Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5' splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5' splice donor site, are required for efficient RNA packaging and dimerization.     

A template RNA entry channel in the fingers domain of the poliovirus polymerase

Positive-strand RNA viruses within the Picornaviridae family express an RNA-dependent RNA polymerase, 3D(pol), that is required for viral RNA replication. Structures of 3D(pol) from poliovirus, coxsackievirus, human rhinoviruses, and other picornaviruses reveal a putative template RNA entry channel on the surface of the enzyme fingers domain. Basic amino acids and tyrosine residues along this entry channel are predicted to form ionic and base stacking interactions with the viral RNA template as it enters the polymerase active site. We generated a series of alanine substitution mutations at these residues in the poliovirus polymerase and assayed their effects on template RNA binding, RNA synthesis initiation, rates of RNA elongation, elongation complex (EC) stability, and virus growth. The results show that basic residues K125, R128, and R188 are important for template RNA binding, while tyrosines Y118 and Y148 are required for efficient initiation of RNA synthesis and for EC stability. Alanine substitutions of tyrosines 118 and 148 at the tip of the 3D(pol) pinky finger drastically decreased the rate of initiation as well as EC stability, but without affecting template RNA binding or RNA elongation rates. Viable poliovirus was recovered from HeLa cells transfected with mutant RNAs; however, mutations that dramatically inhibited template RNA binding (K125A-K126A and R188A), RNA synthesis initiation (Y118A, Y148A), or EC stability (Y118A, Y148A) were not stably maintained in progeny virus. These data identify key residues within the template RNA entry channel and begin to define their distinct mechanistic roles within RNA ECs.     

The coat protein leads the way: an update on basic and applied studies with the Brome mosaic virus coat protein

The Brome mosaic virus (BMV) coat protein (CP) accompanies the three BMV genomic RNAs and the subgenomic RNA into and out of cells in an infection cycle. In addition to serving as a protective shell for all of the BMV RNAs, CP plays regulatory roles during the infection process that are mediated through specific binding of RNA elements in the BMV genome. One regulatory RNA element is the B box present in the 5' untranslated region (UTR) of BMV RNA1 and RNA2 that play important roles in the formation of the BMV replication factory, as well as the regulation of translation. A second element is within the tRNA-like 3' UTR of all BMV RNAs that is required for efficient RNA replication. The BMV CP can also encapsidate ligand-coated metal nanoparticles to form virus-like particles (VLPs). This update summarizes the interaction between the BMV CP and RNAs that can regulate RNA synthesis, translation and RNA encapsidation, as well as the formation of VLPs.     

The tobacco mosaic virus assembly origin RNA. Functional characteristics defined by directed mutagenesis

The in vitro reassembly of tobacco mosaic virus (TMV) begins with the specific recognition by the viral coat protein disk aggregate of an internal TMV RNA sequence, known as the assembly origin (Oa). This RNA sequence contains a putative stem-loop structure (loop 1), believed to be the target for disk binding in assembly initiation, which has the characteristic sequence AAGAAGUCG exposed as a single strand at its apex. We show that a 75-base RNA sequence encompassing loop 1 is sufficient to direct the encapsidation by TMV coat protein disks of a heterologous RNA fragment. This RNA sequence and structure, which is sufficient to elicit TMV assembly in vitro, was explored by site-directed mutagenesis. Structure analysis of the RNA identified mutations that appear to effect assembly via a perturbation in RNA structure, rather than by a direct effect on coat protein binding. The binding of the loop 1 apex RNA sequence to coat protein disks was shown to be due primarily to its regularly repeated G residues. Sequences such as (UUG)3 and (GUG)3 are equally effective at initiating assembly, indicating that the other bases are less functionally constrained. However, substitution of the sequences (CCG)3, (CUG)3 or (UCG)3 reduced the assembly initiation rate, indicating that C residues are unfavourable for assembly. Two additional RNA sequences within the 75-base Oa sequence, both of the form (NNG)3, may play subsidiary roles in disk binding. RNA structure plays an important part in permitting selective protein-RNA recognition, since altering the RNA folding close to the apex of the loop 1 stem reduces the rate of disk binding, as does shortening the stem itself. Whereas the RNA sequence making up the hairpin does not in general affect the specificity of the protein-RNA interaction, it is required to present the apex signal sequence in a special conformation. Mechanisms for this are discussed.     

