scholarly journals Ribavirin Causes Error Catastrophe during Hantaan Virus Replication

2003 ◽  
Vol 77 (1) ◽  
pp. 481-488 ◽  
Author(s):  
William E. Severson ◽  
Connie S. Schmaljohn ◽  
Ali Javadian ◽  
Colleen B. Jonsson
Keyword(s):  
Mechanism Of Action ◽  
Inhibitory Effect ◽  
Viral Rna ◽  
Hantaan Virus ◽  
Slight Reduction ◽  
Mrna Levels ◽  
N Protein ◽  
High Mutation Frequency ◽  
Error Catastrophe ◽  
Infected Cells

ABSTRACT Except for ribavirin, no other antiviral drugs for treating hantaviral diseases have been identified. It is well established that ribavirin will inhibit the production of infectious Hantaan virus (HTNV); however, its mechanism of action is unknown. To characterize the inhibitory effect of ribavirin on HTNV, the levels of viral RNAs, proteins, and infectious particles were measured for 3 days posttreatment of HTNV-infected Vero E6 cells. HTNV-infected cells treated with ribavirin showed a slight reduction in the levels of cRNA, viral RNA, and mRNA populations on the first day postinfection. The amount of cRNA and viral RNA increased to that observed for untreated HTNV-infected cells on day 2, whereas mRNA levels were more greatly reduced on days 2 and 3. Despite the finding of S-segment mRNA, albeit low, three of the viral proteins—nucleocapsid (N) protein and glycoproteins G1 and G2—could not be detected by immunohistochemistry in ribavirin-treated cells. To test the hypothesis that these effects were caused by incorporation of ribavirin into nascent RNA and a resultant “error catastrophe” was occurring, we cloned and sequenced the S-segment cRNA/mRNA from ribavirin-treated or untreated cells from day 3. We found a high mutation frequency (9.5/1,000 nucleotides) in viral RNA synthesized in the presence of ribavirin. Hence, the transcripts produced in the presence of the drug were not functional. These results suggest that ribavirin's mechanism of action lies in challenging the fidelity of the hantavirus polymerase, which causes error catastrophe.

scholarly journals Activation ofTomato Bushy Stunt VirusRNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by PhospholipidsIn Vitro

2015 ◽  
Vol 89 (10) ◽  
pp. 5714-5723 ◽  
Author(s):  
Judit Pogany ◽  
Peter D. Nagy
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Viral Rna ◽  
Infected Cells ◽  
Membrane Bound ◽  
Replicase Complex ◽  
Rna Interaction ◽  
Positive Strand Rna

ABSTRACTSimilar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of theTomato bushy stunt virus(TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRpin vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activationin vitro. In contrast, phosphatidylglycerol (PG) shows a strong and dominant inhibitory effect onin vitroRdRp activation. We also demonstrate that PE and PC stimulate RdRp-viral plus-strand RNA [(+)RNA] interaction, while PG inhibits the binding of the viral RNA to the RdRp. Based on the stimulatory versus inhibitory roles of various phospholipids in tombusvirus RdRp activation, we propose that the lipid composition of targeted subcellular membranes might be utilized by tombusviruses to regulate new VRC assembly during the course of infection.IMPORTANCEThe virus-coded RNA-dependent RNA polymerase (RdRp), which is responsible for synthesizing the viral RNA progeny in infected cells of several positive-strand RNA viruses, is initially inactive. This strategy is likely to avoid viral RNA synthesis in the cytosol that would rapidly lead to induction of RNA-triggered cellular antiviral responses. During the assembly of the membrane-bound replicase complex, the viral RdRp becomes activated through an incompletely understood process that makes the RdRp capable of RNA synthesis. By using TBSV RdRp, we show that the co-opted cellular Hsp70 chaperone and neutral phospholipids facilitate RdRp activationin vitro. In contrast, phosphatidylglycerol (PG) has a dominant inhibitory effect onin vitroRdRp activation and RdRp-viral RNA interaction, suggesting that the membranous microdomain surrounding the RdRp greatly affects its ability for RNA synthesis. Thus, the activation of the viral RdRp likely depends on multiple host components in infected cells.

scholarly journals Antiviral action of nitric oxide on dengue virus type 2 replication

