Showing papers on "RNA-dependent RNA polymerase published in 1993"

Evolution and Taxonomy of Positive-Strand RNA Viruses: Implications of Comparative Analysis of Amino Acid Sequences

Abstract: Despite the rapid mutational change that is typical of positive-strand RNA viruses, enzymes mediating the replication and expression of virus genomes contain arrays of conserved sequence motifs. Proteins with such motifs include RNA-dependent RNA polymerase, putative RNA helicase, chymotrypsin-like and papain-like proteases, and methyltransferases. The genes for these proteins form partially conserved modules in large subsets of viruses. A concept of the virus genome as a relatively evolutionarily stable "core" of housekeeping genes accompanied by a much more flexible "shell" consisting mostly of genes coding for virion components and various accessory proteins is discussed. Shuffling of the "shell" genes including genome reorganization and recombination between remote groups of viruses is considered to be one of the major factors of virus evolution. Multiple alignments for the conserved viral proteins were constructed and used to generate the respective phylogenetic trees. Based primarily on the tentative phylogeny for the RNA-dependent RNA polymerase, which is the only universally conserved protein of positive-strand RNA viruses, three large classes of viruses, each consisting of distinct smaller divisions, were delineated. A strong correlation was observed between this grouping and the tentative phylogenies for the other conserved proteins as well as the arrangement of genes encoding these proteins in the virus genome. A comparable correlation with the polymerase phylogeny was not found for genes encoding virion components or for genome expression strategies. It is surmised that several types of arrangement of the "shell" genes as well as basic mechanisms of expression could have evolved independently in different evolutionary lineages. The grouping revealed by phylogenetic analysis may provide the basis for revision of virus classification, and phylogenetic taxonomy of positive-strand RNA viruses is outlined. Some of the phylogenetically derived divisions of positive-strand RNA viruses also include double-stranded RNA viruses, indicating that in certain cases the type of genome nucleic acid may not be a reliable taxonomic criterion for viruses. Hypothetical evolutionary scenarios for positive-strand RNA viruses are proposed. It is hypothesized that all positive-strand RNA viruses and some related double-stranded RNA viruses could have evolved from a common ancestor virus that contained genes for RNA-dependent RNA polymerase, a chymotrypsin-related protease that also functioned as the capsid protein, and possibly an RNA helicase.

The rule of six, a basic feature for efficient replication of Sendai virus defective interfering RNA.

Abstract: The addition of the hepatitis delta virus genomic ribozyme to the 3' end sequence of a Sendai virus defective interfering RNA (DI-H4) allowed the reproducible and efficient replication of this RNA by the viral functions expressed from cloned genes when the DI RNA was synthesized from plasmid. Limited nucleotide additions or deletions (+7 to -7 nucleotides) in the DI RNA sequence were then made at five different sites, and the different RNA derivatives were tested for their abilities to replicate. Efficient replication was observed only when the total nucleotide number was conserved, regardless of the modifications, or when the addition of a total of 6 nucleotides was made. The replicated RNAs were shown to be properly enveloped into virus particles. It is concluded that, to form a proper template for efficient replication, the Sendai virus RNA must contain a total number of nucleotides which is a multiple of 6. This was interpreted as the need for the nucleocapsid protein to contact exactly 6 nucleotides.

Poliovirus RNA synthesis utilizes an RNP complex formed around the 5'-end of viral RNA.

Abstract: The structure of a ribonucleoprotein complex formed at the 5'-end of poliovirus RNA was investigated. This complex involves the first 90 nucleotides of poliovirus genome which fold into a cloverleaf-like structure and interact with both uncleaved 3CD, the viral protease-polymerase precursor, and a 36 kDa ribosome-associated cellular protein. The cellular protein is required for complex formation and interacts with unpaired bases in one stem-loop of the cloverleaf RNA. Amino acids within the 3C protease which are important for RNA binding were identified by site-directed mutagenesis and the crystal structure of a related protease was used to model the RNA binding domain within the viral 3CD protein. The physiologic importance of the ribonucleic-protein complex is suggested by the finding that mutations that disrupt complex formation abolish RNA replication but do not affect RNA translation or stability. Based on these structural and functional findings we propose a model for the initiation of poliovirus RNA synthesis where an initiation complex consisting of 3CD, a cellular protein, and the 5'-end of the positive strand RNA catalyzes in trans the initiation of synthesis of new positive stranded RNA.

