Replication of virus-like particles in Penicillium stoloniferum mycelia

1974 ◽  
Vol 20 (2) ◽  
pp. 113-117 ◽  
Author(s):  
R. W. Detroy ◽  
E. B. Lillehoj ◽  
C. W. Hesseltine
Keyword(s):  
Nucleic Acid ◽  
Rna Synthesis ◽  
Fungal Growth ◽  
Viral Rna ◽  
Direct Extraction ◽  
Viral Dsrna ◽  
Virus Like Particles ◽  
Absorption Determination ◽  
Penicillium Stoloniferum ◽  
Cellulose Column

Direct extraction of nucleic acid from mycelium of Penicillium stoloniferum yielded five viral double-stranded ribonucleic acid (dsRNA) components, transfer RNA (tRNA), and a deoxyribonuclease-sensitive fraction of deoxyribonucleic acid. Analysis of fungal cells from a submerged fermentation demonstrated detectable viral dsRNA and both types of virus-like particles (VLP) at 36 h with an increase in viral RNA synthesis for both VLP parallel to fungal growth up to 72 h followed by mycelial autolysis and loss of viral RNA. Replication of nucleic acid from VLP was measured by (i) incorporation of 14C-2-uracil into viral dsRNA, (ii) purification of viral dsRNA by cellulose column chromatography and subsequent ultraviolet absorption determination, and (iii) analysis of nucleic acid species by poly-acrylamide gel electrophoresis.

scholarly journals Physiologic RNA Targets and Refined Sequence Specificity of Coronavirus EndoU

2020 ◽  
Author(s):  
Rachel Ancar ◽  
Yize Li ◽  
Eveline Kindler ◽  
Daphne A. Cooper ◽  
Monica Ransom ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Viral Rna ◽  
Host Responses ◽  
Regulatory Sequences ◽  
Sequence Specificity ◽  
Rnase L ◽  
Viral Dsrna ◽  
Negative Strand ◽  
Rna Targets

ABSTRACTCoronavirus EndoU inhibits dsRNA-activated antiviral responses; however, the physiologic RNA substrates of EndoU are unknown. In this study, we used mouse hepatitis virus (MHV)-infected bone-marrow-derived macrophage (BMM) and cyclic phosphate cDNA sequencing to identify the RNA targets of EndoU. EndoU targeted viral RNA, cleaving the 3′ side of pyrimidines with a strong preference for U⬇A and C⬇A sequences (endoY⬇A). EndoU-dependent cleavage was detected in every region of MHV RNA, from the 5′ NTR to the 3′ NTR, including transcriptional regulatory sequences (TRS). Cleavage at two CA dinucleotides immediately adjacent to the MHV poly(A) tail suggest a mechanism to suppress negative-strand RNA synthesis and the accumulation of viral dsRNA. MHV with EndoU (EndoUmut) or 2′-5′ phosphodiesterase (PDEmut) mutations provoked the activation of RNase L in BMM, with corresponding cleavage of RNAs by RNase L. The physiologic targets of EndoU are viral RNA templates required for negative-strand RNA synthesis and dsRNA accumulation.ImpactCoronavirus EndoU cleaves U⬇A and C⬇A sequences (endoY⬇A) within viral (+) strand RNA to evade dsRNA-activated host responses.

scholarly journals Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

2015 ◽  
Vol 89 (9) ◽  
pp. 5148-5153 ◽  
Author(s):  
Priya Luthra ◽  
David S. Jordan ◽  
Daisy W. Leung ◽  
Gaya K. Amarasinghe ◽  
Christopher F. Basler
Keyword(s):  
Rna Synthesis ◽  
Ebola Virus ◽  
Mutational Analysis ◽  
Cytoplasmic Dynein ◽  
Viral Rna ◽  
Interferon Production ◽  
Dynein Light Chain ◽  
High Affinity ◽  
Functional Consequences ◽  
Oligomerization Domain

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

scholarly journals Synthetic Histidine-Rich Peptides Inhibit Candida Species and Other Fungi In Vitro: Role of Endocytosis and Treatment Implications