2006 ◽  
Vol 87 (10) ◽  
pp. 3003-3011 ◽  
Author(s):  
Ratree Takhampunya ◽  
R. Padmanabhan ◽  
Sukathida Ubol
Keyword(s):  
Nitric Oxide ◽  
Dengue Virus ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Viral Rna ◽  
Protein Accumulation ◽  
Rdrp Activity ◽  
No Donor ◽  
Infected Cells

Recently, nitric oxide (NO) has been shown to suppress dengue virus (DENV) RNA and protein accumulation in infected cells. In this report, the potential target of the inhibitory effect of NO was studied at the molecular level. The NO donor, S-nitroso-N-acetylpenicillamine (SNAP), showed an inhibitory effect on RNA accumulation at around 8–14 h post-infection, which corresponded to the step of viral RNA synthesis in the DENV life cycle. The activity of the viral replicase isolated from SNAP-treated DENV-2-infected cells was suppressed significantly compared with that of the negative-control N-acetyl-dl-penicillamine (NAP)-treated cells. Further investigations on the molecular target of NO action showed that the activity of recombinant DENV-2 NS5 in negative-strand RNA synthesis was affected in the presence of 5 mM SNAP in in vitro RNA-dependent RNA polymerase (RdRp) assays, whereas the RNA helicase activity of DENV-2 NS3 was not inhibited up to a concentration of 15 mM SNAP. These results suggest that the inhibitory effect of NO on DENV infection is partly via inhibition of the RdRp activity, which then downregulates viral RNA synthesis.

scholarly journals The Putative Helicase of the Coronavirus Mouse Hepatitis Virus Is Processed from the Replicase Gene Polyprotein and Localizes in Complexes That Are Active in Viral RNA Synthesis

1999 ◽  
Vol 73 (8) ◽  
pp. 6862-6871 ◽  
Author(s):  
Mark R. Denison ◽  
Willy J. M. Spaan ◽  
Yvonne van der Meer ◽  
C. Anne Gibson ◽  
Amy C. Sims ◽  
...  
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Rna Helicase ◽  
Viral Rna ◽  
Replicase Gene ◽  
Reading Frame ◽  
N Protein ◽  
Amino Terminal ◽  
Infected Cells

ABSTRACT The coronavirus mouse hepatitis virus (MHV) translates its replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed to be involved in viral RNA synthesis. Antibodies directed against polypeptides encoded by open reading frame 1b were used to characterize the expression and processing of the MHV helicase and to define the relationship of helicase to the viral nucleocapsid protein (N) and to sites of viral RNA synthesis in MHV-infected cells. The antihelicase antibodies detected a 67-kDa protein in MHV-infected cells that was translated and processed throughout the virus life cycle. Processing of the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known inhibitor of the MHV 3C-like proteinase. When infected cells were probed for helicase by immunofluorescence laser confocal microscopy, the protein was detected in patterns that varied from punctate perinuclear complexes to large structures that occupied much of the cell cytoplasm. Dual-labeling studies of infected cells for helicase and bromo-UTP-labeled RNA demonstrated that the vast majority of helicase-containing complexes were active in viral RNA synthesis. Dual-labeling studies for helicase and the MHV N protein showed that the two proteins almost completely colocalized, indicating that N was associated with the helicase-containing complexes. This study demonstrates that the putative RNA helicase is closely associated with MHV RNA synthesis and suggests that complexes containing helicase, N, and new viral RNA are the viral replication complexes.

scholarly journals Actin-Modulating Protein Cofilin Is Involved in the Formation of Measles Virus Ribonucleoprotein Complex at the Perinuclear Region

2015 ◽  
Vol 89 (20) ◽  
pp. 10524-10531 ◽  
Author(s):  
Ritsuko Koga ◽  
Yukihiko Sugita ◽  
Takeshi Noda ◽  
Yusuke Yanagi ◽  
Shinji Ohno
Keyword(s):  
Viral Genome ◽  
Measles Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Host Protein ◽  
Cellular Proteins ◽  
N Protein ◽  
Phosphorylated Form ◽  
Rnp Complex ◽  
Infected Cells

