Article

Inverted Complementary Terminal Sequences in Single-Stranded RNAs and SnapBack RNAs from Vesicular Stomatitis Defective Interfering Particles

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Abstract

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.

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... on December 20, 2020 by guest http://mmbr.asm.org/ Downloaded from tures (42,43). Similar circularization of Sindbis virus RNA (15) and Sendai virus DI RNAs (26) as well as the segmented bunyavirus RNA (12) has been detected. ...
... Such circularization suggests complementarity at the termini of these RNAs. In some cases, the RNA circles included "panhandle" structures, suggesting extensive inverted complementary sequences at the RNA termini (26,42,43). ...
... A large variety of DI particles of VSV have now been generated (44,46). Several groups have noted the ability of RNA molecules from these DI particles to rapidly self-anneal from a few to as much as 80% (30,40,42,43). These results indicate that the RNA may have long regions of nucleic acid homology that are covalently linked within one molecule. ...
... Separation and quantitation of the relative amounts of error oligonucleotide and the two consensus oligonucleotides allowed us to estimate the in vitro base substitution frequency at this highly conserved position. A sequence exactly homologous to the DI transcript is present at the 5' end of the DI genome and also at the 5' terminus of the genome of infectious virus from which this DI particle was generated (29,31,36,37,40,48,(49)(50)(51)57). We describe methods for isolating this sequence from complete viral and DI particle genomes with complementary synthetic DNA oligonucleotides, for determining relative amounts of error and consensus oligonucleotides, and for sequencing of error oligonucleotide. ...
... 57, 1986 222 STEINHAUER AND HOLLAND genomes. The RNA sequence at the 5' end of the VSV genome has exact sequence homology to the DI polymerase product (36,37,40,(48)(49)(50)(51)57). However, the very complex oligonucleotide maps produced by intact viral and DI genomes (5, 22; see Fig. 8A) cause high backgrounds in greatly overexposed two-dimensional gels, which make it difficult to isolate error oligonucleotides cleanly as was done with DI particle polymerase product. ...
... The 3' end of this DI particle (which codes for DI transcript) is the exact complement of the 5' end for approximately 50 nucleotides. Heating of DI particle RNA and quick cooling in high-salt solution results in panhandle or stem-type molecules in which the complementary termini are base paired (29,31,40,47,48). We isolated RNA from DI particles labeled in vivo with 32p, generated base-paired stems, and treated these with single-strand-specific nuclease(s) as described for DNA-RNA hybrids. ...
Article
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Methods are described which allow direct quantitation and sequence analysis of base substitution levels at predetermined single nucleotide positions in cloned pools of an RNA virus genome or in its RNA transcripts in vitro. Base substitution frequencies for vesicular stomatitis virus (VSV) at one highly conserved site examined were reproducible and extremely high, averaging between 10(-4) and 4 X 10(-4) substitutions per base incorporated at this single site. If polymerase error frequencies averaged as high at all other sites in the 11-kilobase VSV genome, then every member of a cloned VSV population would differ from most other genomes in that clone at a number of different nucleotide positions. The preservation of a consensus sequence in such variable RNA virus genomes then could only result from strong biological selection (in a single host or multihost environment) for the most fit and competitive representatives of extremely heterogeneous virus populations.
... A corollary of these properties is that DI particle RNAs that have plus-sense 3'-terminal sequences have complementary 3'and 5'-terminal sequences. These structures have been identified by hybridization and by direct sequencing (23,25,29). Models for the genesis of this latter and most abundant type of DI particle have been proposed which involve a copy back of the 5'-terminal sequences (9,17) starting at a specific internal RNA polymerase recognition site approximately 48 nucleotides away from the 5' end (29). ...
... Earlier studies by electron microscopy on the structure of hairpin DI particle RNAs by Perrault and Leavitt (23) suggested that nicked and denatured hairpin RNAs could enter into circular and multimer structures. The authors interpreted these results as indicating that the DI particle RNAs had the structure: A A' ...
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The nucleotide sequence of the region which covalently links the complementary strands of the "snapback" RNA of vesicular stomatitis virus, DI011, is (Formula: see text). Both strands of the defective interfering (DI) particle RNA were complementary for their full length and were covalently linked by a single phosphate group. Because the strands were exactly the same length and complementary, template strand and daughter strand nucleocapsids generated during replication of DI 011 were undistinguishable on the basis of sequence, a property not shared by other types of DI particle RNAs. Treatment of the RNA with RNase T1 in high-ionic-strength solutions cleaved the RNA only between positions 1 and 1'. These results and the availability of the guanosine residue in position 1' to kethoxal, a reagent that specifically derivatizes guanosines of single-stranded RNA, suggest that steric constraints keep a small portion of the "turnaround" region in an open configuration. The sequence of the turnaround region was not related in any obvious way to the sequences at the 3' and 5' termini and limited the number of possible models for the origin of this type of DI particle RNA. Two models for the genesis of DI 011 RNA are discussed. We favor one in which the progenitor DI 011 RNA was generated by replication across a nascent replication fork.
... It should be pointed out that this suggestion is based on a minimum of genomic sequence rearrangements. As mentioned above, the following paper (Perrault & Leavitt, 1977) presents evidence that both ends of the snap-back duplex RNAs as well as other single-stranded VSV DI RNAs contain small inverted complementary sequences. Thus, the extent of genomic sequence rearrangements in DI RNAs by recombination and/or other unknown mechanisms may be more extensive than indicated by this simple model. ...
... If we make the above assumption that all snap-back molecules originate from the 5' end of the genome RNA then each of these probably contains a common crosslink corresponding to the original 5' end of the minus strand genome RNA linked to the 3' end of its plus strand complement (Fig. 4). As discussed in the following paper (Perrault & Leavitt, 1977) the cross-link in snap-back molecules probably occurs between a set of complementary sequences which most likely correspond to conserved terminal sequences in VSV DI and B RNAs. Thus, this cross-link could conceivably be a regular feature of DI and possibly B minus strand RNA replication. ...
Article
VSV defective interfering particles of various sizes and from several independent sources frequently contain plus and minus strand RNA. In many cases some of the complementary strands are covalently linked as snap-back molecules. Infectious particles on the other hand package little or no plus strands. Snap-back molecules from the three different sources examined so far vary in size but appear to conform to the same overall linear duplex structure with cross-links at the ends only. They each contain a base sequence which is a subset of the next larger one and appear to correspond to unique sequences in the L cistron of the genome. Possible origins for these snap-back molecules are discussed.
... More complex defective RNAs, with several deletions and duplications, have been described for some plant RNA viruses, e.g., tomato bushy stunt virus (Hillman et al., 1987;White and Morris, 1994), and a defective RNA of influenza virus consisted of a mosaic of sequences from RNA segments 1 and 3 (Fields and Winter, 1982). Snap-back RNAs have been described for several animal RNA viruses, e.g., defective RNAs of vesicular stomatitis virus (Perrault and Leavitt, 1977) and poliovirus full-length ()-and ()-strands covalently linked at one end (Young et al., 1985). However, a combination of a mosaic of sequences from ()-and ()strands and a snap-back structure, such as that found in O. novo-ulmi Ld RNA-10, has not been described previously. ...