2006 ◽  
Vol 50 (8) ◽  
pp. 2797-2805 ◽  
Author(s):  
Jingsong Zhu ◽  
Paul W. Luther ◽  
Qixin Leng ◽  
A. James Mixson
Keyword(s):  
Nucleic Acid ◽  
Antifungal Activity ◽  
Antifungal Agents ◽  
Fungal Growth ◽  
Nucleic Acid Delivery ◽  
Histatin 5 ◽  
Ph Buffering ◽  
Endosomal Acidification

ABSTRACT A family of histidine-rich peptides, histatins, is secreted by the parotid gland in mammals and exhibits marked inhibitory activity against a number of Candida species. We were particularly interested in the mechanism by which histidine-rich peptides inhibit fungal growth, because our laboratory has synthesized a variety of such peptides for drug and nucleic acid delivery. In contrast to naturally occurring peptides that are linear, peptides made on synthesizers can be varied with respect to their degrees of branching. Using this technology, we explored whether histidine-lysine (HK) polymers of different complexities and degrees of branching affect the growth of several species of Candida. Polymers with higher degrees of branching were progressively more effective against Candida albicans, with the four-branched polymer, H2K4b, most effective. Furthermore, H2K4b accumulated efficiently in C. albicans, which may indicate its ability to transport other antifungal agents intracellularly. Although H2K4b had greater antifungal activity than histatin 5, their mechanisms were similar. Toxicity in C. albicans induced by histatin 5 or branched HK peptides was markedly reduced by 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate, an inhibitor of anion channels. We also determined that bafilomycin A1, an inhibitor of endosomal acidification, significantly decreased the antifungal activity of H2K4b. This suggests that the pH-buffering and subsequent endosomal-disrupting properties of histidine-rich peptides have a role in their antifungal activity. Moreover, the ability of the histidine component of these peptides to disrupt endosomes, which allows their escape from the lysosomal pathway, may explain why these peptides are both effective antifungal agents and nucleic acid delivery carriers.

Plant Viral RNA Synthesis in Cell-Free Systems

1994 ◽  
Vol 32 (1) ◽  
pp. 311-335 ◽  
Author(s):  
M de Graaff ◽  
E M J Jaspars
Keyword(s):  
Rna Synthesis ◽  
Viral Rna

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

Analyse des Vermehrungsmechanismus des Newcastle disease Virus (NDV) mit Hilfe verschiedener Inhibitoren

1967 ◽  
Vol 22 (12) ◽  
pp. 1319-1330 ◽  
Author(s):  
Werner Schäfer ◽  
Liselotte Pister ◽  
Rita Schneider
Keyword(s):  
Chick Embryo ◽  
Viral Antigen ◽  
Rna Synthesis ◽  
Protein S ◽  
Viral Rna ◽  
Chick Embryo Fibroblast ◽  
Sensitive Material ◽  
Sensitive Phase ◽  
Antigenic Material ◽  
Structural Antigen

The reproduction of NDV in chick-embryo-fibroblast cultures was studied with 6-Azauridine, 8-Azaguanine, Parafluorophenylalanine (FPA) and Puromycine as inhibitors. The results suggest that no virus initiated FPA-sensitive material is needed for the uncoating of the infecting particles, and that viral parental RNA is able to induce the formation of protein (s) needed for viral RNA-synthesis (“RNA-protein“) as well as the production of viral structural antigen (s). Further antigenic material appears after the beginning of new viral RNA-synthesis. The “RNA-protein (s)“become (s) detectable between 2 and 3 hours after infection and is (are) stable in its function over several hours. According to the formation of viral antigenic material parental viral RNA can act as a messenger longer than 9 hours. The capacity for the production of hemagglutinating units appears after the viral antigen producing capacity, when viral RNA can already be synthesized. This capacity is separated from that to produce plaque forming particles by a FPA-sensitive phase. The character of the corresponding FPA-sensititve material is unknown.

scholarly journals Encapsidation of Viral RNA inPicornavirales: Studies on Cowpea Mosaic Virus Demonstrate Dependence on Viral Replication