ABSTRACTIn measles virus (MV)-infected cells, the ribonucleoprotein (RNP) complex, comprised of the viral genome and the nucleocapsid (N) protein, phosphoprotein (P protein), and large protein, assembles at the perinuclear region and synthesizes viral RNAs. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, interacts with the MV N protein and aids in the formation of the RNP complex. Knockdown of cofilin using the short hairpin RNA reduces the formation of the RNP complex after MV infection and that of the RNP complex-like structure after plasmid-mediated expression of MV N and P proteins. A lower level of formation of the RNP complex results in the reduction of viral RNA synthesis. Cofilin phosphorylation on the serine residue at position 3, an enzymatically inactive form, is increased after MV infection and the phosphorylated form of cofilin is preferentially included in the complex. These results indicate that cofilin plays an important role in MV replication by increasing formation of the RNP complex and viral RNA synthesis.IMPORTANCEMany RNA viruses induce within infected cells the structure called the ribonucleoprotein (RNP) complex in which viral RNA synthesis occurs. It is comprised of the viral genome and proteins that include the viral RNA polymerase. The cellular proteins involved in the formation of the RNP complex are largely unknown. In this report, we show that cofilin, an actin-modulating host protein, binds to the measles virus (MV) nucleocapsid protein and plays an important role in the formation of the MV RNP complex and MV RNA synthesis. The level of the phosphorylated form of cofilin, enzymatically inactive, is increased after MV infection, and the phosphorylated form is preferentially associated with the RNP complex. Our findings determined with cofilin will help us better understand the mechanism by which the RNP complex is formed in virus-infected cells and develop new antiviral drugs targeting the RNP complex.

scholarly journals Mechanism of Action for Respiratory Syncytial Virus Inhibitor RSV604

2014 ◽  
Vol 59 (2) ◽  
pp. 1080-1087 ◽  
Author(s):  
SreeRupa Challa ◽  
Andrew D. Scott ◽  
Olga Yuzhakov ◽  
Ying Zhou ◽  
Choi Lai Tiong-Yip ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Mechanism Of Action ◽  
Rna Synthesis ◽  
Virus Assembly ◽  
Protein Localization ◽  
Viral Rna ◽  
Subgenomic Replicon ◽  
N Protein ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACTRespiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children and other high-risk populations. RSV nucleoprotein (N) is essential for virus assembly and replication as part of the viral ribonucleoprotein (RNP) complex. RSV604 was a putative N inhibitor in phase 2 clinical trials whose molecular mechanism of action (MoA) was not well understood. This study investigated the cell line-dependent potency of RSV604 and demonstrated its direct binding to the N proteinin vitro, providing the first evidence of direct target engagement for this class of inhibitors reported to date. The affinity of RSV604 N binding was not affected by RSV604 resistance mutations in the N protein. RSV604 engaged in two different MoAs in HeLa cells, inhibiting both RSV RNA synthesis and the infectivity of released virus. The lack of inhibition of viral RNA synthesis in some cell lines explained the cell-type-dependent potency of the inhibitor. RSV604 did not inhibit viral RNA synthesis in the RSV subgenomic replicon cells or in the cell-free RNP assay, suggesting that it might act prior to viral replication complex formation. RSV604 did not alter N protein localization in the infected cells. Taken together, these results provide new insights leading to an understanding of the MoAs of RSV604 and other similar N inhibitors.

scholarly journals Activity of Ribavirin against Hantaan Virus Correlates with Production of Ribavirin-5′-Triphosphate, Not with Inhibition of IMP Dehydrogenase

2006 ◽  
Vol 51 (1) ◽  
pp. 84-88 ◽  
Author(s):  
Yanjie Sun ◽  
Dong-Hoon Chung ◽  
Yong-Kyu Chu ◽  
Colleen B. Jonsson ◽  
William B. Parker
Keyword(s):  
Mechanism Of Action ◽  
Broad Spectrum ◽  
Antiviral Agent ◽  
Viral Rna ◽  
Hantaan Virus ◽  
Small Decrease ◽  
Secondary Importance ◽  
Imp Dehydrogenase ◽  
Dose Dependent ◽  
Primary Action

ABSTRACT Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5′-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5′-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 μg/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 μg/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.

