic effects of a large number of nsSNPs have not been characterized. Although several methods have been developed to predict the effects of nsSNPs as "disease" or "neutral," there is still a need for development of methods with improved prediction accuracies. We, therefore, developed a support vector machine (SVM) based method named Hansa which uses a novel set of discriminatory features to classify nsSNPs into disease (pathogenic) and benign (neutral) types. Validation studies on a benchmark dataset and further on an independent dataset of well-characterized known disease and neutral mutations show that Hansa outperforms the other known methods. For example, fivefold cross-validation studies using the benchmark HumVar dataset reveal that at the false positive rate (FPR) of 20% Hansa yields a true positive rate (TPR) of 82% that is about 10% higher than the best-known method. Hansa is available in the form of a web server at http://hansa.cdfd.org.in:8080.