Article
The nucleotide sequences of 2 of the 10 mitochondrial double-stranded (ds) RNA segments in a diseased isolate, Log 1/3-8d2 (Ld), of Ophiostoma novo-ulmi, RNA-7 (1057 nucleotides) and RNA-10 (317-330 nucleotides), have been determined. Both RNAs are A-U-rich, but in Southern and Northern blots, no hybridization with mitochondrial DNA or RNA could be detected. Only very short open reading frames were found in both RNAs. As most of its sequence is unrelated to any of the other Ld dsRNAs, RNA-7 may be regarded as a satellite RNA. Northern blotting detected a full-length single-stranded (ss) form of RNA-7 in nucleic acid extracts from Ld. The 5'- and 3'-terminal 39 nucleotides of ssRNA-7 are imperfect inverted complementary repeats of each other, which could cause ssRNA-7 to form a panhandle structure. In addition, the 5'-terminal nucleotides 1-28 and 3'-terminal nucleotides 1032-1057 of ssRNA-7 each contained inverted complementary sequences, allowing the possibility for each terminus to form separate stem-loop structures. The combination of these two structural features has not been found previously in any dsRNA or ssRNA virus. RNA-10 was shown to have an unusual structure, consisting of a mosaic of sequences derived from regions of the 5'- and 3'-termini, or just the 5'-terminus, of RNA-7, RNA-10 has a high degree of inverted complementarity, with the potential to be folded into a very stable hairpin structure. A model for the formation of RNA-10 is presented, involving replicase-driven strand switching between (-)-strand and (+)-strand templates during RNA synthesis, followed by utilization of the nascent strand as a primer and template to form a snap-back RNA.
... On the basis of these studies, it was concluded that if these viral RNAs have complementary terminal sequences, they involve very few nucleotides. These results were surprising since the genomes of many defective interfering (DI) particles of VSV and Sendai virus contain easily recognizable complementary terminal sequences (11,17). ...
Article
Full-text available
The nucleotide sequences at the 5' and 3' termini of RNA isolated from the New Jersey serotype of vesicular stomatitis virus [vsV(NJ)] and two of its defective interfering (DI) particles have been determined. The sequence differs from that previously demonstrated for the RNA from the Indiana serotype of VSV at only 1 of the first 17 positions from the 3' terminus and at only 2 of the first 17 positions from the 5' terminus. The 5'-terminal sequence of VSV(NJ) RNA is the complement of the 3'-terminal sequence, and duplexes which are 20 bases long and contain the 3' and 5' termini have been isolated from this RNA. The RNAs isolated from DI particles of VSV(NJ) have the same base sequences as do the RNAs from the parental virus. These results are in sharp contrast to those obtained with the Indiana serotype of VSV and its DI particles, in which the 3'-terminal sequences differ in 3 positions within the first 17. However, with both serotypes, the 3'-terminal sequence of the DI RNA is the complement of the 5'-terminal sequence of the RNA from the infectious virus. These findings suggest that the 3' and 5' RNA termini are highly conserved in both serotypes and that the 3' terminus of DI RNA is ultimately derived by copying the 5' end of the VSV genome, as recently proposed (D. Kolakofsky, M. Leppert, and L. Kort, in B. W. J. Mahy and R. D. Barry, ed., Negative-Strand Virus and the Host Cell, 1977; M. Leppert, L. Kort, and D. Kolakofsky, Cell 12:539-552, 1977; A. S. Huang, Bacteriol. Rev. 41:811-8218 1977).
... Success of VSV replication can be influenced by the formation of DI particles. DIs arise as a result of one or more RNA recombination events at a variety of genomic sites (Colonno et al., 1977;Perrault and Leavitt, 1978). These truncated genomes can still be encapsidated and form virus-like particles. ...
Article
Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV's broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV's neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications.
... In contrast to LACV and Influenza, viral particles of Sendai Virus (SeV) and Measles Virus (MeV) which belong to the group of Mononegavirales contain predominantly linear nucleocapsids because encapsidation with structural proteins prevents formation of double stranded or panhandle structures (Bhella et al. 2004;Gerlier and Lyles 2011;Loney et al. 2009). But SeV and VSV were found to produce defective interfering (DI) viral genomes during replication (Kolakofsky 1976;Lazzarini et al. 1981;Perrault and Leavitt 1978). Three kinds of DI genomes were identified: DI genomes with internal deletions, 5 promoter duplications with completely complementary 5 -3 ends, and hairpin DI genomes "snap back", consisting of a dsRNA hairpin of 100-1000 bp (Fig. 1). ...
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Initiating the immune response to invading pathogens, the innate immune system is constituted of immune receptors (pattern recognition receptors, PRR) that sense microbe-associated molecular patterns (MAMPs). Detection of pathogens triggers intracellular defense mechanisms, such as the secretion of cytokines or chemokines to alarm neighboring cells and attract or activate immune cells. The innate immune response to viruses is mostly based on PRRs that detect the unusual structure, modification or location of viral nucleic acids. Most of the highly pathogenic and emerging viruses are RNA genome-based viruses, which can give rise to zoonotic and epidemic diseases or cause viral hemorrhagic fever. As viral RNA is located in the same compartment as host RNA, PRRs in the cytosol have to discriminate between viral and endogenous RNA by virtue of their structure or modification. This challenging task is taken on by the homologous cytosolic DExD/H-box family helicases RIG-I and MDA5, which control the innate immune response to most RNA viruses. This review focuses on the molecular basis for RIG-I like receptor (RLR) activation by synthetic and natural ligands and will discuss controversial ligand definitions.
... These mvRNAs consist of only the extreme 59 and 39 promoter regions, resulting in DVGs of less than 100 nucleotides in length that are recognized by retinoic acid-inducible gene-I (RIG-I) to induce an exaggerated interferon (IFN) response (26,27). Other common DVGs are the so-called copy-back DVGs, where the 59 end is duplicated in reverse complement, creating complementary stem-loop structures (28)(29)(30)(31). ...
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... Copy-back and snap-back DVGs are rearranged genomes in which a sequence is duplicated in reverse complement to create theoretical stem-like structures (panhandle structures for copy-back DVGs or hairpin structures for snap-back DVGs) 41-43 . Copy-back DVGs have been reported in many negative-sense (ns) RNA viruses and are generated from the 5′ end of the genome in a process that produces DVGs with complementary 3′ and 5′ termini 44,45 . If the complementary region of the DVG comprises almost the entire sequence, the DVG can be further characterized as a snap-back DVG 43 (Fig. 1c). ...
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Viruses survive often harsh host environments, yet we know little about the strategies they utilize to adapt and subsist given their limited genomic resources. We are beginning to appreciate the surprising versatility of viral genomes and how replication-competent and -defective virus variants can provide means for adaptation, immune escape and virus perpetuation. This Review summarizes current knowledge of the types of defective viral genomes generated during the replication of RNA viruses and the functions that they carry out. We highlight the universality and diversity of defective viral genomes during infections and discuss their predicted role in maintaining a fit virus population, their impact on human and animal health, and their potential to be harnessed as antiviral tools. This Review describes recent findings on the biogenesis and the role of defective viral genomes during replication of RNA viruses and discusses their impact on viral dynamics and evolution.
... As a consequence, like copyback DVGs, snapback DVGs possess terminal complementarity, but the length of the complementary region in the latter is much longer and the loop is reduced to only a few nucleotides. Both copyback and snapback DVGs have been described for several families of negative strand RNA viruses, including segmented [2,9,[11][12][13] and non-segmented viruses [14][15][16]. Copyback DVGs appear to be the predominant species of DVGs found in several members of the paramyxoviruses, such as Sendai virus, measles virus, and parainfluenza virus, and most of our current knowledge about copyback DVGs stems from research of these viruses [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31]. In theory, copyback DVGs should be generated both during replication of the genome, when replication starts at the antigenomic 3'end containing the trailer (tr) region, and during formation of the antisense genome, which starts at the genomic 3'end containing the leader region. ...
