scholarly journals Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented intrans

2001 ◽  
Vol 75 (17) ◽  
pp. 7791-7802 ◽  
Author(s):  
Claus W. Grassmann ◽  
Olaf Isken ◽  
Norbert Tautz ◽  
Sven-Erik Behrens
Keyword(s):  
Viral Replication ◽  
Proteolytic Processing ◽  
Host Cells ◽  
Molar Ratio ◽  
Replication Capacity ◽  
Coding Region ◽  
Positive Strand ◽  
Reading Frame ◽  
Diarrhea Virus ◽  
Positive Strand Rna

ABSTRACT The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cisor in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to becis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.

scholarly journals Characterization of an Autonomous Subgenomic Pestivirus RNA Replicon

1998 ◽  
Vol 72 (3) ◽  
pp. 2364-2372 ◽  
Author(s):  
Sven-Erik Behrens ◽  
Claus W. Grassmann ◽  
Heinz-Jürgen Thiel ◽  
Gregor Meyers ◽  
Norbert Tautz
Keyword(s):  
Mammalian Cells ◽  
Rna Replication ◽  
Full Length ◽  
Host Cells ◽  
Rnase Protection ◽  
Positive Strand ◽  
Negative Strand ◽  
Diarrhea Virus ◽  
Positive Strand Rna

ABSTRACT As an initial approach to define the requirements for the replication of bovine viral diarrhea virus (BVDV), a member of theFlaviviridae family with a positive-strand RNA genome, full-length genomic and subgenomic RNAs were originated by in vitro transcription of diverse BVDV cDNA constructs and transfected into eucaryotic host cells. RNA replication was measured either directly by an RNase protection method or by monitoring the synthesis of viral protein. When full-length BVDV cRNA was initially applied, the synthesis of negative-strand RNA intermediates as well as progeny positive-strand RNA was detected posttransfection in the cytoplasm of the host cells. Compared to the negative-strand RNA intermediate, an excess of positive-strand RNA was synthesized. Surprisingly, a subgenomic RNA molecule, DI9c, corresponding to a previously characterized defective interfering particle, was found to support both steps of RNA replication in the absence of a helper virus as well, thus functioning as an autonomous replicon. DI9c comprises the 5′ and 3′ untranslated regions of the BVDV genome and the coding regions of the autoprotease Npro and the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. Most interestingly, the NS2 polypeptide was thus determined to be nonessential for RNA replication. As expected, deletion of the genomic 3′ end as well as abolition of the catalytic function of the virus-encoded serine protease resulted in DI9c molecules that were unable to replicate. Deletion of the entire Npro gene also destroyed the ability of DI9c molecules to replicate. On the other hand, DI9c derivatives in which the 5′ third of the Npro gene was fused to a ubiquitin gene, allowing the proteolytic release of NS3 in trans, turned out to be replication competent. These results suggest that the RNA sequence located at the 5′ end of the open reading frame exerts an essential role during BVDV replication. Replication of DI9c and DI9c derivatives was found not to be limited to host cells of bovine origin, indicating that cellular factors functioning as potential parts of the viral replication machinery are well conserved between different mammalian cells. Our data provide an important step toward the ready identification and characterization of viral factors and genomic elements involved in the life cycle of pestiviruses. The implications for other Flaviviridae and, in particular, the BVDV-related human hepatitis C virus are discussed.

scholarly journals BK Polyomavirus MicroRNA Levels in Exosomes Are Modulated by Non-Coding Control Region Activity and Down-Regulate Viral Replication When Delivered to Non-Infected Cells Prior to Infection

Viruses ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 466 ◽  
Author(s):  
Francesco Martelli ◽  
Zongsong Wu ◽  
Serena Delbue ◽  
Fabian Weissbach ◽  
Maria Giulioli ◽  
...  
Keyword(s):  
Viral Replication ◽  
Point Mutations ◽  
T Antigen ◽  
Host Cells ◽  
Replication Capacity ◽  
Viral Gene ◽  
New Host ◽  
Infected Cells ◽  
Multiple Sclerosis Patients ◽  
Coding Control

