scholarly journals Nodamura Virus Nonstructural Protein B2 Can Enhance Viral RNA Accumulation in both Mammalian and Insect Cells

2004 ◽  
Vol 78 (12) ◽  
pp. 6698-6704 ◽  
Author(s):  
Kyle L. Johnson ◽  
B. Duane Price ◽  
Lance D. Eckerle ◽  
L. Andrew Ball
Keyword(s):  
Rna Interference ◽  
Cell Lines ◽  
Nonstructural Protein ◽  
Viral Rna ◽  
Host Cells ◽  
Flock House Virus ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Rna Accumulation ◽  
Nodamura Virus

ABSTRACT During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.

scholarly journals ORF1a-Encoded Replicase Subunits Are Involved in the Membrane Association of the Arterivirus Replication Complex

1998 ◽  
Vol 72 (8) ◽  
pp. 6689-6698 ◽  
Author(s):  
Yvonne van der Meer ◽  
Hans van Tol ◽  
Jacomine Krijnse Locker ◽  
Eric J. Snijder
Keyword(s):  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Membrane Association ◽  
Nonstructural Proteins ◽  
Reading Frame ◽  
Replication Complex ◽  
Viral Proteases ◽  
Double Label ◽  
Membrane Bound ◽  
Triton X

ABSTRACT Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.

scholarly journals Mapping the Nonstructural Protein Interaction Network of Porcine Reproductive and Respiratory Syndrome Virus

2018 ◽  
Vol 92 (24) ◽  
Author(s):  
Jiangwei Song ◽  
Yuanyuan Liu ◽  
Peng Gao ◽  
Yunhao Hu ◽  
Yue Chai ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rna Virus ◽  
Nonstructural Protein ◽  
Transmembrane Proteins ◽  
Interaction Network ◽  
Host Cells ◽  
Respiratory Syndrome Virus ◽  
Reading Frame ◽  
Interaction Map ◽  
Transcription Complexes

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) is a positive-stranded RNA virus belonging to the familyArteriviridae. Synthesis of the viral RNA is directed by replication/transcription complexes (RTC) that are mainly composed of a network of PRRSV nonstructural proteins (nsps) and likely cellular proteins. Here, we mapped the interaction network among PRRSV nsps by using yeast two-hybrid screening in conjunction with coimmunoprecipitation (co-IP) and cotransfection assays. We identified a total of 24 novel interactions and found that the interactions were centered on open reading frame 1b (ORF1b)-encoded nsps that were mainly connected by the transmembrane proteins nsp2, nsp3, and nsp5. Interestingly, the interactions of the core enzymes nsp9 and nsp10 with transmembrane proteins did not occur in a straightforward manner, as they worked in the co-IP assay but were poorly capable of finding each other within intact mammalian cells. Further proof that they can interact within cells required the engineering of N-terminal truncations of both nsp9 and nsp10. However, despite the poor colocalization relationship in cotransfected cells, both nsp9 and nsp10 came together with membrane proteins (e.g., nsp2) at the viral replication and transcription complexes (RTC) in PRRSV-infected cells. Thus, our results indicate the existence of a complex interaction network among PRRSV nsps and raise the possibility that the recruitment of key replicase proteins to membrane-associated nsps may involve some regulatory mechanisms during infection.IMPORTANCESynthesis of PRRSV RNAs within host cells depends on the efficient and correct assembly of RTC that takes places on modified intracellular membranes. As an important step toward dissecting this poorly understood event, we investigated the interaction network among PRRSV nsps. Our studies established a comprehensive interaction map for PRRSV nsps and revealed important players within the network. The results also highlight the likely existence of a regulated recruitment of the PRRSV core enzymes nsp9 and nsp10 to viral membrane nsps during PRRSV RTC assembly.

scholarly journals Dynamics of Protein Accumulation from the 3′ End of Viral RNA Are Different from Those in the Rest of the Genome in Potato Virus A Infection

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Shreya Saha ◽  
Anders Hafren ◽  
Kristiina Mäkinen
Keyword(s):  
Potato Virus ◽  
Viral Protein ◽  
Natural Infection ◽  
Ectopic Expression ◽  
Viral Rna ◽  
Protein Accumulation ◽  
Particle Assembly ◽  
Reading Frame ◽  
Potato Virus A ◽  
Rna Accumulation