Crystal structure of bacteriophage T7 RNA polymerase at 3.3 Å resolution

Abstract: The crystal structure of T7 RNA polymerase reveals a molecule organized around a cleft that can accommodate a double-stranded DNA template A portion (approximately 45%) of the molecule displays extensive structural homology to the polymerase domain of Klenow fragment and more limited homology to the human immunodeficiency virus HIV-1 reverse transcriptase A comparison of the structures and sequences of these polymerases identifies structural elements that may be responsible for discriminating between ribonucleotide and deoxyribonucleotide substrates, and RNA and DNA templates The relative locations of the catalytic site and a specific promoter recognition residue allow the orientation of the polymerase on the template to be defined

Initiation of transcription by RNA polymerase II: a multi-step process

Abstract: Publisher Summary This chapter discusses the initiation of transcription of protein-coding genes. The RNA polymerase II(RNAPII) of the yeast Saccharomyces cerevisiae has been a useful prototype in the study of eukaryotic RNAPII, as features such as function and subunit structure have been highly conserved. The yeast RNAPII is composed of 11 polypeptides with apparent masses ranging from 220 to 10 kDa. This is in contrast to eukaryotic cells, which contain three distinct RNA polymerases, each containing from 8 to 14 polypeptides and responsible for transcribing its own set of genes: RNA polymerase I (RNAPI), which transcribes ribosomal RNA; RNAPII, the RNA polymerase of protein-coding, or class 11, genes; and RNAPIII, which transcribes 5-S rRNA and tRNA genes. In this chapter, only RNAPII is discussed, and only as it pertains to transcription initiation.

Novel mechanism for reverse transcription in hepatitis B viruses.

Abstract: Reverse transcription of all retroviruses and most retroid elements requires tRNA as a primer for DNA synthesis. However, in hepatitis B viruses the viral polymerase itself acts as a primer for reverse transcription (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992). We have now demonstrated that in order to prime DNA synthesis, the polymerase binds to an RNA hairpin, which then serves as a template for the formation of a short DNA primer that is covalently linked to protein. Following its synthesis, the nascent DNA strand apparently dissociates from its template and reanneals with complementary sequences at the 3' end of the RNA genome, where DNA synthesis continues. Since this RNA hairpin also functions as a packaging signal for viral RNA, hepadnaviruses have adopted a replication strategy that relies on the same signal for two biochemically distinct events, RNA packaging and reverse transcription. This mechanism is without precedent among all known retroid elements and among other viruses and bacteriophages that use protein as a primer for RNA or DNA synthesis. It could provide an effective target for antiviral therapy, which is required for the treatment of more than 300 million carriers of hepatitis B virus.

Method and reagent for inhibiting viral replication.

Abstract: Enzymatic RNA molecules which specifically cleave a picornavirus RNA, an immunodeficiency virus RNA in a gene required for viral replication, e.g., the vif, nef, tat or rev gene regions, RNA of a hepatitis virus, T-cell leukemia virus RNA, RNA of a hepatitis C virus, mRNA or rhRNA of a cytomegalovirus, an influenza virus RNA, and a herpes simplex virus mRNA molecule; and methods and reagents useful in selection and optimization of ribozymes. In particular, the enzymatic RNA molecule is formed in a hammerhead motif, in the motif of a hairpin, hepatitis delta virus, group I intron or RNaseP-like RNA. The figure is a diagrammatic representation of an enzymatic RNA molecule in the hammerhead motif.

Semliki Forest virus expression system: production of conditionally infectious recombinant particles.