New antiviral pathway that mediates hepatitis C virus replicon interferon sensitivity through ADAR1

While many clinical hepatitis C virus (HCV) infections are resistant to alpha interferon (IFN-alpha) therapy, subgenomic in vitro self-replicating HCV RNAs (HCV replicons) are characterized by marked IFN-alpha sensitivity. IFN-alpha treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through double-stranded RNA-activated protein kinase (PKR) and also through a new pathway involving RNA editing by an adenosine deaminase that acts on double-stranded RNA (ADAR1). More than 200 genes are induced by IFN-alpha, and yet only a few are attributed with an antiviral role. We show that inhibition of both PKR and ADAR1 by the addition of adenovirus-associated RNA stimulates replicon expression and reduces the amount of inosine recovered from RNA in replicon cells. Small inhibitory RNA, specific for ADAR1, stimulated the replicon 40-fold, indicating that ADAR1 has a role in limiting replication of the viral RNA. This is the first report of ADAR's involvement in a potent antiviral pathway and its action to specifically eliminate HCV RNA through adenosine to inosine editing. These results may explain successful HCV replicon clearance by IFN-alpha in vitro and may provide a promising new therapeutic strategy for HCV as well as other viral infections.     

Transient RNA-protein interactions in RNA folding

The RNA folding trajectory features numerous off-pathway folding traps, which represent conformations that are often equally as stable as the native functional ones. Therefore, the conversion between these off-pathway structures and the native correctly folded ones is the critical step in RNA folding. This process, referred to as RNA refolding, is slow, and is represented by a transition state that has a characteristic high free energy. Because this kinetically limiting process occurs in vivo, proteins (called RNA chaperones) have evolved that facilitate the (re)folding of RNA molecules. Here, we present an overview of how proteins interact with RNA molecules in order to achieve properly folded states. In this respect, the discrimination between static and transient interactions is crucial, as different proteins have evolved a multitude of mechanisms for RNA remodeling. For RNA chaperones that act in a sequence-unspecific manner and without the use of external sources of energy, such as ATP, transient RNA-protein interactions represent the basis of the mode of action. By presenting stretches of positively charged amino acids that are positioned in defined spatial configurations, RNA chaperones enable the RNA backbone, via transient electrostatic interactions, to sample a wider conformational space that opens the route for efficient refolding reactions.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.     

Antiviral protein APOBEC3G localizes to ribonucleoprotein complexes found in P bodies and stress granules

Members of the APOBEC (apolipoprotein B mRNA-editing enzyme catalytic polypeptide 1-like) family of cytidine deaminases inhibit host cell genome invasion by exogenous retroviruses and endogenous retrotransposons. Because these enzymes can edit DNA or RNA and potentially mutate cellular targets, their activities are presumably regulated; for instance, APOBEC3G (A3G) recruitment into high-molecular-weight ribonucleoprotein (RNP) complexes has been shown to suppress its enzymatic activity. We used tandem affinity purification together with mass spectrometry (MS) to identify protein components within A3G-containing RNPs. We report that numerous cellular RNA-binding proteins with diverse roles in RNA function, metabolism, and fate determination are present in A3G RNPs but that most interactions with A3G are mediated via binding to shared RNAs. Confocal microscopy demonstrated that substantial quantities of A3G localize to cytoplasmic microdomains that are known as P bodies and stress granules (SGs) and are established sites of RNA storage and metabolism. Indeed, subjecting cells to stress induces the rapid redistribution of A3G and a number of P-body proteins to SGs. Among these proteins are Argonaute 1 (Ago1) and Argonaute 2 (Ago2), factors that are important for RNA silencing and whose interactions with A3G are resistant to RNase treatment. Together, these findings reveal that A3G associates with RNPs that are found throughout the cytosol as well as in discrete microdomains. We also speculate that the interplay between A3G, RNA-silencing pathways, and cellular sites of RNA metabolism may contribute to A3G's role as an inhibitor of retroelement mobility and as a possible regulator of cellular RNA function.     

Application of pyronin Y(G) in cytochemistry of nucleic acids

Chinese hamster ovary (CHO) cells or isolated nuclei were stained with pyronin Y(PY) and analyzed by absorption or fluorescence microscopy, as well as by flow cytometry. Specificity of the staining reaction was assayed by testing sensitivity of the stainable material to RNase or DNase. The colored complexes detected by light absorption in fixed cells stained with PY are nonfluorescent and are most likely the products of condensation of single-stranded (ss) RNA by PY; the poly(rA) and poly(rA,rG) are the most sensitive to condensation. The products of PY interaction with double-stranded (ds) nucleic acids are fluorescent and can be detected in cells by cytofluorometry. PY used alone stains both DNA and RNA, and the staining capabilities of these nucleic acids vary depending upon the PY concentration at equilibrium; at a concentration above 330 microM, the RNA stainability decreases, perhaps due to its denaturation and condensation caused by the dye. In the presence of Hoechst 33342, PY can specifically stain RNA in fixed cells or isolated cell nuclei. Because only complexes of PY with ds RNA are fluorescent, this dye can be used as a probe of RNA conformation, e.g., to monitor denaturation of RNA in situ. The RNA stainability of mitotic cells is about 25% lower than that of cells in G2 phase, which indicates that during mitosis proportionately less cellular RNA is in the ds conformation. The advantages and limitations of the two cytochemical methods for DNA/RNA detection, one based on the use of Hoechst 33342 and PY, and another employing the metachromatic properties of acridine orange, are compared.     