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DNA synthesized after infection of simian tissue culture cells (BSC-1 or CV-1) with human adenovirus type 2 or 5 or with simian adenovirus 7 was characterized. It was demonstrated that as much as 40% of the virus-specific DNA in nuclei of infected monkey cells consists of subgenomic pieces. No subgenomic viral DNA species were detected in the nuclei of human (HeLa) cells infected with these adenovirus types. Restriction analysis showed that these short viral DNA molecules contain normal amounts of the sequences from the ends of the viral genome, whereas internal regions are underrepresented. The production of subgenomic DNAs is not correlated with semipermissive infection. Although adenovirus types 2 and 5 are restricted in monkey cells, these cells are fully permissive for simian adenovirus 7. HR404, an adenovirus type 5 mutant which is not restricted in monkey cells, produced the same percentage of subgenomic DNAs as did its wild type (restricted) parent, and coinfection of monkey cells with adenovirus type 5 DNAs. The array of predominant size classes among the heterogeneously sized short DNAs is serotype specific. Extensive plaque purification and comparison of wild-type adenovirus type 5 with several viral mutants indicated that the distribution of aberrant sizes of DNA is characteristic of the virus and not a result of random replicative errors and then enrichment of particular species.
Article
RNA genomes from standard vesicular stomatitis virus and two defective interfering (DI) particles dI 0.33 (DI-T) and DI 0.52, were purified and digested with RNase T1. The resulting oligonucleotides were labeled at the 5' end with [32P]ATP and separated by two-dimensional electrophoresis in polyacrylamide gels. All of the major oligonucleotides containing 20 or more nucleotides were sequenced. Those oligonucleotides that were thought to be in common by their migration on polyacrylamide gels actually did have identical sequences. Those oligonucleotides thought to be unique to the DI RNAs either differed by only one nucleotide from oligonucleotides of the standard RNA or contained new sequences which were complementary to known sequences at the 5' end. These data indicate that RNAs from DI particles are not simple deletions but contain point mutations and additional complementary sequences.
Article
The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesized an aberrant polyadenylated mRNA in vivo and in vitro. No normal glycoprotein message could be detected among the in vivo transcription products. The abnormal RNA contained a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message. The polyadenylate is located at the 3' end of the molecule and is most probably encoded by the remnant polymerase gene polyadenylation signal. This aberrant RNA may be synthesized because of either a failure to terminate transcription at the end of the glycoprotein gene or an inability to process an abnormal polycistronic precursor.
Article
We have sequenced (via a product RNA) the 3' RNA terminus of a defective interfering particle that was generated from the standard virus isolated from a culture of BHK-21 cells persistently infected with vesicular stomatitis virus for over 5 years. By hybridization and RNA sequencing, seven mutations were identified in the 46 nucleotides at the terminus of this defective-interfering-particle RNA. It is likely that these mutations are a reflection of altered protein-nucleic acid interactions that the virus has evolved to maintain its persistently infected carrier state in vitro.
Article
We have characterized the genome sequences represented in two defective interfering particles derived from the heat-resistant strain of vesicular stomatitis virus by means of end-labeling and hybridization techniques. Both defective particle RNAs, which differ slightly in size, contain 5'-end sequences identical to each other and to that of the standard infectious virus genome, for at least the first approximately 55 bases. In contrast, the 3'-end sequences of these two RNAs are different. The 3'-end sequence of the smaller RNA is identical to that of the standard genome for at least the first 48 bases. The 3'-end sequence of the larger RNA is an inverted complement of its 5' end for approximately 65 bases. The bulk of the sequences in both RNAs is derived from the 3' half of the standard genome. We also show that the two defective particles differ in vitro transcription and in vivo replication properties. These results provide direct evidence for the presence of internal genome deletions in defective interfering particles of negative-stranded RNA animal viruses and demonstrate the existence of at least two distinct classes of these particles.
Article
The electron microscopy of nucleic acid molecules has become a routine tool in the analysis of genome structure and function. This is primarily due to the availability of simple reliable methods for the visualization of single- and double-stranded DNA and RNA molecules, of RNA-DNA hybrids, and of nucleic acid-protein complexes. Most of these methods were developed by using viral genomes because these nucleic acid molecules were of considerable physical homogeneity and could be produced in abundant quantities
Chapter
There are five families of RNA viruses in which the negative strand is sequestered in the extracellular virion. Viruses of two of these families, the Rhabdoviridae and the Paramyxoviridae, have unitary linear genomes, whereas viruses of the other three families, Arenaviridae, Bunyaviridae, and Orthomyxoviridae, have segmented genomes comprising, respectively, two, three, and seven or eight subunits. The informational macromolecules that comprise the genomes of rhabdoviruses and paramyxoviruses are among the largest functional RNA molecules and are exceeded in size only by those of the plus-strand coronaviruses. Reanney (1982, 1984) has calculated that the upper size limit for any RNA virus genome cannot be much in excess of 17,600 nucleotides (mol. wt. ≈5.7 × 106) as a consequence of the low copying fidelity of RNA polymerases. The segmentation of the genomes of the other negative-strand viruses may be a consequence of such constraints on molecular size or a device for decoupling the transcription of individual genes. Whatever the reason, the genetic properties of the segmented-genome viruses differ substantially from those of the unsegmented-genome viruses, because variation in the former is generated by reassortment of genome subunits as well as by mutation. Mutation is the sole mechanism of variation in unsegmented-genome viruses, since the intramolecular recombination observed with positivestrand RNA viruses does not seem to be permissible for any negativestrand RNA virus.
Chapter
Rhabdoviruses are relatively simple, membrane-enveloped viruses containing a single-stranded RNA genome. The genomic RNA is the negative sense—i.e., complementary to the messenger RNAs (mRNAs)—and is noninfectious. The virus particles must therefore contain an RNA-dependent RNA polymerase to generate the mRNAs (Baltimore et al., 1970). Rhabdoviruses have a bacilliform, bullet-, or cone-shaped morphology and are known to infect vertebrates, invertebrates, and plants. The composition of various rhabdoviruses has been reviewed by McSharry (1979). The virus particles contain a helical, nucleocapsid core composed of the genomic RNA and protein. Generally, three proteins termed N (nucleocapsid), NS (originally indicating nonstructural), and L (large) are found to be associated with the nucleocapsid. An additional matrix (M) protein lies within the membrane envelope, perhaps interacting both with the membrane and the nucleocapsid core. A single glycoprotein (G) species spans the membrane and forms the spikes on the surface of the virus particle.
Chapter
The negative-strand RNA genome of vesicular stomatitis virus (VSV) serves as template for two types of RNA-synthesis reactions, transcription and replication. A major distinction between the two RNA-synthesis processes is a requirement for protein synthesis. Transcription, the synthesis of the leader RNA and five discrete monocistronic messenger RNAs (mRNAs), does not require protein synthesis. Replication, the production of full-length copies of the viral RNA, requires continuous synthesis of viral proteins. This chapter will focus on the question of what newly synthesized protein or proteins are required to effect and maintain the transition from transcription to replication of the negative-strand RNA. Reviews of negative-strand virus RNA replication, including discussions of the nature of the template and specific sequences involved in replication, were presented by Wertz (1980), Lazzarini et al. (1981), and Ball and Wertz (1981). This chapter will concentrate on work that has been carried out since these previous reviews.
Chapter
This introductory chapter is an attempt to combine our knowledge of the general features of viral replication studied at the molecular level (usually in cell culture) with some information on the course and outcome of infection (insofar as it can be studied) in the whole animal. A comprehensive review of molecular virologic studies would be so diverse that it would be beyond the scope of this chapter and would probably be superfluous for the majority of readers. However, some remarks on the underlying principles of the immune response to viral infection and on viral pathogenesis seem to be appropriate, along with some specific comments on integration of viral DNA into cell genomes and on defective interfering particles. Paradoxically, the host’s immune response to virus infection can play several roles: it can provide both short- and long-term assistance in combating the infection, but also may be at least partially responsible for development of disease symptoms.
Chapter
This chapter will emphasize recently derived knowledge concerning the nature of defective interfering (DI) particles of RNA animal viruses, their biological origins and functions, and their involvement in long-term persistent infections. We will not attempt to review all of the DI literature, and we will confine ourselves to DI particles of RNA viruses. The previous review by Huang and Baltimore (1977) amply documents the occurrence and behavior of DI particles in a wide variety of DNA and RNA viruses and discusses their biological effects, and a very thorough recent review of rhabdovirus DI particles by Reichmann and Schnitzlein (1978) provides excellent in-depth coverage of many areas not covered by the present chapter as well as some alternate viewpoints of areas which are considered here. We will omit DNA virus DI particles from extensive consideration because of space limitations and because they are generally less well characterized at present.
