scholarly journals Intraspecies Host Specificity of a Single-Stranded RNA Virus Infecting a Marine Photosynthetic Protist Is Determined at the Early Steps of Infection

2006 ◽  
Vol 81 (3) ◽  
pp. 1372-1378 ◽  
Author(s):  
Hiroyuki Mizumoto ◽  
Yuji Tomaru ◽  
Yoshitake Takao ◽  
Yoko Shirai ◽  
Keizo Nagasaki
Keyword(s):  
Host Specificity ◽  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Progeny Virus ◽  
Virus Suspension ◽  
Virus Propagation ◽  
Protein Coding ◽  
Transmission Electron ◽  
A Cell

ABSTRACT Viruses are extremely abundant in seawater and are believed to be significant pathogens to photosynthetic protists (microalgae). Recently, several novel RNA viruses were found to infect marine photosynthetic protists; one of them is HcRNAV, which infects Heterocapsa circularisquama (Dinophyceae). There are two distinct ecotypes of HcRNAV with complementary intraspecies host ranges. Nucleotide sequence comparison between them revealed remarkable differences in the coat protein coding gene resulting in a high frequency of amino acid substitutions. However, the detailed mechanism supporting this intraspecies host specificity is still unknown. In this study, virus inoculation experiments were conducted with compatible and incompatible host-virus combinations to investigate the mechanism determining intraspecies host specificity. Cells were infected by adding a virus suspension directly to a host culture or by transfecting viral RNA into host cells by particle bombardment. Virus propagation was monitored by Northern blot analysis with a negative-strand-specific RNA probe, transmission electron microscopy, and a cell lysis assay. With compatible host-virus combinations, propagation of infectious progeny occurred regardless of the inoculation method used. When incompatible combinations were used, direct addition of a virus suspension did not even result in viral RNA replication, while in host cells transfected with viral RNA, infective progeny virus particles with a host range encoded by the imported viral RNA were propagated. This indicates that the intraspecies host specificity of HcRNAV is determined by the upstream events of virus infection. This is the first report describing the reproductive steps of an RNA virus infecting a photosynthetic protist at the molecular level.

scholarly journals Viral RNA at Two Stages of Reovirus Infection Is Required for the Induction of Necroptosis

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Angela K. Berger ◽  
Bradley E. Hiller ◽  
Deepti Thete ◽  
Anthony J. Snyder ◽  
Encarnacion Perez ◽  
...  
Keyword(s):  
Cell Death ◽  
Immune System ◽  
Virus Infection ◽  
De Novo ◽  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Type I ◽  
Tissue Destruction ◽  
Reovirus Infection

ABSTRACT Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the de novo synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus. IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.

scholarly journals Role of an Internal and Two 3′-Terminal RNA Elements in Assembly of Tombusvirus Replicase

2005 ◽  
Vol 79 (16) ◽  
pp. 10608-10618 ◽  
Author(s):  
Zivile Panaviene ◽  
Tadas Panavas ◽  
Peter D. Nagy
Keyword(s):  
Rna Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Replication Proteins ◽  
Rna Sequences ◽  
Alternative Structures ◽  
Recognition Element ◽  
Rna Elements

ABSTRACT Plus-strand RNA virus replication requires the assembly of the viral replicase complexes on intracellular membranes in the host cells. The replicase of Cucumber necrosis virus (CNV), a tombusvirus, contains the viral p33 and p92 replication proteins and possible host factors. In addition, the assembly of CNV replicase is stimulated in the presence of plus-stranded viral RNA (Z. Panaviene et al., J. Virol. 78:8254-8263, 2004). To define cis-acting viral RNA sequences that stimulate replicase assembly, we performed a systematic deletion approach with a model tombusvirus replicon RNA in Saccharomyces cerevisiae, which also coexpressed p33 and p92 replication proteins. In vitro replicase assays performed with purified CNV replicase preparations from yeast revealed critical roles for three RNA elements in CNV replicase assembly: the internal p33 recognition element (p33RE), the replication silencer element (RSE), and the 3′-terminal minus-strand initiation promoter (gPR). Deletion or mutagenesis of these elements reduced the activity of the CNV replicase to a minimal level. In addition to the primary sequences of gPR, RSE, and p33RE, formation of two alternative structures among these elements may also play a role in replicase assembly. Altogether, the role of multiple RNA elements in tombusvirus replicase assembly could be an important factor to ensure fidelity of template selection during replication.

scholarly journals Potentially translated sequences determine protein-coding potential of RNAs in cellular organisms