Abstract: In the recently developed Semliki Forest virus (SFV) DNA expression system, recombinant RNA encoding the viral replicase, and helper RNA molecules encoding the structural proteins needed for virus assembly are cotransfected into cells. Since the helper RNA lacks the sequence needed for its packaging into nucleocapsids, only recombinant RNAs should be packaged. We have found, however, that small amounts of replication-proficient SFV particles can still be produced. Here we describe the construction of a helper variant with a mutation in the gene encoding the viral spike protein such that its product cannot undergo normal proteolytic processing to activate viral entry functions. Hence, the recombinant stock is noninfectious, but may be activated by cleavage with chymotrypsin. When recombinant virus produced with the new helper was examined in a variety of assays, including sensitive animal tests, we were unable to detect any replication-competent SFV particles. We therefore conclude that this conditional expression system meets extremely stringent biosafety requirements.

The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor 4A is required for RNA binding and ATP hydrolysis.

Abstract: eIF-4A is a eukaryotic translation initiation factor that is required for mRNA binding to ribosomes. It exhibits single-stranded RNA-dependent ATPase activity, and in combination with a second initiation factor, eIF-4B, it exhibits duplex RNA helicase activity. eIF-4A is the prototype of a large family of proteins termed the DEAD box protein family, whose members share nine highly conserved amino acid regions. The functions of several of these conserved regions in eIF-4A have previously been assigned to ATP binding, ATPase, and helicase activities. To define the RNA-binding region of eIF-4A, a UV-induced cross-linking assay was used to analyze binding of mutant eIF-4A proteins to RNA. Mutants carrying mutations in the ATP-binding region (AXXXXGKT), ATPase region (DEAD), helicase region (SAT), and the most carboxy-terminal conserved region of the DEAD family, HRIGRXXR, were tested for RNA cross-linking. We show that mutations, either conservative or not, in any one of the three arginines in the HRIGRXXR sequence drastically reduced eIF-4A cross-linking to RNA. In addition, all the mutations in the HRIGRXXR region abrogate RNA helicase activity. Some but not all of these mutations affect ATP binding and ATPase activity. This is consistent with the hypothesis that the HRIGRXXR region is involved in the ATP hydrolysis reaction and would explain the coupling of ATPase and RNA-binding/helicase activities. Our results show that the HRIGRXXR region, which is QRXGRXXR or QXXGRXXR in the RNA and DNA helicases of the helicase superfamily II, is involved in ATP hydrolysis-dependent RNA interaction during unwinding. We also show that mutations in other regions of eIF-4A that abolish ATPase activity sharply decrease eIF-4A cross-linking to RNA. A model is proposed in which eIF-4A first binds ATP, resulting in a change in eIF-4A conformation which allows RNA binding that is dependent on the HRIGRXXR region. Binding of RNA induces ATP hydrolysis, leading to a more stable interaction with RNA. This process is then linked to unwinding of duplex RNA in the presence of eIF-4B.

RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis.

Abstract: The genomic RNA of human astrovirus was sequenced and found to contain 6797 nt organized into three open reading frames (1a, 1b, and 2). A potential ribosomal frameshift site identified in the overlap region of open reading frames 1a and 1b consists of a "shifty" heptanucleotide and an RNA stem-loop structure that closely resemble those at the gag-pro junction of some retroviruses. This translation frame-shift may result in the suppression of in-frame amber termination at the end of open reading frame 1a and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase. Comparative sequence analysis indicated that the protease and polymerase of astrovirus are only distantly related to the respective enzymes of other positive-strand RNA viruses. The astrovirus polyprotein lacks the RNA helicase domain typical of other positive-strand RNA viruses of similar genome size. The genomic organization and expression strategy of astrovirus, with the protease and the polymerase brought together by predicted frameshift, most closely resembled those of plant leuteoviruses. Specific features of the sequence and genomic organization support the classification of astroviruses as an additional family of positive-strand RNA viruses, designated Astroviridae.

An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation.