Article
Viruses usurp host cell pathways for different stages of their infection. Understanding virus-host interaction will be invaluable to elucidate molecular mechanisms of virus infection and to identify drug targets. In order to identify such critical cellular genes in vesicular stomatitis virus (VSV, a model non-segmented negative strand RNA virus) infection, we developed a stable cell line constitutively expressing replication proteins of VSV. Attempts to establish a cell line replicating a sub-genomic replicon was not successful because of induction of interferon response by replication of viral genomic analog. Subsequently, we used siRNA technology and conducted a genome-wide siRNA screen in HeLa cells to identify host factors regulating VSV infection. A total of 23,000 human genes were knocked down individually, and their effect on viral infection was interrogated using a high-throughput cell-based assay. Our study identified several previously unknown host proteins required for VSV infection. Bioinformatics analysis predicted enrichment of several biological functions among these proteins and some of them are commonly utilized by other pathogens such as human immune deficiency virus (HIV), hepatitis C virus (HCV) and Influenza virus. We also noted that 35% of these genes (25 out of 72) are required for lymphocytic choriomeningitis virus (LCMV) and human parainfluenza virus type 3 (HPIV3) infection, suggesting evolutionary conserved mechanisms of virus-host interactions. Further studies focusing on host coatomer complex 1 (COPI) identified a role of COPI in early stage of VSV infection. The effect of COPI is mediated at the level of viral RNA synthesis. COPI functions are required not only for VSV but also for LCMV and HPIV. ADP ribosylation factor 1 (ARF1), the immediate upstream modulator of COPI was found as a required factor for VSV RNA synthesis. ARF1 is activated by the Golgi-associated brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1) which was found to be a critical determinant of VSV RNA synthesis. These studies suggested that the components of the cellular secretory pathway are required for VSV RNA synthesis. Adviser: Asit K. Pattnaik
Article
RNA genomes fromstandard vesicular stomatitis virusandtwo defective interfering (DI)particles, DI0.33(DI-T) andDI0.52, were purified anddigested withRNaseT1.Theresulting oligonucleotides were labeled atthe5'endwith (32P)ATP andseparated bytwo-dimensional electrophoresis inpolyacrylamide gels. Allofthemajoroligonucleotides containing 20or more nucleotides were sequenced. Thoseoligonucleotides that were thought tobeincommon bytheir migration on polyacrylamide gels actually didhaveidentical sequences.Those oligonucleotides thought tobeunique totheDIRNAseither differed byonly one nucleotide fromoligonucleotides ofthestandard RNA orcontained new sequences which were complementary toknownsequencesatthe5'end.Thesedataindicate thatRNAsfromDIparticles arenotsimple deletions butcontain point mutations andadditional complementary sequences. Vesicular stomatitis virus (VSV) isanegative- strand RNA virus. Thereplication strategy of this virus entails synthesis ofafull-length plus- strand antigenomic RNA fromthegenomic RNA,whichinturncodes forother full-length minusstrands, someofwhichserve asmRNA templates whereas others arepackaged into progeny virions (26). During this process ofRNA replication, deletion mutants, nameddefective interfering (DI)particles, aregenerated (8). MostoftheDIgenomesofVSV havebeen mappedtothe5'endofthegenome. Thegen- eration eventofthese deleted RNAshasbeen hypothesized tooccurwhenthereplicase is copying theantigenomic RNA intogenomic RNA (7,14). Atsomepoint thereplicase is hypothesized tojumpfromtheantigenomic tem- plate tothenascent chain, creating adeleted RNAwithself-complementary ends. Sequencing oftheendsoftheDIRNAsderived fromthe5' endofthegenomehasdemonstrated thatthey
Article
The 3′ terminal sequences of four different DI particle RNAs ranging in size from 10S to 30S have been determined directly using rapid RNA sequencing methods or deduced, in the case of the fourth DI RNA, from the complementary sequence of a small RNA transcribed from this part of the genome (Schubert et al., 1978). One DI particle (DI 011) contains covalently linked genomic and antigenomic RNA. The 5′ end of this RNA is identical to that of VSV RNA, as determined by annealing for at least 1 kb, as well as to the other DI particle RNAs used in this study. The 3′ ends of the other three DI particle RNAs are exact copies of the common 5′ terminal sequence for 48 nucleotides in two cases and 45 nucleotides in the third. Beyond these complementary regions the sequences are different for each DI RNA. The fact that these regions differ in length by only three nucleotides, despite the wide differences in the overall size of the DI particle RNAs, indicates that if these DIs were formed by the copy-back mechanisms similar to those proposed by Leppert, Kort and Kolakofsky (1977) and Huang (1977), a specific recognition site for the RNA polymerase must be involved in copying the 5′ terminus. We determined the 5′ terminal sequence from position 43–48 at the end of the complementary region and found it to be 5′-GGUCUU-3′. This hexamer is also part of other highly conserved terminal RNA polymerase initiation sites (Keene et al., 1978; Keene, Schubert and Lazzarini, 1979) and may be a specific internal RNA polymerase recognition site. We conclude that this sequence is one of the elements involved in the genesis of DI particle chromosomes containing short complementary sequences at their termini. The ability of the polymerase to resume synthesis at or near a specific recognition site is discussed.
Article
Full-text available
Multicellular organisms evolved efficient host-defense mechanisms to sense viruses and to block their replication and spread. Invertebrates and plants mainly rely on RNA interference (RNAi) for antiviral defense. In mammals, the initiation of antiviral defense mechanisms is largely based on the detection of viral nucleic acids by innate receptors: retinoic acid-inducible gene I (RIG-I)-like helicases (RLHs) and Toll-like receptors (TLRs). RLHs play a pivotal role in sensing viral RNA and DNA in the cytoplasm of cells. RLHs, like Dicer of the RNAi pathway, belong to the phylogenetically conserved DExD/H-box family of helicases. Unlike TLRs, RLHs are functional in all somatic cells. Activation of RIG-I triggers antiviral responses including type I interferon (IFN), inflammasome activation and proapoptotic signaling. Here, we provide a comprehensive overview of the current literature on the ligand structures detected by RIG-I, and conclude with the molecular definition of the RIG-I ligand: short double-stranded blunt-end 5'-triphosphate RNA. The recent information on the RIG-I ligand now allows the design of short double-stranded RNA (dsRNA) oligonucleotides that are ideally suited alone or in combination with small-interfering RNA (siRNA) for the treatment of viral infection and cancer.
Article
Sendai virus and VSV minus strand genome RNAs, labeled specifically at their 3' ends with RNA ligase, were used as probes to detect leader RNA--that is, short transcripts (approximately 50 nucleotides) complementary to the exact 3' end of the minus strand genome. These probes have allowed the detection of plus strand leader RNAs in both Sendai virus and VSV-infected cells as well as in the virion transcriptase reactions. The use of a similar probe, prepared from the self-complementary ends of DI genome RNA and containing the 3' end of the plus strand antigenome RNA, has allowed the detection of a minus strand leader RNA of identical size in VSV-infected cells. Since the presence of DI genomes could not be detected by analytical sucrose gradient centrifugation in these VSV-infected cells, this minus strand leader RNA is apparently synthesized on the template formed by the exact 3' end of the antigenome RNA.