ABSTRACT One large open reading frame (ORF) encodes 10 potyviral proteins. We compared the accumulation of cylindrical inclusion (CI) protein from the middle, coat protein (CP) from the 3′end, and Renilla luciferase (RLUC) from two distinct locations in potato virus A (PVA) RNA. 5′ RLUC was expressed from an rluc gene inserted between the P1 and helper component proteinase (HCPro) cistrons, and 3′ RLUC was expressed from the gene inserted between the RNA polymerase and CP cistrons. Viral protein and RNA accumulation were quantitated (i) when expressed from PVA RNA in the presence of ectopically expressed genome-linked viral protein (VPg) and auxiliary proteins and (ii) at different time points during natural infection. The rate and timing of 3′ RLUC and CP accumulation were found to be different from those of 5′ RLUC and CI. Ectopic expression of VPg boosted PVA RNA, 3′ RLUC, and, together with HCPro, CP accumulation, whereas 5′ RLUC and CI accumulation remained unaffected regardless of the increased viral RNA amount. In natural infection, the rate of the noteworthy minute early accumulation of 3′ RLUC accelerated toward the end of infection. 5′ RLUC accumulation, which was already pronounced at 2 days postinfection, increased moderately and stabilized to a constant level by day 5, whereas PVA RNA and CP levels continued to increase throughout the infection. We propose that these observations connect with the mechanisms by which potyvirus infection limits CP accumulation during early infection and specifically supports its accumulation late in infection, but follow-up studies are required to understand the mechanism of how this occurs. IMPORTANCE The results of this study suggest that the dynamics of potyviral protein accumulation are regulated differentially from the 3′ end of viral RNA than from the rest of the genome, the significance of which would be to satisfy the needs of replication early and particle assembly late in infection.

scholarly journals Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation

2006 ◽  
Vol 80 (1) ◽  
pp. 85-94 ◽  
Author(s):  
Beau J. Fenner ◽  
Rekha Thiagarajan ◽  
Hui Kheng Chua ◽  
Jimmy Kwang
Keyword(s):  
Rna Interference ◽  
Reverse Genetics ◽  
Nonstructural Protein ◽  
Juvenile Fish ◽  
Nervous Necrosis Virus ◽  
Rna Transfection ◽  
B2 Rna ◽  
Rna Accumulation ◽  
High Level ◽  
Late Stages

ABSTRACT Betanodaviruses are small positive-sense bipartite RNA viruses that infect a wide variety of fish species and are notorious for causing lethal outbreaks in juvenile fish hatcheries worldwide. The function of a small nonstructural protein, B2, encoded by the subgenomic RNA3 of betanodaviruses, has remained obscure. Greasy grouper nervous necrosis virus, a betanodavirus model, was used to develop a facile DNA-based reverse genetics system that recapitulated the virus infection cycle, and we used this system to show that B2 is a small nonstructural protein that is essential for high level accumulation of viral RNA1 after RNA transfection of fish, mammalian, and avian cells. The defect in RNA1 accumulation in a B2 mutant was partially complemented by supplying B2 RNA in trans. Confocal analysis of the cellular distribution of B2 indicated that B2 is able to enter the nucleus and accumulates there during the late stages of GGNNV infection. Using human HeLa cells as a cellular RNA interference model, we found that B2 could efficiently antagonize RNA interference, which is a property shared by the distantly related alphanodavirus B2 proteins. This function provides appears to provide an explanation, at least in part, for why B2 mutant RNA1 is severely impaired in its intracellular accumulation.

scholarly journals Betanodavirus B2 Is an RNA Interference Antagonist That Facilitates Intracellular Viral RNA Accumulation

2007 ◽  
Vol 81 (9) ◽  
pp. 4909-4909 ◽  
Author(s):  
Beau J. Fenner ◽  
Rekha Thiagarajan ◽  
Hui Kheng Chua ◽  
Jimmy Kwang
Keyword(s):  
Rna Interference ◽  
Viral Rna ◽  
Rna Accumulation

scholarly journals Quantitative Analysis of the Hepatitis C Virus Replication Complex

2005 ◽  
Vol 79 (21) ◽  
pp. 13594-13605 ◽  
Author(s):  
Doris Quinkert ◽  
Ralf Bartenschlager ◽  
Volker Lohmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Synthesis ◽  
Nonstructural Protein ◽  
Viral Proteins ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Positive Strand ◽  
Negative Strand

ABSTRACT The hepatitis C virus (HCV) encodes a large polyprotein; therefore, all viral proteins are produced in equimolar amounts regardless of their function. The aim of our study was to determine the ratio of nonstructural proteins to RNA that is required for HCV RNA replication. We analyzed Huh-7 cells harboring full-length HCV genomes or subgenomic replicons and found in all cases a >1,000-fold excess of HCV proteins over positive- and negative-strand RNA. To examine whether all nonstructural protein copies are involved in RNA synthesis, we isolated active HCV replication complexes from replicon cells and examined them for their content of viral RNA and proteins before and after treatment with protease and/or nuclease. In vitro replicase activity, as well as almost the entire negative- and positive-strand RNA, was resistant to nuclease treatment, whereas <5% of the nonstructural proteins were protected from protease digest but accounted for the full in vitro replicase activity. In consequence, only a minor fraction of the HCV nonstructural proteins was actively involved in RNA synthesis at a given time point but, due to the high amounts present in replicon cells, still representing a huge excess compared to the viral RNA. Based on the comparison of nuclease-resistant viral RNA to protease-resistant viral proteins, we estimate that an active HCV replicase complex consists of one negative-strand RNA, two to ten positive-strand RNAs, and several hundred nonstructural protein copies, which might be required as structural components of the vesicular compartments that are the site of HCV replication.