RNA binding efficacy of theophylline,theobromine and caffeine

The binding of naturally occurring methylxanthines such as theophylline, theobromine and caffeine to nucleic acids are reckoned to be pivotal as they are able to modulate the cellular activities. We explore the interaction of yeast RNA binding efficacy of the above xanthine derivatives by using UV absorption differential spectroscopy and Fourier Transform Infrared (FTIR) spectroscopy. Both the analyses show discrimination in their binding affinity to RNA. The differential UV-spectrum at P/D 3.3 reveals the greater RNA binding activity for theophylline (85 +/- 5%), whereas moderate and comparatively less binding activity for theobromine (45 +/- 5%) and caffeine (30 +/- 5%) and the binding activity was found to depend on concentration of the drugs. In FTIR analysis we observed changes in the amino group (NH) of RNA complexed by drugs, where the NH band is found to become very broad, indicating hydrogen bonding (H-bonding) with theophylline (3343.4 cm(-1)), theobromine (3379.8 cm(-1)) and caffeine (3343 cm(-1)) as compared to the free RNA (3341.6 cm(-1)). Furthermore in RNA-theophylline complex, it is observed that the carbonyl (C=O) vibration frequency (nu(C=O)) of both drug (nu(C=O)=1718, 1666 cm(-1)) as well as RNA (nu(C=O)=1699, 1658 cm(-1)) disappeared and a new vibration band appeared around 1703 cm(-1), indicating that the C=O and NH groups of drug and RNA are effectively involved in H-bonding. Whereas in RNA-theobromine and RNA-caffeine complexes, we found very little changes in C=O frequency and only broadening of the NH band of RNA due to complexation is observed in these groups. The changes in the vibrations of G-C/A-U bands and other bending frequencies are discussed. Thus the discrimination in the binding affinity of methylxanthines with RNA molecule shows that strong RNA binding drugs like theophylline can selectively be delivered to RNA targets of microbial pathogens having the mechanism of RNA catalysis.     

A simple method for electrophoretic selection and fractionation of poly(A)-containing RNA and poly(A).

A simple procedure, useful for quantitative and qualitative assays of poly(A)-containing RNA and poly(A), as well as for preparative purposes, is described. Glass-fiber filters with immobilized poly(U), a well-known technique for absorption of poly(A)-containing RNA, is combined with electrophoresis in a gel slab of agarose. In front of each of the two troughs in a gel slab, glass-fiber filters are inserted, one of which is impregnated with poly(U). Two identical RNA samples, e.g., split samples of total RNA from salivary glands of Chironomus tentans, are applied to the troughs and are moved electrophoretically across two different filters. The electrophoresis is conducted under conditions which promote the formation of duplexes between absorbed poly(U) and moving poly(A). While the passage of RNA chains across the control filter may take place essentially freely, RNA molecules that contain poly(A) hybridize with poly(U) fixed in the glass-fiber filter and become trapped there. The difference between resulting gel profiles [pattern of the total RNA minus the pattern of RNA not containing poly(A)] yields the electrophoretic distribution of poly(A)-containing RNA. In addition, poly(A)-containing RNA can be eluted from the poly(U) filter with formamide and subjected to electrophoresis without a subsequent precipitation in ethanol. No measurable quantities of ribosomal RNA or tRNA are retained on the poly(U) glass-fiber filters. The hybridization technique enables a quantitative retention of poly(A) molecules representing a wide range of chain lengths.     

Plant small nuclear RNAs. Nucleolar U3 snRNA is present in plants: partial characterization

Nuclei, isolated from a number of plant species by either of two independent, newly developed methods, regularly contained a common set of low-molecular-mass RNAs. Partial characterization of these RNAs, based on cell fractionation, polyacrylamide gel electrophoretic and chemical sequencing techniques, as well as comparison with literature data, revealed that, in addition to tRNA, 5S RNA and 5.8S RNA, plant nuclei contain two families of low-molecular-mass RNAs, that are counterparts of vertebrate U1 and U5 RNAs respectively, and three individual low-molecular-mass RNA species. One of these may be related to vertebrate U6 RNA. The two others are true eukaryotic U2 and U3 RNAs, respectively, on the basis of the following lines of evidence obtained from analyses of broad bean nuclear RNAs. The 3'-end portion (121 nucleotides sequenced) of broad bean U2 RNA shows a nearly perfect sequence homology with that of authentic pea U2 RNA. Broad bean U3 RNA is localized in the nucleolus and its 3'-end portion (164 nucleotides sequenced) (a) shows sequence homology with that of both rat U3 RNA (48%) and Dictyostelium D2 RNA (39%), (b) has a secondary structure which fits perfectly that proposed for both rat U3 RNA and Dictyostelium D2 RNA, and (c) contains the specific sequence which, in a model based on the primary structure of rat U3 RNA, is supposed to be involved in the processing of eukaryotic 32S pre-ribosomal RNA. This is the first report on the occurrence in plants of nucleolar U3 RNA.     

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.