Article
Molecular biology applied toward understanding viral diseases has come of age, and the time will come when questions of viral pathogenesis can be answered by biochemical analysis of animal viruses and their infected cells. This is not, however, a one-way street. Experience has taught that in designing experiments which attempt to answer questions related to disease processes new information is often gathered that contributes significantly to our thinking about general problems in molecular biology. This two-way exchange between molecular biology and viral pathogenesis is illustrated here by recent examples. One conclusion is that the study of inverted terminal complementary sequences may have potential impact in several important areas. For viral diseases, these studies may reveal the underlying principles by which certain defective genomes can compete and successfully inhibit synthesis of standard virus. For molecular biology, these studies may reveal the link between linear viral genomes that can circularize and the mechanism by which their nucleic acids are replicated. For generating defective interfering (DI) genomes, the various models on the presence of inverted complementary termini on linear molecules, preclude random recombination between RNA molecules as a means of generating DI genomes. This type of model predicts that the DI genomes will arise only from one or the other end of the genome. Complementary or extragenomic sequences arise by hairpin formation during synthesis. How internal hairpin loops are made during nucleic acid replication is not clearly understood, but such events are thought to occur when the reverse transcriptase functions in vitro in the absence of actinomycin D. Therefore, these studies on the genomes of VSV DI particles, which were initiated because of the intrinsic relevance of interference to viral diseases, are likely to lead to answers basic for the replication of nucleic acids and for the generation of deleted and inverted sequences.
Article
Vesicular stomatitis virus (VSV) and defective interfering (DI) particle RNAs were labeled at their 3' ends by using RNA ligase and cytidine 3',5'-bis[32P]phosphate. The RNAs were subjected to partial digestion with alkali and analyzed by oligonucleotide fingerprinting in two dimensions. VSV and DI particle RNAs have complete sequence homology for the first eight bases from the 3' end. The following four positions contain three mismatched nucleotides in which guanosine residues in one strand are replaced by uridine residues in the other. There is again complete homology for the next five bases (positions 13-17). The locations of purine residues within the sequence were confirmed by partial digestion with RNase T1 and RNase U2 and separation by size on 20% acrylamide gels. The latter method also indicated that sequences of VSV and DI particle RNAs diverge beyond the 18th nucleotide from the 3' termini.
Article
We have sequenced the endogenous RNA polymerase product produced by disrupted purified virions of vesicular stomatitis virus defective interfering particles by using the newer one-dimensional rapid gel sequencing techniques and confirming this with a modified two-dimensional gel vectoring technique. The sequence of this 46-nucleotide RNA is: 5'(pp)pACGAAGACCACAAAACCA-GAUAAAAAAUAAAAACCACAAGAGGG(U)COH3'. We infer that this sequence is identical to the sequence at the 5' end of infectious vesicular stomatitis virus RNA and is complementary to the sequence of the 3'-OH terminus of this defective interfering particle genome RNA.
Article
The ability of defective-interfering (DI) particles of vesicular stomatitis virus (VSV) to induce interferon was studied in relation to the amount of snapback [±] double-stranded sequences in their RNA. Five DI particles propagated in BHK-21 cells were analyzed: two DI particles generated by undiluted passages of cloned wild-type VSV (Indiana); two DI particles generated by serial undiluted passages of culture fluid from L cells persistently infected with VSV; and DI-011, a DI particle with [±] snapback RNA, which is known to be a potent inducer of interferon. Induction of interferon in L cells by these DI particles was not proportional to the amount of [±] sequences in their RNA. DI-011 (26 to 37% [±] RNA sequences) induced a significant amount of interferon at a multiplicity of infection of one DI particle per cell. In contrast, the two DI particles from wild-type VSV (43 to 54% [±] RNA sequences) were 20- to 30-fold less efficient inducers of interferon than DI-011. Furthermore, the two DI particles (1 to 4% [±] RNA sequences) generated from L cell carrier cultures were only slightly less efficient inducers of interferon than the wild-type DI particles. The data also indicate that a population of DI particles which contains [±] RNA is not selected in L cells persistently infected with VSV.
Article
A small RNA, containing approximately 50 nucleotides, is synthesized by cells coinfected with standard vesicular stomatitis virus and its defective interfering (DI) particles. Infection of cells by standard virus or DI particles alone does not lead to synthesis of significant amounts of small RNA. The RNA is initiated at its 5' end with (p)ppXp and is not polyadenylylated at the 3' end despite a content of 51% adenosine. It has sequences complementary to the genome of a DI particle. The synthesis of the small RNA correlates with the replication of the genome of DI particles with molar ratio small RNA/genome RNA of DI particles greater than 50. When replication of DI genomes is prevented by the addition of cycloheximide or prior UV irradiation of DI particles, small RNA is not synthesized in coinfected cells. These results indicate that the small RNA is not the result of transcriptional initiation and that it may relate to interference mediated by DI particles.
Article
We have determined the nucleotide sequence for the first 50 nucleotides at the 5′ terminus of vesicular stomatitis virus (VSV) genome RNA. This sequence is identical to that of the in vitro RNA polymerase product synthesized by defective Interfering (DI) particles of VSV. These results confirm previous conclusions regarding DI and standard viral terminal sequences based on hybridization studies and earlier sequencing of the DI polymerase product RNA.
Article
UV inactivation of vesicular stomatitis virus and its defective interfering (DI) particles was measured in order to obtain the target size for interference. In the case of DI particles whose genomes mapped at the 5' end of the virion RNA, this target size corresponded to the entire DI particle RNA molecule regardless of whether it amounted to 10, 30, or 50% of the viral genome. These data were interpreted as demonstrating that both termini of the DI particle RNAs were required for their replication and for interference with virion RNA replication. The unique heat-resistant DI particle, with an RNA molecule corresponding to the 3' half of the viral genome, exhibited an inactivation target size of approximately 42% of its RNA molecule with respect to both homotypic and heterotypic interference. Unlike other DI particles, this particle interfered with virion primary transcription. The unusual inactivation target size of the heat-resistant DI particle was interpreted as being a compromise between the requirements for replication of its genome and those for interference with virion primary transcription.
Article
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Human-mouse somatic cell hybrids were made between adenine phosphoribosyltransferase-deficient mouse L cells and a strain of human primary fibroblasts and selected in medium containing alanosine and adenine (J. A. Tischfield and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 71 :45-49, 1974). These hybrids were tested for the generation of defective interfering (DI) particles of vesicular stomatitis virus to determine whether or not a host gene controls the induction of DI particles. None of the seven independently arising hybrid clones tested generated detectable DI particles during 13 successive undiluted passages. In addition, the parental human cells also failed to generate DI particles. In contrast, the parental mouse cells generated a detectable level of DI particles during continuous passage. Thus, failure to generate DI particles appears to act in a dominant fashion in these hybrids. Human chromosome 16 and adenine phosphoribosyltransferase were present, as a direct consequence of the selection system, in all of the hybrid clones that failed to generate DI particles. It was the only human chromosome observed in the cells of every hybrid clone. This was verified by both isozyme and karyotype analyses. After hybrids were back-selected (with 2,6-diaminopurine) for loss of human adenine phosphoribosyltransferase and chromosome 16, they gained the ability to generate DI particles. Replication of DI particles already present in virus stocks, however, was normal in all of the hybrid clones and the parental human cells. This suggests that the induction, but not the replication, of DI particles is affected by the human genome and that a factor on human chromosome 16 seems to selectively suppress the mouse cell's ability to generate DI particles in the hybrids. These results support the idea that the induction of DI particles is controlled in part by host cell function(s), as suggested previously (C. Y. Kang and R. Allen, J. Virol. 25 :202-206, 1978).
Article
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Poliovirus RNA stimulates imcorporation of 35S from both [35S]methionine and formyl-[35S]methionyl-tRNAfMet in cell-free systems derived from HeLa cells or from poliovirus-infected HeLa cells. The largest product formed under the direction of the viral RNA is the same size as the polyprotein thought to represent translation of the entire RNA. Synthesis of this polyprotein and other large products was stimulated greatly by increasing the salt concentration during the reaction from the optimum for initiation (90 mM) to the optimum for elongation (155 mM). Only one initiation peptide could be identified, and a tryptic digest of the product contained mainly peptides that cochromatographed with peptides from authentic viral proteins. The RNA from a deletion mutant of poliovirus initiated protein synthesis at the same site used by standard RNA and programmed synthesis of an appropriately deleted set of polypeptides. The results strongly support the model of translation of poliovirus RNA from a single initiation site into a continuous polyprotein that is cleaved to form the functional proteins. It is suggested that uninfected HeLa cell extracts can carry out the cleavages of nascent polyprotein.