2021 ◽  
Author(s):  
Yusuke Suenaga ◽  
Mamoru Kato ◽  
Momoko Nagai ◽  
Kazuma Nakatani ◽  
Hiroyuki Kogashi ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Virus ◽  
Host Cells ◽  
Rna Sequences ◽  
Protein Coding ◽  
Functional Peptides ◽  
Definition Of ◽  
Virus Genomes ◽  
Sequence Characteristics ◽  
Coding Potential

AbstractRNA sequence characteristics determine whether their transcripts are coding or noncoding. Recent studies have shown that, paradoxical to the definition of noncoding RNA, several long noncoding RNAs (lncRNAs) translate functional peptides/proteins. However, the characteristics of RNA sequences that distinguish such newly identified coding transcripts from lncRNAs remain largely unknown. In this study, we found that potentially translated sequences in RNAs determine the protein-coding potential of RNAs in cellular organisms. We defined the potentially translated island (PTI) score as the fraction of the length of the longest potentially translated region among all regions. To analyze its relationship with protein-coding potential, we calculated the PTI scores in 3.4 million RNA transcripts from 100 cellular organisms, including 5 bacteria, 10 archaea, and 85 eukaryotes, as well as 105 positive-sense single-strand RNA virus genomes. In bacteria and archaea, coding and noncoding transcripts exclusively presented high and low PTI scores, respectively, whereas those of eukaryotic coding and noncoding transcripts showed relatively broader distributions. The relationship between the PTI score and protein-coding potential was sigmoidal in most eukaryotes; however, it was linear passing through the origin in three distinct eutherian lineages, including humans. The RNA sequences of virus genomes appeared to adapt to translation systems of host organisms by maximizing protein-coding potential in host cells. Hence, the PTIs determined the protein-coding potential of RNAs in cellular organisms. Additionally, coding and noncoding RNA do not exhibit dichotomous sequence characteristics in eukaryotes, instead they exhibit a gradient of protein-coding potential.

scholarly journals N-Myc Downstream-Regulated Gene 1 Restricts Hepatitis C Virus Propagation by Regulating Lipid Droplet Biogenesis and Viral Assembly

2017 ◽  
Vol 92 (2) ◽  
Author(s):  
Cameron J. Schweitzer ◽  
Fang Zhang ◽  
Audrey Boyer ◽  
Kristin Valdez ◽  
Maggie Cam ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Liver Cancer ◽  
Lipid Droplet ◽  
Viral Infections ◽  
Rna Virus ◽  
Viral Assembly ◽  
Hcv Infection ◽  
Host Cells ◽  
Virus Propagation

ABSTRACT Host cells harbor various intrinsic mechanisms to restrict viral infections as a first line of antiviral defense. Viruses have evolved various countermeasures against these antiviral mechanisms. Here we show that N-Myc downstream-regulated gene 1 (NDRG1) limits productive hepatitis C virus (HCV) infection by inhibiting viral assembly. Interestingly, HCV infection downregulates NDRG1 protein and mRNA expression. The loss of NDRG1 increases the size and number of lipid droplets, which are the sites of HCV assembly. HCV suppresses NDRG1 expression by upregulating MYC, which directly inhibits the transcription of NDRG1. The upregulation of MYC also leads to the reduced expression of the NDRG1-specific kinase serum/glucocorticoid-regulated kinase 1 (SGK1), resulting in a markedly diminished phosphorylation of NDRG1. The knockdown of MYC during HCV infection rescues NDRG1 expression and phosphorylation, suggesting that MYC regulates NDRG1 at both the transcriptional and posttranslational levels. Overall, our results suggest that NDRG1 restricts HCV assembly by limiting lipid droplet formation. HCV counteracts this intrinsic antiviral mechanism by downregulating NDRG1 via a MYC-dependent mechanism. IMPORTANCE Hepatitis C virus (HCV) is an enveloped single-stranded RNA virus that targets hepatocytes in the liver. HCV is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and estimates suggest a global prevalence of 2.35%. Up to 80% of acutely infected individuals will develop chronic infection, and as many as 5% eventually progress to liver cancer. An understanding of the mechanisms behind virus-host interactions and viral carcinogenesis is still lacking. The significance of our research is that it identifies a previously unknown relationship between HCV and a known tumor-associated gene. Furthermore, our data point to a new role for this gene in the liver and in lipid metabolism. Thus, HCV infection serves as a great biological model to advance our knowledge of liver functions and the development of liver cancer.