In immunosuppressed patients, BKPyV-variants emerge carrying rearranged non-coding control-regions (rr-NCCRs) that increase early viral gene region (EVGR) expression and replication capacity. BKPyV also encodes microRNAs, which have been reported to downregulate EVGR-encoded large T-antigen transcripts, to decrease viral replication in infected cells and to be secreted in exosomes. To investigate the interplay of NCCR and microRNAs, we compared archetype- and rr-NCCR-BKPyV infection in cell culture. We found that laboratory and clinical rr-NCCR-BKPyV-strains show higher replication rates but significantly lower microRNA levels than archetype virus intracellularly and in exosomes. To investigate whether rr-NCCR or increased EVGR activity modulated microRNA levels, we examined the (sp1-4)NCCR-BKPyV, which has an archetype NCCR-architecture but shows increased EVGR expression due to point mutations inactivating one Sp1 binding site. We found that microRNA levels following (sp1-4)NCCR-BKPyV infection were as low as in rr-NCCR-variants. Thus, NCCR rearrangements are not required for lower miRNA levels. Accordingly, Sp1 siRNA knock-down decreased microRNA levels in archetype BKPyV infection but had no effect on (sp1-4)- or rr-NCCR-BKPyV. However, rr-NCCR-BKPyV replication was downregulated by exosome preparations carrying BKPyV-microRNA prior to infection. To explore the potential relevance in humans, urine samples from 12 natalizumab-treated multiple sclerosis patients were analysed. In 7 patients, rr-NCCR-BKPyV were detected showing high urine BKPyV loads but low microRNAs levels, whereas the opposite was seen in 5 patients with archetype BKPyV. We discuss the results in a dynamic model of BKPyV replication according to NCCR activity and exosome regulation, which integrates immune selection pressure, spread to new host cells and rr-NCCR emergence.

scholarly journals Expression and Processing of Proteins Encoded by the Saccharomyces Retrotransposon Ty5

2001 ◽  
Vol 75 (4) ◽  
pp. 1790-1797 ◽  
Author(s):  
Phillip A. Irwin ◽  
Daniel F. Voytas
Keyword(s):  
Reverse Transcriptase ◽  
Time Course ◽  
Immunoblot Analysis ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Reading Frame ◽  
C Terminus ◽  
Virus Like Particle ◽  
Ionic Detergent ◽  
Time Course Experiment

ABSTRACT Retroelements (retrotransposons and retroviruses) have two genes in common: gag, which specifies structural proteins that form a virus or virus-like particle, andpol, which specifies catalytic proteins required for replication. For many retroelements, gag andpol are present on separate reading frames. Their expression is highly regulated, and the ratio of Gag to Pol is critical for retroelement replication. The Saccharomycesretrotransposon Ty5 contains a single open reading frame, and we characterized Gag and Pol expression by generating transpositionally active Ty5 elements with epitope tags at the N terminus or C terminus or within the integrase coding region. Immunoblot analysis identified two Gag species (Gag-p27 and Gag-p37), reverse transcriptase (Pol-p59), and integrase (Pol-p80), all of which are largely insoluble in the absence of urea or ionic detergent. These proteins result from proteolytic processing of a polyprotein, because elements with mutations in the presumed active site of Ty5 protease express a single tagged protein (Gag-Pol-p182). Protease mutants are also transpositionally inactive. In a time course experiment, we monitored protein expression, proteolytic processing, and transposition of a Ty5 element with identical epitope tags at its N and C termini. Both transposition and the abundance of Gag-p27 increased over time. In contrast, the levels of Gag-p37 and reverse transcriptase peaked after ∼14 h of induction and then gradually decreased. This may be due to differences in stability of Gag-p27 relative to Gag-p37 and reverse transcriptase. The ratio of Ty5 Gag to Pol averaged 5:1 throughout the time course experiment, suggesting that differential protein stability regulates the amounts of these proteins.

scholarly journals Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

2016 ◽  
Vol 113 (8) ◽  
pp. E1064-E1073 ◽  
Author(s):  
Jiantao Zhang ◽  
Zhenlu Zhang ◽  
Vineela Chukkapalli ◽  
Jules A. Nchoutmboube ◽  
Jianhui Li ◽  
...  
Keyword(s):  
Viral Replication ◽  
Rna Viruses ◽  
Critical Role ◽  
Brome Mosaic Virus ◽  
Alternate Host ◽  
Positive Strand ◽  
Cellular Processes ◽  
Replication Sites ◽  
Positive Strand Rna