Article
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Electron microscope and gel electrophoresis studies show that the high-molecular-weight (50 to 70S) RNA extract from Friend virus (FV) is a dimer with the same basic structure previously observed for the RNAs from RD-114 virus, baboon virus, and woolly monkey virus. This observation greatly strengthens the inference that the dimer structure is a general characteristic of the RNAs of all mammalian type C viruses. The FV dimer is slightly less stable than the RNA dimer of woolly monkey virus, which is, in turn, much less stable than those of RD-114 and baboon virus. There are three FV monomer components, small (S), medium (M), and large (L), with molecular lengths of 6.7 +/- 0.6, 7.7 +/- 0.6, and 9.5 +/- 0.6 kilobases, respectively. There are approximately equal amounts of the S and M components and much less of the L component. Most of the dimers are homodimers (SS, MM, and LL). The frequency of heterodimers (SM, SL, ML) is much less than expected for a random assortment model.
Article
Adeno-associated virus linear, single polynucleotide chains contain an inverted terminal repetition which allows the formation of single-stranded circles when the DNA is exposed to annealing conditions. Under appropriate annealing conditions single-stranded circular dimers are formed, the majority of which have two projections separated by 180 ° visible in the electron microscope. We conclude that these projections represent the regions of self-complementarity (inverted terminal repetition) contained within the virus DNA. Measurements of the lengths of the projections indicate that the length of the inverted terminal repetition represents approximately 1.5% of the genome.
Article
The RNA isolated from a new defective interfering particle of vesicular stomatitis virus is 80 to 90% double-stranded. Annealing and electron microscopic analyses indicate that the complementary sequences of this duplex are covalently linked in a hairpin structure. Approximately half of the sequences found in this RNA are normal genomic sequences found in the RNA of the infectious virus. The other half, the complement of the former, are message sequences that are not found in the infectious virus. Within the particle, the RNA exists as a single-stranded ribonucleoprotein complex, 0·85 μm in length. Upon deproteinization the RNA collapses into a self-complementary double stranded duplex, 0·33 μm in length.The possible relevance of covalently linked message and genomic sequences to the mechanism of vesicular stomatitis virus replication is discussed.
Article
The ability of various DI† particles generated by the Indiana serotype of VSV to interfere heterotypically with New Jersey VSV infections in baby hamster kidney cells (BHK-21) and mouse L cells was investigated. All DI particles, with the exception of one (HR), exhibited no heterotypic interference in L cells and approximately 1 to 10% in BHK cells as compared to homotypic interference. In contrast, the HR DI particle interfered to the same extent with infections of both VSV serotypes in both cell lines. Annealing experiments with viral messenger RNAs showed only the HR DI particle RNA to contain sequences complementary to the 13 S to 18 S mRNA population. All other particle RNAs contained nucleotide sequences corresponding to a portion of the 30 S mRNA. Complementarity between How DI particle RNA and 13 S to 18 S mRNAs, observed by Leamnson & Reichmann (1974), was due to impurities. The following conclusions were drawn from these results. 1.(1) The HR DI particle RNA is unique functionally (heterotypic interference) and structurally (complementarity to 13 S to 18 S mRNA).2.(2) The ability to interfere heterotypically is not entirely a function of the DI particle RNA size, since three other RNAs of a comparable size to HR DI particle RNA did not exhibit this property. This ability seems to be due to nucleotide sequences originating from the 13 S to 18 S mRNA cistrons.3.(3) The extent of a residual ability to interfere heterotypically by DI particles other than HR seems to be cell-line-dependent.Based on these conclusions and previous reports in the literature (Leamnson & Reichmann, 1974; Stamminger & Lazzarini, 1974; Roy & Bishop, 1972), a revised map of DI particle RNAs relative to viral cistronic regions is proposed. The map invokes a fixed generation point for all DI particle RNAs (except HR) near the 5′ terminus of the viral RNA, which is postulated to be the 30 S mRNA domain. Sequences of the shorter DI particle RNAs are contained in the sequences of the longer particles. The HR DI RNA has no overlapping nucleotide sequences with the other DI particle RNAs, although a structurally similar replicase recognition sequence can not be excluded. The significance of this model in relation to the origin and interfering ability of DI particles is discussed.
Article
A class of vesicular stomatitis defective interfering virus particles isolated in this laboratory contains mostly double-stranded RNA of approximately 0.7–0.9 × 106 daltons molecular weight. This RNA “snaps-back” or reanneals independently of concentration after heat denaturation but remains denatured if first nicked with ribonuclease. The nicked denatured products are about half the size of the double-stranded RNA and approximately half of them anneal to infectious virion single-stranded RNA. It is proposed that the complementary strands of this double-stranded RNA are cross-linked to each other, probably covalently. The structure can be viewed best as a hairpin or a closed circle base-paired along most of its length.
Article
When passaged at high multiplicity, four strains of Sendai virus all showed evidence that they contained defective interfering (DI) particles. RNA isolated from nucleocapsids of cells infected with the high multiplicity passage stocks was found to consist of only minor amounts of nondefective genome length RNA and major amounts of smaller RNAs, the DI-RNAs. These DI-RNAs were found to have unusual and variable sedimentation properties in sucrose gradients, but were found to represent unique segments of the viral genome by length measurements in the electron microscope and by hybridization. A striking feature of the DI-RNAs is their ability to form circular structures, indicating that the ends of the DI-RNA are complementary. The implications of this finding in terms of the mechanism of genome replication is discussed.
Article
Purified virions of vesicular stomatitis virus (VSV) are capable of synthesizing two distinct types of virus-specific RNA in vitro. The first consists of several viral mRNAs which have been previously shown to contain the blocked 5' terminal sequence GpppApApCpApGp and 3' terminal poly(A). The second type of RNA has an unblocked 5' terminus and does not contain poly(A) stretches long enough to bind to oligo (dT)-cellulose columns. It migrates in 20% polyacrylamide gels as a single homogeneous peak with an estimated chain length of 68 nucleotides. Base analysis demonstrated that this small RNA molecule is composed of 48% AMP, 20% CMP, 11% GMP, and 21% UMP. The 5' terminal sequence of the small RNA is ppApCpGp, which appears to be complementary to the 3' terminal sequence of the VSV genome RNA (...PypGpU). These results indicate that this small RNA molecule probably represents the intitiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.
Article
VSV defective interfering particles of various sizes and from several independent sources frequently contain plus and minus strand RNA. In many cases some of the complementary strands are covalently linked as snap-back molecules. Infectious particles on the other hand package little or no plus strands. Snap-back molecules from the three different sources examined so far vary in size but appear to conform to the same overall linear duplex structure with cross-links at the ends only. They each contain a base sequence which is a subset of the next larger one and appear to correspond to unique sequences in the L cistron of the genome. Possible origins for these snap-back molecules are discussed.
Article
La Crosse (LAC) virions purified by velocity and equilibrium gradient centrifugation contained three single-stranded RNA species. The three segments had sedimentation coefficients of 31S, 25S, and 12S by sodium dodecyl sulfate-sucrose gradient centrifugation. By comparison with other viral and cellular RNA species, the LAC viral RNAs had molecular weights of 2.9 x 10(6), 1.8 x 10(6), and 0.4 x 10(6). Phenol-sodium dodecyl sulfate-extracted LAC virion RNA was not infectious for BHK-21 cell cultures under conditions in which Sindbis viral RNA was infectious. Treatment of LAC virus with the nonionic detergent Triton X-100 and salt released three nucleocapsid structures, each containing one species of virion RNA. The nucleocapsids had sedimenation coefficients of 115S, 90S, and 65S. Negative-contrast electron microscopy of the nucleocapsids indicated that they were convoluted, supercoiled, and apparently circular. They had a mean diameter of 10 to 12 nm and modal lengths of 200, 510, and 700 nm (some were even longer). By chemical and enzymatic analysis of purified viral RNA, one type of 5' nucleotide (pppAp) present in the proportion of one per RNA segment was identified. After periodate oxidation, each virion RNA species was labeled by reduction with [3H]sodium borohydride. Taken together, these results suggest that although the nucleocapsids appear as closed loops, the viral RNA has free 5' and 3' ends and is, therefore, not circular.