scholarly journals Development of an HTS Assay for the Search of Anti-influenza Agents Targeting the Interaction of Viral RNA with the NS1 Protein

2008 ◽  
Vol 13 (7) ◽  
pp. 581-590 ◽  
Author(s):  
Marta Maroto ◽  
Yolanda Fernandez ◽  
Juan Ortin ◽  
Fernando Pelaez ◽  
M. Angerles Cabello
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Cytopathic Effect ◽  
Nonstructural Protein ◽  
Specific Interaction ◽  
Chemical Compounds ◽  
Viral Rna ◽  
Ns1 Protein ◽  
Rna Molecules ◽  
Novel Target

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate® technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate® wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs. ( Journal of Biomolecular Screening 2008:581-590)

scholarly journals Effects of Gag Mutation and Processing on Retroviral Dimeric RNA Maturation

2006 ◽  
Vol 80 (3) ◽  
pp. 1242-1249 ◽  
Author(s):  
William Fu ◽  
Que Dang ◽  
Kunio Nagashima ◽  
Eric O. Freed ◽  
Vinay K. Pathak ◽  
...  
Keyword(s):  
Viral Protein ◽  
Leukemia Virus ◽  
Viral Rna ◽  
Host Cells ◽  
Protein Cleavage ◽  
Rna Packaging ◽  
Virus Rna ◽  
Rna Dimer ◽  
Rna Maturation ◽  
Hiv 1

ABSTRACT After their release from host cells, most retroviral particles undergo a maturation process, which includes viral protein cleavage, core condensation, and increased stability of the viral RNA dimer. Inactivating the viral protease prevents protein cleavage; the resulting virions lack condensed cores and contain fragile RNA dimers. Therefore, protein cleavage is linked to virion morphological change and increased stability of the RNA dimer. However, it is unclear whether protein cleavage is sufficient for mediating virus RNA maturation. We have observed a novel phenotype in a murine leukemia virus capsid mutant, which has normal virion production, viral protein cleavage, and RNA packaging. However, this mutant also has immature virion morphology and contains a fragile RNA dimer, which is reminiscent of protease-deficient mutants. To our knowledge, this mutant provides the first evidence that Gag cleavage alone is not sufficient to promote RNA dimer maturation. To extend our study further, we examined a well-defined human immunodeficiency virus type 1 (HIV-1) Gag mutant that lacks a functional PTAP motif and produces immature virions without major defects in viral protein cleavage. We found that the viral RNA dimer in the PTAP mutant is more fragile and unstable compared with those from wild-type HIV-1. Based on the results of experiments using two different Gag mutants from two distinct retroviruses, we conclude that Gag cleavage is not sufficient for promoting RNA dimer maturation, and we propose that there is a link between the maturation of virion morphology and the viral RNA dimer.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 2 Interacts with a Host Protein Complex Involved in Mitochondrial Biogenesis and Intracellular Signaling

2009 ◽  
Vol 83 (19) ◽  
pp. 10314-10318 ◽  
Author(s):  
Cromwell T. Cornillez-Ty ◽  
Lujian Liao ◽  
John R. Yates ◽  
Peter Kuhn ◽  
Michael J. Buchmeier
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Intracellular Signaling ◽  
Nonstructural Protein ◽  
Host Protein ◽  
Nonstructural Proteins ◽  
Host Proteins ◽  
Catalytic Activities ◽  
Nonstructural Protein 2 ◽  
Host Signaling ◽  
Prohibitin 1

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) generates 16 nonstructural proteins (nsp's) through proteolytic cleavage of a large precursor protein. Although several nsp's exhibit catalytic activities that are important for viral replication and transcription, other nsp's have less clearly defined roles during an infection. In order to gain a better understanding of their functions, we attempted to identify host proteins that interact with nsp's during SARS-CoV infections. For nsp2, we identified an interaction with two host proteins, prohibitin 1 (PHB1) and PHB2. Our results suggest that nsp2 may be involved in the disruption of intracellular host signaling during SARS-CoV infections.

scholarly journals Roles of the AX4GKS and Arginine-Rich Motifs of Hepatitis C Virus RNA Helicase in ATP- and Viral RNA-Binding Activity

2000 ◽  
Vol 74 (20) ◽  
pp. 9732-9737 ◽  
Author(s):  
Shin C. Chang ◽  
Ju-Chien Cheng ◽  
Yi-Hen Kou ◽  
Chuan-Hong Kao ◽  
Chiung-Hui Chiu ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Viral Rna ◽  
Atp Binding ◽  
Rna Unwinding ◽  
Arginine Rich Motif

ABSTRACT The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed inEscherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX4GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX4GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the γ-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

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