Prevention of Gestational Diabetes Mellitus (GDM) and Probiotics: Mechanism of Action: A Review

2020 ◽  
Vol 16 (6) ◽  
pp. 538-545 ◽  
Author(s):  
Aziz Homayouni ◽  
Nahal Bagheri ◽  
Sakineh Mohammad-Alizadeh-Charandabi ◽  
Neda Kashani ◽  
Noshin Mobaraki-Asl ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Gestational Diabetes ◽  
Gut Microbiota ◽  
Gestational Diabetes Mellitus ◽  
Intestinal Permeability ◽  
Mechanism Of Action ◽  
Economic Cost ◽  
Slight Reduction ◽  
Future Health ◽  
Keywords Probiotics

Background: : Gestational Diabetes Mellitus (GDM) is a health problem that is increasing around the world. Introduction:: Prevention of GDM, rather than treatment, could have several benefits in terms of both health and economic cost. Even a slight reduction in maternal glucose in non-diabetic women, particularly in women at high risk for GDM, may have significant benefits for pregnancy results and the future health of off-springs. Probiotics are a relatively new intervention, which are assessed by mothers’ metabolism, and can reduce blood sugar levels, prevent gestational diabetes and reduce the maternal and fetal complications resulting from it. The aim of this study was to review the studies on the prevention of gestational diabetes and assess the potential beneficial effects of probiotics on gestational diabetes and their possible mechanism of action. Method:: Articles compiled through clinical trials indexed in PubMed, Science Direct, Cochran, and Medlib between 2000 and 2017, with the keywords probiotics, prevention, and gestational diabetes mellitus were selected. Result:: Considering the potential of probiotics in the modulation of gut microbiota, naturalization increases intestinal permeability, regulation of pro-inflammatory mediators’ secretion and thereby controlling local and systemic inflammation results in decreasing intestinal permeability, enhancing the immune system. It likely has the ability to prevent or control diabetes during pregnancy although confirmatory studies are still needed. Conclusion:: Experimental and clinical evidence support the supposition that the modulation of the gut microbiota via probiotic microorganisms could be effective in the prevention of gestational diabetes mellitus.

scholarly journals Atlas der SARS-CoV-2-RNA-Protein-Interaktionen in infizierten Zellen

BIOspektrum ◽  
2021 ◽  
Vol 27 (4) ◽  
pp. 376-379
Author(s):  
Nora Schmidt ◽  
Mathias Munschauer
Keyword(s):  
Mass Spectrometry ◽  
Viral Rna ◽  
Defense Strategies ◽  
Pharmacological Inhibition ◽  
Novel Therapeutics ◽  
Contact Sites ◽  
Human Proteins ◽  
Infected Cells

AbstractUsing RNA antisense purification and mass spectrometry, we identified more than 100 human proteins that directly and specifically bind SARS-CoV-2 RNA in infected cells. To gain insights into the functions of selected RNA interactors, we applied genetic perturbation and pharmacological inhibition experiments, and mapped the contact sites on the viral RNA. This led to the identification of host dependency factors and defense strategies, which can guide the design of novel therapeutics against SARS-CoV-2.

scholarly journals Extended Interactions between HIV-1 Viral RNA and tRNALys3 Are Important to Maintain Viral RNA Integrity

2020 ◽  
Vol 22 (1) ◽  
pp. 58
Author(s):  
Thomas Gremminger ◽  
Zhenwei Song ◽  
Juan Ji ◽  
Avery Foster ◽  
Kexin Weng ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Potential Interaction ◽  
Viral Rna ◽  
Primer Binding Site ◽  
Rna Integrity ◽  
Immunodeficiency Virus ◽  
Infected Cells ◽  
Extended Interaction ◽  
Hiv 1

The reverse transcription of the human immunodeficiency virus 1 (HIV-1) initiates upon annealing of the 3′-18-nt of tRNALys3 onto the primer binding site (PBS) in viral RNA (vRNA). Additional intermolecular interactions between tRNALys3 and vRNA have been reported, but their functions remain unclear. Here, we show that abolishing one potential interaction, the A-rich loop: tRNALys3 anticodon interaction in the HIV-1 MAL strain, led to a decrease in viral infectivity and reduced the synthesis of reverse transcription products in newly infected cells. In vitro biophysical and functional experiments revealed that disruption of the extended interaction resulted in an increased affinity for reverse transcriptase (RT) and enhanced primer extension efficiency. In the absence of deoxyribose nucleoside triphosphates (dNTPs), vRNA was degraded by the RNaseH activity of RT, and the degradation rate was slower in the complex with the extended interaction. Consistently, the loss of vRNA integrity was detected in virions containing A-rich loop mutations. Similar results were observed in the HIV-1 NL4.3 strain, and we show that the nucleocapsid (NC) protein is necessary to promote the extended vRNA: tRNALys3 interactions in vitro. In summary, our data revealed that the additional intermolecular interaction between tRNALys3 and vRNA is likely a conserved mechanism among various HIV-1 strains and protects the vRNA from RNaseH degradation in mature virions.

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