Article
PARAMYXOVIRUSES are assuming increasing importance as possible causative agents of several human diseases1,2. To date little is known about their mode of replication, although it has been suggested that the nucleocapsid replicates without extensive dissociation of the protein from the nucleic acid component3. Previous reports on the structure of nucleocapsids extracted from virions or infected cells, with or without further purification, have shown by electron microscopy that the molecules are linear, and although short pieces can be found, the majority are about 1.0 µm long4. We report here the occurrence of rare circular forms of measles nucleocapsid as well as linear molecules of greater than normal length.
Article
Oligonucleotide fingerprinting on slabs of polyacrylamide gel (De Wachter & Fiers, 1972) has been used to investigate the sequence relationships between the 42 S RNA genome of standard Semliki Forest virus and the intracellular virus-specified single-stranded RNAs isolated from baby hampster kidney cells infected with standard virus and co-infected with standard virus and defective-interfering particles.For the intracellular RNAs of standard virus-infected cells these studies show that the nucleotide sequence of the 26 S RNA, which acts as messenger RNA for the structural proteins of the virus particle [8] and [9], is located at the 3′ end of the 42 S RNA and that the 38 S and 33 S RNAs which appear as minor species on RNA polyacrylamide gels run under non-denaturing conditions, are conformational variants of the 42 S and 26 S RNAs, respectively.Studies on the two RNAs of defective-interfering particles isolated from cells co-infected with standard virus and defective-interfering particles indicate extensive sequence homology between the defective-interfering RNAs, and show that both RNAs contain nucleotide sequences present in both the structural and non-structural genes of the 42 S RNA. Furthermore, by determining the order (5′→3′) of the characteristic oligonucleotides of the 42 S RNA, and by digestion with snake venom exonuclease, it is concluded that the nucleotide sequences of the defective-interfering RNAs are derived from the 5′ and 3′ extremities of the 42 S RNA; about 75% from the 5′ end and 25% from the 3′ end. The significance of this observation in relation to the genesis of defective-interfering particles and to the mechanism of interference by the defective-interfering RNAs is discussed.
Article
Uukuniemi virus, a proposed member of the new large Bunyavirus group of arboviruses, has an interesting structure which differs in many respects from that of other enveloped RNA viruses. The surface structure reveals a clustering of the two glycoproteins into distinct capsomere-like subunits, arranged in a T = 12 icosahedral lattice. The genome is found in at least three pieces that appear to represent unique virus-specific RNAs. The ribonucleoproteins corresponding to the RNA species are circular, according to electron micrographs. The virion contains an RNA-dependent RNA polymerase suggesting that Uukuniemi virus is a negative-strand virus. This paper summarizes the data obtained on the structure of Uukuniemi virus and discusses its relationship to other members of the Bunyavirus group.
Article
A strain of the Indiana serotype of vesicular stomatitis virus (VSV) designated HR-LT produces a defective particle which differs from the T particle described by others. This “long T” particle is approximately one-half the length of the infectious B particle and contains a single-stranded RNA having a sedimentation coefficient of 30 S. Infection of L cells with the HR-LT strain of VSV results in the intracellular accumulation of viral nucleoprotein structures similar to nucleoprotein structures derived from mature B and “long T” particles. In the infected cell, virus-specific RNA species with sedimentation coefficients of 30 S and 15 S appear to be associated with polyribosomes.
Article
Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.
Article
The vesicular stomatitis virus-specific messenger RNA fraction of infected HeLa cell polysomes has been identified by (1) its removal from the polysome region of sucrose gradients by EDTA treatment, and (2) its sensitivity to ribonuclease. This mRNA fraction anneals to virion RNA and is a collection of several species of small RNA molecules of the approximate size distribution appropriate to code for the virus-specific polypeptides found in infected cells.The base composition of the mRNA region of sucrose gradients is lower in U and higher and A than would be predicted for RNA complementary to total virion RNA. This finding, along with labeling kinetics and ribonuclease resistance of structures found in the polysome region of sucrose gradients, gives rise to two possible working models for the mechanism of vesicular stomatitis virus mRNA synthesis.
Article
The kinetics of RNA synthesis by vesicular stomatitis virus particles have been examined. Product RNA is synthesized in association with template RNA and subsequently released as free species having molecular weights of between 2 and 10 × 105. The viral RNA is conserved intact during synthesis. Two defective particles (VSV-II and VSV-III) have been characterized. The viral particles do not have the RNA-directed, DNA-directed or hybrid RNA-DNA-directed DNA polymerase that oneogenic viruses possess.
Article
The small defective T particle of vesicular stomatitis virus (VSV-111) has no detectable enzyme activity when assayed at 31 C, although qualitatively it possesses all the virion proteins found in complete VSV-1 virions. With VSV-1 transcription product ribonucleic acid (RNA), it is shown that the VSV-111 RNA is identical to part of the VSV-1 genome. Evidence is also presented to support the idea that in vitro VSV-1 transcription is sequential.
Article
Exposure of vesicular stomatitis virus-infected Chinese hamster ovary cells to cycloheximide results in the complete transcription of virion ribonucleic acid (RNA) into only 28S and 13 to 15S viral-specific RNA species. These RNA are identical to viral messenger RNA by the following criteria: size, single-strandedness, complementarity to virion RNA, and formation of messenger ribonucleoproteins. This transcription represents the intracellular enzymatic activity of the virion-associated polymerase and is shown to be dependent on input multiplicity. Intracellular transcription differs from in vitro polymerase activity in having a temperature optimum of 34 to 37 C and in synthesizing 28S as well as 13 to 15S messenger RNA species. Addition of interfering quantities of defective T particles to these cycloheximide-treated cells, either an hour before or at the same time as standard B particles of vesicular stomatitis virus, does not alter the rate of transcription nor does it change the sucrose gradient pattern of the viral RNA species. From these results it is concluded that the RNA of defective T particles does not serve a transcriptive function and probably interferes through the replicative mechanism for virion RNA synthesis.
Article
The wild-type strain of vesicular stomatitis virus (VSV) contains in its complete virion (VSV-1, B particles) a minus strand RNA. The principle defective particle of the wild-type strain (VSV-111, T particles) contains a shorter minus strand, homologous to part of the VSV-1 genome. Neither virion contains any detectable complementary (plus) strand RNA. In contrast, a preparation of a heat-resistant (HR) strain of VSV containing defective virions was found to contain both plus (21%) and minus strand RNA, present in several distinct size classes. It was found that the RNA in the HR virion preparation was at least 94% single-stranded and principally (96%) in ribonucleoprotein complexes. On extraction the plus and minus strand RNA species partially annealed to give a population of double- and multistranded RNA species. A small amount of RNA polymerase activity was associated with the HR defective virus preparation.
Article
All defective T particles of Indiana serotype vesicular stomatitis virus contain an RNA polymerase activity. This polymerase activity is particle bound and like the B virion transcriptase requires detergent-disruption for activation. It requires magnesium rather than manganese as a cofactor and all four nucleoside triphosphates as substrate. Unlike the B virion transcriptase, it gives linear kinetics of RNA synthesis for only about 1 hr. It makes only small amounts of product RNA, and this product consists of extremely short chains of RNA which anneal poorly to the T particle template RNA. Nearly 40% of the 3H-adenylate incorporated by T particle polymerase is present in poly (A) tracts. This poly (A) synthesizing activity of T particles differs from the B virion activity in producing short tracts of poly (A) which are not associated with large messenger RNA molecules.
