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Journal of Virology Nov 1979Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol...
Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.
Topics: Defective Viruses; Genes, Viral; Nucleoproteins; Orthomyxoviridae; Ribonucleoproteins; Viral Interference; Viral Proteins
PubMed: 501805
DOI: 10.1128/JVI.32.2.697-702.1979 -
Journal of Virology Apr 1985Three of six independently derived defective interfering (DI) particles of Sindbis virus generated by high-multiplicity passaging in cultured cells have tRNAAsp...
Three of six independently derived defective interfering (DI) particles of Sindbis virus generated by high-multiplicity passaging in cultured cells have tRNAAsp sequences at the 5' terminus of their RNAs (Monroe and Schlesinger, J. Virol. 49:865-872, 1984). In the present work, we found that the 5'-terminal sequences of the three tRNAAsp-negative DI RNAs were all derived from viral genomic RNA. One DI RNA sample had the same 5'-terminal sequence as the standard genome. The DI RNAs from another DI particle preparation were heterogeneous at the 5' terminus, with the sequence being either that of the standard 5' end or rearrangements of regions near the 5' end. The sequence of the 5' terminus of the third DI RNA sample consisted of the 5' terminus of the subgenomic 26S mRNA with a deletion from nucleotides 24 to 67 of the 26S RNA sequence. These data showed that the 5'-terminal nucleotides can undergo extensive variations and that the RNA is still replicated by virus-specific enzymes. DI RNAs of Sindbis virus evolve from larger to smaller species. In the two cases in which we followed the evolution of DI RNAs, the appearance of tRNAAsp-positive molecules occurred at the same time as did the emergence of the smaller species of DI RNAs. In pairwise competition experiments, one of the tRNAAsp-positive DI RNAs proved to be the most effective DI RNA, but under identical conditions, a second tRNAAsp-positive DI RNA was unable to compete with the tRNAAsp-negative DIs. Therefore, the tRNAAsp sequence at the 5' terminus of a Sindbis DI RNA is not the primary factor in determining which DI RNA becomes the predominant species in a population of DI RNA molecules.
Topics: Aspartic Acid; Base Sequence; Defective Viruses; RNA, Transfer; RNA, Viral; Sindbis Virus; Viral Interference; Virus Replication
PubMed: 3973983
DOI: 10.1128/JVI.54.1.38-44.1985 -
Proceedings of the National Academy of... Feb 1991An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the...
An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.
Topics: Animals; Antibodies, Monoclonal; Cell Line; Cloning, Molecular; DNA, Viral; Defective Viruses; Genetic Vectors; Microscopy, Electron; Plasmids; RNA, Viral; Vesicular stomatitis Indiana virus; Viral Proteins; Virus Replication
PubMed: 1847519
DOI: 10.1073/pnas.88.4.1379 -
Journal of Virology Apr 1984To develop a highly sensitive and direct assay for defective interfering (DI) particles of vesicular stomatitis virus (VSV), we reverse transcribed RNA from DI particles...
To develop a highly sensitive and direct assay for defective interfering (DI) particles of vesicular stomatitis virus (VSV), we reverse transcribed RNA from DI particles and cloned the DNA in pBR322 and used it as hybridization probes. At the lower limit, cDNA of about 850 nucleotides detected 150 pg of VSV RNA. For differentiation of hybridizable sequences found in the RNA of DI particles from complementary or identical sequences in the L mRNA or standard genomic RNA of VSV, RNA obtained from mouse brains was first separated by size, blotted onto nitrocellulose, and then hybridized to in vitro-labeled cDNA probe. Genomic VSV, DI, or L mRNA sequences from one-half of the brain of an infected mouse were detectable, whereas uninfected mice failed to react with this specific probe. When mice were infected intranasally with 10(8) PFU of standard VSV, most of them died between days 6 and 7, and the detection of standard genomic RNA correlated with paralysis and death. DI RNA was not detected in these mice. When mice were infected with 10(8) PFU of standard VSV together with an equivalent amount of DI particles, similar results were obtained. When fewer DI particles were inoculated together with standard virus, significant protection of mice occurred together with the detection of DI RNA. These results indicate that DI particles are protective in vivo and that the details of the virus-host interaction may resemble the cyclic growth patterns in cell cultures for standard VSV and its DI particles.
Topics: Animals; Brain; DNA; Defective Viruses; Electrophoresis, Agar Gel; Female; Male; Mice; Nucleic Acid Hybridization; RNA, Viral; Vesicular stomatitis Indiana virus; Viral Interference; Virus Diseases
PubMed: 6321804
DOI: 10.1128/JVI.50.1.86-91.1984 -
Journal of Virology Jan 1981An erythroleukemia cell clone, 7C, which failed to produce reverse transcriptase-containing virions or infectious virus, was found to produce noninfectious virus...