Abstract: Selective encapsidation of hepatitis B virus (HBV) genomic RNA within cytoplasmic core particles requires recognition of the cis-encapsidation signal, (termed epsilon) located at the 5' end of genomic RNA By transfecting plasmids expressing chimeric RNAs bearing HBV sequences fused to lacZ, we have mapped the minimal region of epsilon to the 5' 94 nucleotides (nt) of genomic RNA Enzymatic probing of the RNA secondary structure in this region (by using either in vitro transcripts or RNA extracted from HBV core particles) reveals a stem-loop structure containing a lower stem, a 6-nt bulge, an upper stem with a single unpaired U residue, and a 6-nt loop The functional role of this structure in encapsidation was explored by examining the effects of mutations in epsilon on encapsidation of RNA in vivo These studies reveal that (i) in the lower stem, base pairing but not specific primary sequence is required for function; (ii) there is no requirement for base pairing in the lower portion of the upper stem, but base pairing elsewhere in this stem contributes to packaging efficiency; (iii) the presence of the 6-nt bulge, but not its primary sequence, is important for function; and (iv) specific nucleotide sequences in the loop and in regions of the upper stem are critical for RNA encapsidation

The RNA world : the nature of modern RNA suggests a prebiotic RNA world

Abstract: THE ORIGINS OF RNA AND RNA AT THE ORIGIN 1 Setting the Stage The History, Chemistry, and Geobiology behind RNA 2 Progress toward Understanding the Origin of the RNA World 3 Protocells: Genetic Polymers Inside Membrane Vesicles BUILDING A FUNCTIONAL RNA 4 Riboswitches and the RNA World 5 Catalytic Strategies of Self-cleaving Ribozymes: Relics of an RNA World? 6 How the Group I Intron Works: A Case Study of RNA Structure and Function EXITING THE ANCIENT RNA WORLD - SYNTHETASES AND RIBOSOMES 7 RNA, Lipids, and Membranes 8 Aminoacyl tRNA Synthetases: From the RNA World to the Theater of Proteins 9 The Roles of RNA in the Synthesis of Protein 10 Evolution of Ribosomes and Translation from an RNA World RICHNESS OF RNA ROLES IN MODERN RNA WORLD 11 The RNA World 12 The Ever-Growing World of Small Nuclear Ribonucleoproteins 13 Spliceosome Structure and Function 14 Urdine Insertion/Deletion RNA Editing as a Paradigm for Site-specific Modifications of RNA Molecules 15 Telomerase RNA 16 The Shapely mRNA: Knotting Ventured, Knotting Gained RNA CONTINUES TO TRIUMPH OVER DNA 17 Group II Introns: Ribozymes that Splice RNA and Invade DNA 18 SINEs and LINEs: Troublemakers, Saboteurs, Benefactors, Ancestors 19 The Biology of Short RNAs 20 Versatile Roles of Small RNA Regulators in Bacteria 21 Large Noncoding RNAs in Mammalian Gene Dosage Regulation EMERGING TOOLS 22 Predicting RNA Secondary Structure 23 A Modular and Hierarchical Approach for All-Atom RNA Modeling 24 Automated In Vitro Selection and Microarray Applications for Functional RNA Sequences 25 RNA Folding, Unfolding, and Dynamics, One Molecule at a Time Appendix Index

Viral amplification of recombinant messenger RNA in transgenic plants

Abstract: A novel method of over expressing genes in plants is provided. This method is based on the RNA amplification properties of plus strand RNA viruses of plants. A chimeric multicistronic gene is constructed containing a plant promoter, viral replication origins, a viral movement protein gene, and one or more foreign genes under control of viral subgenomic promoters. Plants containing one or more of these recombinant RNA transcripts are inoculated with helper virus. In the presence of helper virus, recombinant transcripts are replicated producing high levels of foreign gene RNA. Sequences are provided for the high level expression of the enzyme chloramphenicol acetyltransferase in tobacco plants by replicon RNA amplification with helper viruses and movement protein genes derived from the tobamovirus group.

The RNA binding site of bacteriophage MS2 coat protein.

Abstract: The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat

A closely related group of RNA-dependent RNA polymerases from double-stranded RNA viruses.