Article
The defective interfering particles of vesicular stomatitis virus are analogous to deletion mutants since they contain only part of the single-stranded, unfragmented viral RNA genome (Huang, 1973). The sequences contained in the RNA of DI† particles of varying size classes were studied in relation to viral cistrons by annealing with complementary viral messenger RNA species. For these studies, DI particle RNAs corresponding to and of the viral genome were purified from cells infected with a temperature-sensitive, and a heat-resistant mutant, respectively. In the course of isolating the wild type DI particles it was found that the size of their RNA varied, depending on the origin of the inoculum. Three different wild type DI particle RNA species were selected that were generated consistently by wild type inocula from three different laboratories, and which corresponded to approximately to of the viral genome. These RNA species were annealed with purified viral mRNA of the 30 S and 18 to 13 S size classes. The following conclusions were drawn from the results: 1.(1) The two mRNA size classes contained unique nucleotide sequences, which in combination were complementary to the entire viral genome.2.(2) The long DI particle RNA from the heat-resistant mutant contained nucleotide sequences complementary to all mRNA species of the 18 S to 13 S size class, but very few if any to the 30 S class.3.(3) In contrast, all the shorter DI particle RNA species contained either exclusively or predominantly sequences complemenary to the 30 S mRNA. The longest DI particle RNA of this group also contained some sequences complementary to the 18 S to 13 S class. Annealing experiments involving mixtures of these DI particle RNAs with the 30 S mRNA suggested that the longer RNA species contained all the nucleotide sequences of the shorter species.On the basis of these conclusions, two alternate maps of the DI particle RNAs relative to cistronic regions were constructed. The first map invokes a fixed initiation point for all particle RNAs containing nucleotide sequences in the 30 S mRNA domain and a variable termination point. This map is not compatible with hypothetical recognition sequences of substantial length common to all DI particles. The second map postulates short overlapping sequences near the intercistronic border. This interpretation would place the common site at the non-equivalent RNA ends (in relation to 5′ and 3′ termini) of the long particle and the short particles, respectively. Other hypotheses were also proposed. The possibility that the unique capacity of the long DI particle for heterotypic interference may be due to either its different site on the viral genome or its content of the entire viral cistron was considered.
Article
Viral mRNA isolated from infected cells and the virion RNA isolated from two classes of defective interfering particles have been analyzed by RNA-RNA duplex reactions. The results show that the RNA of both defective interfering particles is viral, not host in origin. The RNA isolated from the two defective particles represents homogeneous populations of molecules containing only part of the genetic information present in the whole VSV genome. Annealing competition experiments indicate that if any overlap exists between the two, it is less than 220 nucleotides. We conclude from the data presented that a rudimentary physical map of the VSV and DI particle genomes is {A figure is presented} Our results suggest that there is not a single specific site that is required for autointerference.
Article
Using a formamide-urea modification of the Kleinschmidt spreading technique, the contour length of 40–45 S vesicular stomatitis virus (VSV) RNA was determined to be 3.1 μm. Molecules isolated from defective T particles were found to be 1.1 μm in length. The results correspond well with previously published data on the molecular weight of the viral genome. Additional molecules were observed which may represent RNA species from other intermediate-sized defective particles.
Article
Denaturation and renaturation of the adenovirus-2 chromosome (a duplex rod) generates single-stranded circles of unit length. These circles can be opened into linear DNA molecules by digestion with exonuclease III, indicating that hydrogen bonding between the two ends of an adenovirus strand is responsible for maintaining the rod in a circular state. The formation of adenovirus single-stranded circles, and their sensitivity to exonuclease III, indicate that the mature adenovirus-2 DNA molecule contains an inverted terminal repetition. That is, the base sequence at one end of the molecule is inverted and appears again at the other end of the molecule. This is the first example of such a structure, and its function is unknown.
Article
At least 70% of plus or minus strands of adenovirus-associated virus DNA contain self-complementary sequences at or near their termini. Self-annealing of these sequences generates circular molecules that are closed by duplex, hydrogen-bonded segments. The self-annealed segments are sensitive to exonuclease III and have a thermal stability comparable to that of double-stranded DNA molecules. Length measurements of double-stranded adenovirus-associated virus DNA molecules show a bimodal distribution, with the larger component being 10% shorter than SV40 DNA. The presence of self-complementary terminal sequences in single-stranded molecules of viral DNA has been observed previously only with DNA from adenoviruses. It is thus especially notable that adenovirus-associated virus replication is unconditionally dependent on a helper adenovirus. A possible role for terminal self-complementary sequences in viral DNA replication is suggested.
Article
The effect of diazepam (Valium) on vestibular compensation was studied in the cat. Twelve hemilabyrinthectomized cats were given diazepam (0.4 mg/kg/day — physiological dose; 1.6 mg/kg/day — toxic dose) chronically during the compensation period. Thirteen additional hemilabyrinthectomized animals were used as controls. The neural activity of the medial vestibular nucleus was monitored as well as the observations made on nystagmus and postural co-ordination. The results indicate that diazepam does not delay vestibular compensation in the cat.
Article
A unique form of terminal redundancy has been observed in DNA molecules extracted from several human adenovirus serotypes. Electron microscopic studies reveal that single-stranded circular molecules are formed when native DNA is denatured and then annealed. Temperatures approaching the T(m) of native DNA are required to convert circles to linear molecules, indicating a high degree of self-complementarity between terminal base sequences of DNA strands. Single-stranded circles are not generated if a limited number of nucleotides (2-4%) are removed from the 3' ends of native DNA by digestion with Escherichia coli exonuclease III before denaturation and annealing. The lenght of the redundant segment appears to differ among major serotypic groups, and a possible association between increased length of the redundant segment and increased oncogenic capability of virus serotype is suggested. Evidence for the configuration of the duplex closure region of circular molecules is also presented.
Article
Sedimentation behavior is used to follow strand combination in T7 DNA and to analyze the nature of the product formed. Complementary single strands of DNA apparently combine under any condition where the DNA double helix is stable. The rate of strand combination is most strongly influenced by the ionic strength and the degree of folding of the single strands; the nature of the product formed is determined by the degree of folding of the reactants. Temperature affects the reaction primarily through its influence on folding, except at temperatures near Tm, where its effect on the stability of base pairing becomes dominant.
Article
Defective interfering particles of New Jersey serotype VSV were found to contain very little or no virion transcriptase activity. Furthermore no VSV transcriptase activity was observed in cells treated with cycloheximide and infected with DI. Cells infected with DI together with standard infectious virus and cycloheximide showed little or no DI interference with standard virion primary transcription, whereas double infection in the absence of cycloheximide exhibited strong DI interference with replication of standard virus genome RNA. We present a model of DI interference based upon competition at the level of viral RNA replication.
I972). A unique form of terminal redundancy in adenovirus DNA molecules
  • C F Garon
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GARON, C. F., BERRY, K. & ROSE, J. g. (I972). A unique form of terminal redundancy in adenovirus DNA molecules. Proceedings of the National Academy of Sciences of the United States of America 69, 239~-2395.
In vitro replication of RNA tumor viruses
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VILLA-KOMAROFF, L., GUTTMAN, N., BALTIMORE, D. & LODISH, H. F. 0975). Complete translation of poliovirus RNA in a eukaryotic cell-free system. Proceedings of the National Academy of Sciences of the United States of America 7z, 4157-416I.
Adenovirus-2 DNA contains an inverted terminal repetition
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WOLESON, J. & DRESSLER, D. (I972). Adenovirus-2 DNA contains an inverted terminal repetition. Proceedings of the National Academy of Sciences of the United States of America 69, 3o54-3o57. (Received I2 May I977)