An erythroleukemia cell clone, 7C, which failed to produce reverse transcriptase-containing virions or infectious virus, was found to produce noninfectious virus particles by gradient banding of [3H]leucine- and [3H]uridine-labeled virions. The RNA from the 7C virus was shown to consist of the normal 70S size component, which converted to 35S upon heat denaturation. In contrast, the 7C virion proteins showed multiple defects. Analysis of the virion proteins by gel electrophoresis demonstrated that the pr65 gag precursor was incorporated into the 7C virus and that the processing of this precursor was severely diminished. Polymerase proteins pr180gag-pol and pr120pol were also detected in virions, and a third possible polymerase protein, p70, was reduced in size compared to its normal counterpart, p80. Incorporation of the viral gp70 glycoprotein into particles was also reduced 10-fold, despite synthesis and incorporation of gp70 into the 7C cell membrane in normal amounts. Pulse-chase analysis of the synthesis of the viral gag and env proteins in 7C cells showed greatly reduced amounts of pr180gag-pol, pr65gag, p80gag, and p42gag, whereas pr90env, gp70, and spleen focus-forming virus-specific gp55 were synthesized and processed normally. These results suggested that at least one defect in 7C virus was impaired cleavage of gag or pol proteins or both, most likely due to a lack of the appropriate viral protease, and that this lack of cleavage might affect incorporation of gp70 into virus particles.
Topics: Animals; Cells, Cultured; Clone Cells; Defective Viruses; Friend murine leukemia virus; Gene Products, gag; Leukemia, Erythroblastic, Acute; Mice; Neoplasms, Experimental; Protein Precursors; RNA-Directed DNA Polymerase; Viral Envelope Proteins; Viral Proteins
PubMed: 6163868
DOI: 10.1128/JVI.37.1.161-170.1981 -
Journal of Virology May 1974Supercoiled DNA molecules purified from mouse cells infected with high-multiplicity-passaged polyoma virus has a broader size distribution and sediments more slowly than...
Supercoiled DNA molecules purified from mouse cells infected with high-multiplicity-passaged polyoma virus has a broader size distribution and sediments more slowly than DNA derived from low-multiplicity-passaged virus. The shorter DNA molecules are predominately noninfectious. Virus populations containing distinct size classes of defective virus DNA were isolated by growing virus from single cells infected by a defective and nondefective helper virus (infectious center). This technique probably results in the cloning of defective virus particles. By applying the infectious center method to DNA from various fractions of sucrose gradients it has been possible to obtain shorter circular DNA molecules ranging in size from 50 to 95% of the unit-length polyoma DNA molecule. The shorter molecules in any one preparation are homogeneous in size. This class size is retained upon repeated passage of crude viral lysates at high multiplicity. Thus far, all the purified shorter DNA molecules tested appear to be noninfectious and largely resistant to cleavage by the R(1) restriction enzyme. Some of the purified defective molecules have been found to interfere with the production of infectious virus upon co-infection with unit-length infectious polyoma DNA.
Topics: Animals; Cells, Cultured; Centrifugation, Density Gradient; DNA, Viral; Defective Viruses; Mice; Polyomavirus; Viral Plaque Assay; Virus Cultivation
PubMed: 4363255
DOI: 10.1128/JVI.13.5.939-946.1974 -
Virology Dec 1995The host range of individual geminiviruses may be quite narrow, and closely related viruses can exhibit distinct host adaptations. Two such bipartite geminiviruses are...
The host range of individual geminiviruses may be quite narrow, and closely related viruses can exhibit distinct host adaptations. Two such bipartite geminiviruses are bean golden mosaic virus (GBMV) and tomato golden mosaic virus (TGMV). In both, the BL1 and BR1 genes are required for the spread of virus infection in plants. We have investigated the contributions of BL1 and BR1 to host-specific phenotypes of BGMV and TGMV by constructing hybrid viruses in which these coding regions were exchanged. Hybrids were assayed on bean, a good host for BGMV, and Nicotiana benthamiana, a good host for TGMV. A BGMV hybrid having TGMV BL1 and BR1 efficiently infected beans, but elicited attenuated symptoms. In N. benthamiana, this hybrid had slightly increased virulence and DNA accumulation relative to wild-type BGMV. A TGMV hybrid having BGMV BL1 and BR1 was virulent in N. benthamiana, but elicited attenuated symptoms. However, this hybrid exhibited no gain of function in beans relative to wild-type TGMV. Hybrid viruses with TGMV BL1 and BGMV BR1 had severely defective phenotypes in either viral or host background. Although exchanging BL1 and BR1 between BGMV and TGMV did not change host range, some host adaptation of these genes is suggested. However, virus-specific compatibility between BL1 and BR1 is of more importance for viability. Thus, these gene products may act in concert to potentiate virus movement.