Abstract: Probably one of the first proteinaceous enzymes was an RNA-dependent RNA polymerase (RDRP). Although there are several conserved motifs present in the RDRPs of most positive and double-stranded RNA (dsRNA) viruses, the RDRPs of the dsRNA viruses show no detectable sequence similarity outside the conserved motifs. There is now, however, a group of dsRNA viruses of lower eucaryotes whose RDRPs are detectably similar. The origin of this sequence similarity appears to be common descent from one or more noninfectious viruses of a progenitor cell, an origin that predates the differentiation of protozoans and fungi. The cause of this preservation of sequence appears to be constraints placed on the RDRP by the life-style of these viruses--the maintenance of a stable, persistent, noninfectious state.

Ribonuclease Protection Assay

Abstract: Sequence-specific hybridization probes of high specific activity are prepared by cloning the probe sequence downstream of a bacteriophage promoter. The plasmid is cleaved with a restriction enzyme, and the plasmid DNA is transcribed with bacteriophage RNA polymerase, which efficiently transcribes the cloned sequence into a discrete RNA species of known specific activity and high abundance. The RNA is purified by removal of the DNA template, protein, and the unincorporated label. Alternatively, the probe is purified by gel electrophoresis, as described in a support protocol. The probe RNA is hybridized to sample RNAs and the hybridization reactions are treated with ribonuclease to remove free probe, leaving intact fragments of probe annealed to homologous sequences in the sample RNA. These fragments are analyzed by electrophoresis on a sequencing gel and the presence of the target mRNA is revealed by the appearance of an appropriately sized fragment of the probe.

Comparative analysis of the regulation of the interferon-inducible protein kinase PKR by Epstein-Barr virus RNAs EBER-1 and EBER-2 and adenovirus VAI RNA.

Abstract: The interferon-inducible protein kinase PKR interacts with a number of small viral RNA species, including adenovirus VAI RNA and the Epstein-Barr virus-encoded RNA EBER-1 These RNAs bind to PKR and protect protein synthesis from inhibition by double-stranded RNA in the reticulocyte lysate system Using a peptide phosphorylation assay we show here that EBER-1, like VAI, directly inhibits the activation of purified PKR A second Epstein-Barr virus RNA, EBER-2, also regulates PKR EBER-1, EBER-2 and VAI RNA exhibit mutually competitive binding to the native or recombinant enzyme, as assessed by UV crosslinking experiments and filter binding assays The affinities of all three RNAs for PKR in vitro are similar (Kd = ca 03 nM) Since this protein kinase has been proposed to exert a tumour suppressor function in vivo, the ability of EBER-1 to inhibit its activation suggests a role for this small RNA in cell transformation by Epstein-Barr virus

Genetically Engineered Protection Against Viruses in Transgenic Plants

Abstract: Transgenic plants carrying nucleotide sequences derived from plant viruses can exhibit increased resistance to viral disease. Many viral sequences confer some level of either resistance to infection or suppression of disease symptoms (tolerance). These include segments of viral genomes encoding capsid or coat proteins, sequences encoding proteins that are or may be subunits of the viral replicase, sequences incapable of encoding proteins, entire genomes of defective interfering viruses and satellite viruses, and complete genomes of mild strains of virus. The transgene may act on initiation of infection, replication of virus, spread of the infection throughout the plant, and symptom development. More than one of these processes can be impaired by a single transgene derived from a single viral gene. The level of protection ranges from very low to high, while the breadth of protection ranges from very narrow, where protection is only observed against closely related strains of the virus from which the transgene was derived, to moderately broad, extending to other viruses. Data are insufficient to establish a molecular mechanism of resistance for most of the described examples. In addition, although the use of a particular segment of the viral genome confers resistance in one virus-host system, analogous sequences from a different virus in another host may be ineffective.

The 110-kDa polypeptide of spinach plastid DNA-dependent RNA polymerase: single-subunit enzyme or catalytic core of multimeric enzyme complexes?