All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting theCHO2gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes.

scholarly journals Early secretory pathway localization and lack of processing for hepatitis E virus replication protein pORF1

2013 ◽  
Vol 94 (4) ◽  
pp. 807-816 ◽  
Author(s):  
Julia Perttilä ◽  
Pirjo Spuul ◽  
Tero Ahola
Keyword(s):  
Hepatitis E Virus ◽  
Secretory Pathway ◽  
Hepatitis E ◽  
Proteolytic Processing ◽  
Full Length ◽  
Replication Protein ◽  
Positive Strand ◽  
Early Secretory Pathway ◽  
Positive Strand Rna

Hepatitis E virus (HEV) is a positive-strand RNA virus and a major causative agent of acute sporadic and epidemic hepatitis. HEV replication protein is encoded by ORF1 and contains the predicted domains of methyltransferase (MT), protease, macro domain, helicase (HEL) and polymerase (POL). In this study, the full-length protein pORF1 (1693 aa) and six truncated variants were expressed by in vitro translation and in human HeLa and hepatic Huh-7 cells by using several vector systems. The proteins were visualized by three specific antisera directed against the MT, HEL and POL domains. In vitro translation of full-length pORF1 yielded smaller quantities of two fragments. However, these fragments were not observed after pORF1 expression and pulse–chase studies in human cells, and their production was not dependent on the predicted protease domain in pORF1. The weight of evidence supports the proposition that pORF1 is not subjected to specific proteolytic processing, which is unusual among animal positive-strand RNA viruses but common for plant viruses. pORF1 was membrane associated in cells and localized to a perinuclear region, where it partially overlapped with localization of the endoplasmic reticulum (ER) marker BAP31 and was closely interspersed with staining of the ER–Golgi intermediate compartment marker protein ERGIC-53. Co-localization with BAP31 was enhanced by treatment with brefeldin A. Therefore, HEV may utilize modified early secretory pathway membranes for replication.

scholarly journals Molecular Mechanisms for Norovirus Genome Replication

2021 ◽  
Author(s):  
Muhammad Amir Yunus
Keyword(s):  
Molecular Mechanisms ◽  
Rna Viruses ◽  
Open Reading Frames ◽  
Productive Infection ◽  
Genome Replication ◽  
Subgenomic Rna ◽  
Positive Strand ◽  
Reading Frame ◽  
Viral Genome Replication ◽  
Positive Strand Rna

The genomes of positive strand RNA viruses often contain more than one open reading frame. Some of these viruses have evolved novel mechanisms to regulate the synthesis of the other open reading frames that in some cases involved the production of a subgenomic RNA or RNAs. Very often, the presence of the subgenomic RNA is used as indicator for active viral genome replication. Norovirus, a major cause for gastroenteritis as well as with all other caliciviruses follow a typical positive strand RNA viruses genome replication strategy. In addition, noroviruses also produce a subgenomic RNA during their replication in infected cells. Efficient and adequate synthesis of norovirus subgenomic RNA is crucial for successful viral replication and productive infection leading to the generation of infectious viral progeny. This chapter will dissect the significant findings on mechanisms involved in norovirus genome replication as well as focusing on subgenomic RNA production.

scholarly journals Soybean antiviral immunity conferred by dsRNase targets the viral replication complex

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Kazuhiro Ishibashi ◽  
Masayasu Saruta ◽  
Takehiko Shimizu ◽  
Miao Shu ◽  
Toyoaki Anai ◽  
...  
Keyword(s):  
Viral Replication ◽  
Positional Cloning ◽  
Rna Virus ◽  
Soybean Mosaic Virus ◽  
Immune Surveillance ◽  
Virus Multiplication ◽  
Rnase H ◽  
Viral Dsrna ◽  
Positive Strand ◽  
Positive Strand Rna

Abstract Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes.

scholarly journals Essential and Nonessential Elements in the 3′ Nontranslated Region of Bovine Viral Diarrhea Virus