scholarly journals Development of a New Reverse Genetics System for Ebola Virus

mSphere ◽  
2021 ◽  
Vol 6 (3) ◽  
Author(s):  
Tianyu Gan ◽  
Dihan Zhou ◽  
Yi Huang ◽  
Shuqi Xiao ◽  
Ziyue Ma ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Ebola Virus ◽  
Rna Virus ◽  
Case Fatality ◽  
Supporting Cell ◽  
Viral Rna ◽  
Model Systems ◽  
Virus Propagation ◽  
L Proteins ◽  
Subviral Particles

ABSTRACT Ebola virus (EBOV) is a highly pathogenic negative-stranded RNA virus that has caused several deadly endemics in the past decades. EBOV reverse genetics systems are available for studying live viruses under biosafety level 4 (BSL-4) or subviral particles under BSL-2 conditions. However, these systems all require cotransfection of multiple plasmids expressing viral genome and viral proteins essential for EBOV replication, which is technically challenging and unable to naturally mimic virus propagation using the subviral particle. Here, we established a new EBOV reverse genetics system only requiring transfection of a single viral RNA genome into an engineered cell line that stably expresses viral nucleoprotein (NP), viral protein 35 (VP35), VP30, and large (L) proteins and has been fine-tuned for its superior permissiveness for EBOV replication. Using this system, subviral particles expressing viral VP40, glycoprotein (GP), and VP24 could be produced and continuously propagated and eventually infect the entire cell population. We demonstrated the authentic response of the subviral system to antivirals and uncovered that the VP35 amount is critical for optimal virus replication. Furthermore, we showed that fully infectious virions can be efficiently rescued by delivering the full-length EBOV genome into the same supporting cell, and the efficiency is not affected by genome polarity or virus variant specificity. In summary, our work provides a new tool for studying EBOV under different biosafety levels. IMPORTANCE Ebola virus is among the most dangerous viral pathogens, with a case fatality rate of up to 90%. Since 2013, the two largest and most complex Ebola outbreaks in Africa have revealed the lack of investigation on this notorious virus. A reverse genetics system is an important tool for studying viruses by producing mutant viruses or generating safer and convenient model systems. Here, we developed an EBOV life cycle modeling system in which subviral particles can spontaneously propagate in cell culture. In addition, this system can be employed to rescue infectious virions of homologous or heterologous EBOV isolates using either sense or antisense viral RNA genomes. In summary, we developed a new tool for EBOV research.

scholarly journals A Comprehensive Roadmap Towards Generation of Influenza B Reporter Assay Using a Single DNA Polymerase Based Cloning of Reporter RNA Construct

2021 ◽  
Author(s):  
Nandita Kedia ◽  
Saptarshi Banerjee ◽  
Arindam Mondal
Keyword(s):  
Rna Synthesis ◽  
Rna Virus ◽  
Assay System ◽  
Viral Rna ◽  
Host Protein ◽  
Influenza B Virus ◽  
Influenza B ◽  
Reporter Assay ◽  
Virus Propagation ◽  
B Virus

Mini-genome reporter assay is a key tool for conducting RNA virus research. But, procedural complications and lack of adequate literature pose major challenge towards developing these assay systems. Here we present a novel yet generic and simple cloning strategy for construction of influenza B virus reporter RNA template and describe extensive standardization of the reporter RNP/polymerase activity assay for monitoring viral RNA synthesis in infection free setting. Using this assay system, we, for the first time showed the effect of viral protein NS1 and host protein PKC-Delta upon influenza B virus RNA synthesis. Additionally, the assay system showed promising results in evaluating the efficacy of antiviral drugs targeting viral RNA synthesis and virus propagation. Together, this work offers a detailed protocol for standardization of influenza virus mini-genome assay and an excellent tool for screening of host factors and antivirals in a fast, user friendly and high throughput manner.

scholarly journals Autophagy Promotes Replication of Influenza A VirusIn Vitro

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Ruifang Wang ◽  
Yinxing Zhu ◽  
Jiachang Zhao ◽  
Chenwei Ren ◽  
Peng Li ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Rna Synthesis ◽  
Virus Production ◽  
Viral Rna ◽  
Host Cells ◽  
Progeny Virus ◽  
Mtor Signaling Pathway ◽  
Autophagy Pathway ◽  
Progeny Virus Production

ABSTRACTInfluenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP- and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus.IMPORTANCEAutophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP- and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.