Abstract: Highly purified RNA polymerase preparations from spinach chloroplasts contain seven major polypeptides of 150, 145, 110, 102, 80, 75, and 38 kDa. I find that RNA polymerase activity can be separated under defined conditions into three different fractions by heparin-Sepharose chromatography. Immunological analysis has shown that the first fraction contains RNA polymerase activity associated with all seven major polypeptides, and other studies have shown that some of these polypeptides (150, 145, 80, and 38 kDa) are associated with an RNA polymerase similar to the Escherichia coli enzyme. However, similar analyses of the remaining fractions show activity associated only with the 110-kDa polypeptide, suggesting the existence of a second kind of chloroplast RNA polymerase. Samples of this 110-kDa polypeptide purified by SDS/PAGE actively synthesize RNA in a reaction dependent on a supercoiled DNA template and the four ribonucleoside triphosphates. Hence, this polypeptide has all of the properties expected of a single-subunit RNA polymerase of the T7 bacteriophage type.

Characterization of a host protein associated with brome mosaic virus RNA-dependent RNA polymerase.

Abstract: The association of host proteins with viral RNA replication proteins has been reported for a number of (+)-strand RNA viruses. However, little is known about the identity or function of these host proteins in viral replication. In this paper we report the characterization of a host protein associated with the RNA-dependent RNA polymerase (RdRp) from brome mosaic virus (BMV)-infected barley. A host protein was specifically and proportionally enriched with BMV RdRp activity through several purification steps. This RdRp-associated host protein reacted with an antiserum prepared against wheat germ eukaryotic translation initiation factor 3 (eIF-3). The RdRp-associated host protein, the p41 subunit of wheat germ eIF-3, and an antigenically related protein from rabbit reticulocyte lysates were all found to bind with high affinity and specificity to BMV-encoded protein 2a, which is involved in viral RNA replication. Moreover, addition of wheat germ eIF-3 or the p41 subunit from wheat germ to BMV RdRp gave a specific and reproducible 3-fold stimulation of (-)-strand RNA synthesis in vivo. These results suggest that the barley analog of eIF-3 subunit p41, or a closely related protein, associates with BMV RdRp in vivo and is involved in BMV RNA replication. This observation and the established role of translation factors in bacteriophage Q beta RdRp suggest that association with translation factors may be a general feature of RNA replication by (+)-strand RNA viruses.

Cytoplasmic expression of a reporter gene by co-delivery of T7 RNA polymerase and T7 promoter sequence with cationic liposomes

Abstract: Expression of bacteriophage T7 RNA polymerase in mammalian cells can efficiently drive the transcription of a foreign gene controlled by the T7 promoter (Elroy-Stein et al., Proc. Natl. Acad. Sci. USA. 86, 6126-6130, 1989). We have tested the hypothesis that purified T7 RNA polymerase can be co-delivered into mammalian cells together with a reporter gene (chloramphenicol acetyltransferase, CAT) controlled by the T7 promoter (pT7-EMC-CAT) using DC-chol cationic liposomes. Indeed, significant level of CAT activity was observed in human lung adenocarcinoma (A549-1) cells which had been incubated with a complex of T7 RNA polymerase, pT7-EMC-CAT DNA and DC-chol cationic liposomes. The expression was specific in that T3 RNA polymerase could not replace the T7 RNA polymerase, and that co-delivered T7 RNA polymerase did not enhance the expression of a CAT gene controlled by the SV40 early promoter. The system was optimized in terms of enzyme, DNA and liposome concentrations. Time course experiment indicated that the expression of the T7 system was about 8-10 hours sooner than the SV40 system, consistent with the notion that T7 RNA polymerase does not enter into the nucleus and the transcription takes place in the cytoplasm of the transfected cells. The expression of the T7 system was transient; it declined after 30 hours post transfection, probably due to turnover of the phage enzyme in the mammalian cells. The expression system described here should be useful for gene transfer experiments which require a fast but transient expression of a foreign gene. We have also compared our delivery system with a commercial reagent, Lipofectin, which has been used to deliver T3 or T7 RNA polymerase with a reporter plasmid encoding the T3 or T7 promoter.

Extreme resistance to potato virus X infection in plants expressing a modified component of the putative viral replicase.