2018 ◽  
Vol 93 (2) ◽  
Author(s):  
Inga Kruse ◽  
Hadrien Peyret ◽  
Pooja Saxena ◽  
George P. Lomonossoff
Keyword(s):  
Viral Replication ◽  
Mosaic Virus ◽  
Viral Rna ◽  
Cowpea Mosaic Virus ◽  
Rna Packaging ◽  
Functional Link ◽  
Close Coupling ◽  
Genomic Rnas ◽  
Viral Rnas ◽  
Virus Like Particles

ABSTRACTTo elucidate the linkage between replication and encapsidation inPicornavirales, we have taken advantage of the bipartite nature of a plant-infecting member of this order, cowpea mosaic virus (CPMV), to decouple the two processes. RNA-free virus-like particles (empty virus-like particles [eVLPs]) can be generated by transiently coexpressing the RNA-2-encoded coat protein precursor (VP60) with the RNA-1-encoded 24,000-molecular-weight (24K) protease, in the absence of the replication machinery (K. Saunders, F. Sainsbury, and G. P. Lomonossoff, Virology 393:329–337, 2009, https://doi.org/10.1016/j.virol.2009.08.023). We have made use of the ability to produce assembled capsids of CPMV in the absence of replication to examine the putative linkage between RNA replication and packaging in thePicornavirales. We have created a series of mutant RNA-1 and RNA-2 molecules and have assessed the effects of the mutations on both the replication and packaging of the viral RNAs. We demonstrate that mutations that affect replication have a concomitant impact on encapsidation and that RNA-1-mediated replication is required for encapsidation of both RNA-1 and RNA-2. This close coupling between replication and encapsidation provides a means for the specific packaging of viral RNAs. Moreover, we demonstrate that this feature of CPMV can be used to specifically encapsidate custom RNA by placing a sequence of choice between the RNA-2 sequences required for replication.IMPORTANCEThe mechanism whereby members of the orderPicornaviralesspecifically package their genomic RNAs is poorly understood. Research with monopartite members of the order, such as poliovirus, indicated that packaging is linked to replication, although the presence of “packaging signals” along the length of the viral RNA has also been suggested. Thanks to the bipartite nature of the CPMV genome, which allows the manipulation of RNA-1 without modifying RNA-2, we show here that this specificity is due to a functional link between the two processes of viral replication and encapsidation. This has important implications for our understanding of the fundamental molecular biology ofPicornaviralesand opens the door to novel research and therapeutic applications in the field of custom RNA packaging and delivery technologies.

scholarly journals An Alanine-to-Valine Substitution in the Residue 175 of Zika Virus NS2A Protein Affects Viral RNA Synthesis and Attenuates the Virus In Vivo

Viruses ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 547 ◽  
Author(s):  
Silvia Márquez-Jurado ◽  
Aitor Nogales ◽  
Ginés Ávila-Pérez ◽  
Francisco Iborra ◽  
Luis Martínez-Sobrido ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Zika Virus ◽  
Rna Synthesis ◽  
Infectious Clone ◽  
Vero Cells ◽  
Viral Rna ◽  
Single Amino Acid ◽  
Reverse Genetic System ◽  
Infectious Clones

The recent outbreaks of Zika virus (ZIKV), its association with Guillain–Barré syndrome and fetal abnormalities, and the lack of approved vaccines and antivirals, highlight the importance of developing countermeasures to combat ZIKV disease. In this respect, infectious clones constitute excellent tools to accomplish these goals. However, flavivirus infectious clones are often difficult to work with due to the toxicity of some flavivirus sequences in bacteria. To bypass this problem, several alternative approaches have been applied for the generation of ZIKV clones including, among others, in vitro ligation, insertions of introns and using infectious subgenomic amplicons. Here, we report a simple and novel DNA-launched approach based on the use of a bacterial artificial chromosome (BAC) to generate a cDNA clone of Rio Grande do Norte Natal ZIKV strain. The sequence was identified from the brain tissue of an aborted fetus with microcephaly. The BAC clone was fully stable in bacteria and the infectious virus was efficiently recovered in Vero cells through direct delivery of the cDNA clone. The rescued virus yielded high titers in Vero cells and was pathogenic in a validated mouse model (A129 mice) of ZIKV infection. Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. This BAC approach provides a stable and reliable reverse genetic system for ZIKV that will help to identify viral determinants of virulence and facilitate the development of vaccine and therapeutic strategies.

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