Topics: Adaptation, Physiological; DNA, Viral; Defective Viruses; Fabaceae; Geminiviridae; Genes, Viral; Solanum lycopersicum; Open Reading Frames; Phenotype; Plant Viral Movement Proteins; Plants, Medicinal; Plants, Toxic; Nicotiana; Viral Proteins
PubMed: 8553533
DOI: 10.1006/viro.1995.0042 -
Vaccine Nov 2013Defective viral genomes (DVGs) are generated during virus replication. DVGs bearing complementary ends are strong inducers of dendritic cell (DC) maturation and of the...
Defective viral genomes (DVGs) are generated during virus replication. DVGs bearing complementary ends are strong inducers of dendritic cell (DC) maturation and of the expression of antiviral and pro-inflammatory cytokines by triggering signaling of the RIG-I family of intracellular pattern recognition receptors. Our data show that DCs stimulated with virus containing DVGs have an enhanced ability to activate human T cells and can induce adaptive immunity in mice. In addition, we describe the generation of a short Sendai virus (SeV)-derived DVG RNA (DVG-324) that maintains strong immunostimulatory activity in vitro and in vivo. DVG-324 induced high levels of Ifnb expression when transfected into cells and triggered fast expression of pro-inflammatory cytokines and mobilization of dendritic cells when injected into the footpad of mice. Importantly, DVG-324 enhanced the production of antibodies to a prototypic vaccine after a single intramuscular immunization in mice. Notably, the pro-inflammatory cytokine profile induced by DVG-324 was different from that induced by poly I:C, the only viral RNA analog currently used as an immunostimulant in vivo, suggesting a distinct mechanism of action. SeV-derived oligonucleotides represent novel alternatives to be harnessed as potent adjuvants for vaccination.
Topics: Animals; Antibodies, Viral; Cytokines; Defective Viruses; Dendritic Cells; Injections, Intramuscular; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; RNA, Viral; Sendai virus; T-Lymphocytes; Viral Vaccines
PubMed: 24099876
DOI: 10.1016/j.vaccine.2013.09.040 -
Virology Mar 2014Defective RNAs (D RNAs) are small RNA replicons derived from viral RNA genomes. No D RNAs have been found associated with members of the plus-strand RNA virus genus...
Defective RNAs (D RNAs) are small RNA replicons derived from viral RNA genomes. No D RNAs have been found associated with members of the plus-strand RNA virus genus Aureusvirus (family Tombusviridae). Accordingly, we sought to construct a D RNA for the aureusvirus Cucumber leaf spot virus (CLSV) using the known structure of tombusvirus defective interfering RNAs as a guide. An efficiently accumulating CLSV D RNA was generated that contained four non-contiguous regions of the viral genome and this replicon was used as a tool to studying viral cis-acting RNA elements. The results of structural and functional analyses indicated that CLSV contains counterparts for several of the major RNA elements found in tombusviruses. However, although similar, the CLSV D RNA and its components are distinct and provide insights into RNA-based specificity and mechanisms of function.
Topics: Base Sequence; Defective Viruses; Genome, Viral; Molecular Sequence Data; Nucleic Acid Conformation; RNA, Viral; Tombusviridae; Tombusvirus; Viral Proteins; Virus Replication
PubMed: 24606684
DOI: 10.1016/j.virol.2013.12.033 -
Virology Mar 1996Previous studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and...
Previous studies have shown that the protein encoded by herpes simplex virus type 1 (HSV-1) gene UL6 is required for processing and packaging of replicated viral DNA and is a minor component of virions and capsids. In this report, we describe the construction of UL6- HSV-1 mutants with a disrupted UL6 gene using complementing cells and show that they fail to synthesize the UL6 protein or produce infectious virus in noncomplementing cells. The mutants synthesized but failed to process and encapsidate viral DNA and accumulated only immature capsids which lacked the UL6 protein. Immunofluorescence analysis showed that the UL6 protein, when expressed transiently in transfected cells in the absence of other HSV-1 proteins, is localized exclusively to the nucleus. We also investigated an HSV-1 mutant with a defect in gene UL33, the product of which is also thought to be involved in viral DNA processing and packaging. The phenotype of this mutant on noncomplementing cells with regard to failure to process and encapsidate viral DNA, accumulation of immature capsids, and inability to produce infectious virus was the same as that of UL6- viruses. This mutant, however, produced capsids containing the UL6 protein, indicating that association of the UL6 protein with the capsid is independent of the UL33 protein.
Topics: Animals; Capsid; Cell Line; Cell Nucleus; Chlorocebus aethiops; Cricetinae; DNA, Viral; Defective Viruses; Herpesvirus 1, Human; Humans; Microscopy, Electron; Mutagenesis, Insertional; Recombinant Proteins; Vero Cells; Viral Proteins; Virus Replication
PubMed: 8599195
DOI: 10.1006/viro.1996.0098