A myxozoan-like parasite causing xenomas in the brain of the mole, Talpa europaea L., 1758 (Vertebrata, Mammalia)

Parasitology ◽  
2000 ◽  
Vol 121 (5) ◽  
pp. 483-492 ◽  
Author(s):  
C. FRIEDRICH ◽  
E. INGOLIC ◽  
B. FREITAG ◽  
G. KASTBERGER ◽  
V. HOHMANN ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Developmental Stages ◽  
Host Cells ◽  
Brain Capillaries ◽  
Talpa Europaea ◽  
Transmission Electron ◽  
Eukaryotic Organism ◽  
A Cell ◽  
Exact Identification ◽  
The Brain

Light and transmission electron microscopy revealed pericytes of brain capillaries of moles (Talpa europaea L., 1758) as parasitized intracellularly. These host cells were enlarged and of globular or ellipsoid shape, and incorporated a cell-within-cell sequence of primary, secondary and, rarely found, tertiary developmental stages of an eukaryotic organism. Other stages like spores were not discovered either in brain or in other organs. Due to the vertebrate host, and the parasitic cells showing the enveloped state this parasite can be classified as belonging to the Myxozoa rather than Paramyxea. Since spores, which would allow an exact identification of the parasite, could not be detected and mammals are very unusual hosts for Myxozoa, the parasite was designated a myxozoan-like organism.

scholarly journals Growth Characteristics and Intraspecies Host Specificity of a Large Virus Infecting the Dinoflagellate Heterocapsa circularisquama

2003 ◽  
Vol 69 (5) ◽  
pp. 2580-2586 ◽  
Author(s):  
Keizo Nagasaki ◽  
Yuji Tomaru ◽  
Kenji Tarutani ◽  
Noriaki Katanozaka ◽  
Satoshi Yamanaka ◽  
...  
Keyword(s):  
Host Specificity ◽  
Burst Size ◽  
Growth Characteristics ◽  
Host Cells ◽  
Capsid Formation ◽  
Transmission Electron ◽  
Heterocapsa Circularisquama ◽  
Wide Range ◽  
Algal Host ◽  
Large Virus

ABSTRACT The growth characteristics and intraspecies host specificity of Heterocapsa circularisquama virus (HcV), a large icosahedral virus specifically infecting the bivalve-killing dinoflagellate H. circularisquama, were examined. Exponentially growing host cells were more sensitive to HcV than those in the stationary phase, and host cells were more susceptible to HcV infection in the culture when a higher percent of the culture was replaced with fresh medium each day, suggesting an intimate relationship between virus sensitivity and the physiological condition of the host cells. HcV was infective over a wide range of temperatures, 15 to 30°C, and the latent period and burst size were estimated at 40 to 56 h and 1,800 to 2,440 infective particles, respectively. Transmission electron microscopy revealed that capsid formation began within 16 h postinfection, and mature virus particles appeared within 24 h postinfection at 20°C. Compared to Heterosigma akashiwo virus, HcV was more widely infectious to H. circularisquama strains that had been independently isolated in the western part of Japan, and only 5.3% of the host-virus combinations (53 host and 10 viral strains) showed resistance to viral infection. The present results are helpful in understanding the ecology of algal host-virus systems in nature.

scholarly journals The molecular interaction between ML336 and the VEEV non-structural proteins

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Abdrew M. Skidmore ◽  
Dong-Hoon Chung
Keyword(s):  
Large Scale ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Viral Rna ◽  
Antiviral Compound ◽  
Equine Encephalitis Virus ◽  
Direct Acting Antiviral ◽  
A Cell ◽  
Infected Cells ◽  
Direct Acting

Venezuelan Equine Encephalitis Virus (VEEV) is a positive sense RNA virus in the family Togaviridae. VEEV circulates in the Americas, causing occasional large scale epidemics. Our group has previously discovered and described the anti-VEEV compound ML336. We found that ML336 inhibits viral RNA synthesis during infection. This RNA synthesis inhibition is highly specific for VEEV, and ML336 has no effect on the closely related chikungunya virus, or on cellular RNA synthesis. We also found that this activity was maintained in a cell-free viral RNA synthesis system, supporting our hypothesis that ML336 is a direct acting antiviral compound. We recently discovered that treatment with ML336 reduces the amount of double stranded RNA present in infected cells. This reduction supports that ML336 is interfering with the synthesis of viral RNA. This was measured qualitatively with microscopy, and quantitatively with flow cytometry. We have also reported that resistant viral mutants emerge when grown in the presence of inhibitory compounds and these mutations mapped to the N terminal domains of both nsP2 and nsP4. This region of nsP2 has recently been shown to be a helical region which serves as an accessory domain to the viral helicase. However, the region of nsP4 in question currently lacks known function. Based on this genetic data we hypothesized that ML336 and related compounds interact with these two domains to interfere with the activity of the replicase complex. We are currently examining this interaction using ectopically expressed protein.

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