Abstract: Three types of mutation were introduced into the sequence encoding the GDD motif of the putative replicase component of potato virus X (PVX). All three mutations rendered the viral genome completely noninfectious when inoculated into Nicotiana clevelandii or into protoplasts of Nicotiana tabacum (cv. Samsun NN). In order to test whether these negative mutations could inactivate the viral genome in trans, the mutant genes were expressed in transformed N.tabacum (cv. Samsun NN) under control of the 35S RNA promoter of cauliflower mosaic virus and the transformed lines were inoculated with PVX. In 10 lines tested in which the GDD motif was expressed as GAD or GED there was no effect on susceptibility to PVX. In two of four lines transformed to express the ADD form of the conserved motif, the F1 and F2 progeny plants were highly resistant to infection by PVX, although only to strains closely related to the source of the transgene. The resistance was associated with suppression of PVX accumulation in the inoculated and systemic leaves and in protoplasts of the transformed plants, although some low level viral RNA production was observed in the inoculated but not the systemic leaves when the inoculum was as high as 100 or 250 micrograms/ml PVX RNA. These results suggest for a plant virus, as reported previously for Q beta phage, that virus resistance may be engineered by expression of dominant negative mutant forms of viral genes in transformed cells.

Identification of a binding site for the human immunodeficiency virus type 1 nucleocapsid protein

Abstract: The nucleocapsid (NC) protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is important for encapsidation of the virus genome, RNA dimerization, and primer tRNA annealing in vitro. Here we present evidence from gel mobility-shift experiments indicating that NCp7 binds specifically to an RNA sequence. Two complexes were identified in native gels. The more slowly migrating complex contained two RNA molecules and one peptide, while the more rapidly migrating one is composed of one RNA and one peptide. Further, mutational analysis of the RNA shows that the predicted stem and loop structure of stem-loop 1 plays a critical role. Our results show that NCp7 binds to a unique RNA structure within the psi region; in addition, this structure is necessary for RNA dimerization. We propose that NCp7 binds to the RNA via a direct interaction of one zinc-binding motif to stem-loop 1 followed by binding of the other zinc-binding motif to stem-loop 1, stem-loop 2, or the linker region of the second RNA molecule, forming a bridge between the two RNAs.

Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.

Abstract: Packaging of retroviral RNA is attained through the specific recognition of a cis-acting encapsidation site (located near the 5' end of the viral RNA) by components of the Gag precursor protein. Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) are two lentiviruses that lack apparent sequence similarity in their putative encapsidation regions. We used SIV vectors to determine whether HIV-1 particles can recognize the SIV encapsidation site and functionally propagate SIV nucleic acid. SIV nucleic acid was replicated by HIV-1 proteins. Thus, efficient lentivirus pseudotyping can take place at the RNA level. Direct examination of the RNA contents of virus particles indicated that encapsidation of this heterologous RNA is efficient. Characterization of deletion mutants in the untranslated leader region of SIV RNA indicates that only a very short region at the 5' end of the SIV RNA is needed for packaging. Comparison of this region with the corresponding region of HIV-1 reveals that both are marked by secondary structures that are likely to be similar. Thus, it is likely that a similar higher-order RNA structure is required for encapsidation.

Biochemical and genetic evidence for a pseudoknot structure at the 3' terminus of the poliovirus RNA genome and its role in viral RNA amplification.

Abstract: The sequences in the plus-stranded poliovirus RNA genome that dictate the specific amplification of viral RNA in infected cells remain unknown. We have analyzed the structure of the 3' noncoding region of the viral genome by thermodynamic-based structure calculation and by chemical and enzymatic probing of in vitro-synthesized RNAs and provide evidence for the existence of an RNA pseudoknot structure in this region. To explore the functional significance of this structure, revertants of a mutant bearing a lesion in the proposed pseudoknot and exhibiting a temperature-sensitive defect in viral RNA synthesis were isolated and mapped. The results of this genetic analysis established a correlation between the structure of the 3' terminus of the viral RNA and its function in vivo in RNA amplification. Furthermore, phylogenetic analysis indicated that a similar structure could be formed in coxsackievirus B1, a related enterovirus, which further supports a role for the pseudoknot structure in viral RNA amplification in infected cells.