2005 ◽  
Vol 79 (14) ◽  
pp. 9119-9127 ◽  
Author(s):  
Alexander Pankraz ◽  
Heinz-Jürgen Thiel ◽  
Paul Becher
Keyword(s):  
Viral Replication ◽  
Bovine Viral Diarrhea Virus ◽  
Host Cells ◽  
Close Relative ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Nontranslated Region ◽  
Viral Diarrhea Virus ◽  
Efficient Viral Replication

ABSTRACT The 3′ nontranslated region (NTR) of the pestivirus Bovine viral diarrhea virus (BVDV), a close relative of human Hepatitis C virus, consists of three stem-loops which are separated by two single-stranded regions. As in other positive-stranded RNA viruses, the 3′ NTR of pestiviruses is involved in crucial processes of the viral life cycle. While several studies characterized cis-acting elements within the 3′ NTR of a BVDV replicon, there are no studies addressing the significance of these elements in the context of a replicating virus. To examine the functional importance of 3′ NTR elements, a set of 4-base deletions and deletions of each of the three stem-loops were introduced into an infectious BVDV cDNA clone. Emerging mutant viruses were characterized with regard to plaque phenotype, growth kinetics, and synthesis of viral RNA. The results indicated that presence of stem-loop (SL) I and the 3′-terminal part of the single-stranded region between stem-loops I and II are indispensable for pestiviral replication. In contrast, deletions within SL II and SL III as well as absence of either SL II or SL III still allowed efficient viral replication; however, a mutant RNA lacking both SL II and SL III was not infectious. The results of this study provide a detailed map of the essential and nonessential elements within the 3′ NTR of BVDV and contribute to our understanding of sequence and structural elements important for efficient viral replication of pestiviruses in natural host cells.

Tombusviruses target a major crossroad in the endocytic and recycling pathways via co-opting Rab7 small GTPase

2021 ◽  
Author(s):  
Zhike Feng ◽  
Jun-ichi Inaba ◽  
Peter D. Nagy
Keyword(s):  
Viral Replication ◽  
Virus Replication ◽  
Rna Viruses ◽  
Small Gtpase ◽  
Replication Protein ◽  
Positive Strand ◽  
Sorting Nexin ◽  
Retromer Complex ◽  
Infected Cells ◽  
Positive Strand Rna

Positive-strand RNA viruses induce the biogenesis of unique membranous organelles, called viral replication organelles (VROs), which perform virus replication in infected cells. Tombusviruses have been shown to rewire cellular trafficking and metabolic pathways, remodel host membranes and recruit multiple host factors to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) usurp Rab7 small GTPase to facilitate building VROs in the surrogate host yeast and in plants. Depletion of Rab7 small GTPase, which is needed for late endosome and retromer biogenesis, strongly inhibits TBSV and CIRV replication in yeast and in planta. The viral p33 replication protein interacts with Rab7 small GTPase, which results in relocalization of Rab7 into the large VROs. Similar to depletion of Rab7, deletion of either MON1 or CCZ1 heterodymeric GEFs (guanine nucleotide exchange factors) of Rab7, inhibited TBSV repRNA replication in yeast. This suggests that the activated Rab7 has pro-viral functions. We show that the pro-viral function of Rab7 is to facilitate the recruitment of the retromer complex and the endosomal sorting nexin-BAR proteins into VROs. We demonstrate that TBSV p33-driven retargeting Rab7 into VROs results in delivery of several retromer cargos with pro-viral functions. These proteins include lipid enzymes, such as Vps34 PI3K (phosphatidylinositol 3-kinase), PI4Kα-like Stt4 (phosphatidylinositol 4-kinase) and Psd2 phosphatidylserine decarboxylase. In summary, based on these and previous findings, we propose that subversion of Rab7 into VROs allows tombusviruses to reroute endocytic and recycling trafficking to support virus replication. Importance: Replication of positive-strand RNA viruses depends on the biogenesis of viral replication organelles (VROs). However, formation of membranous VROs is not well understood yet. Using tombusviruses and the model host yeast, the authors discovered that the endosomal Rab7 small GTPase is critical for the formation of VROs. Interaction between Rab7 and the TBSV p33 replication protein leads to the recruitment of Rab7 into VROs. TBSV-driven usurping of Rab7 has pro-viral functions through facilitating the delivery of co-opted retromer complex, sorting nexin-BAR proteins and lipid enzymes into VROs to create optimal milieu for virus replication. These results open up the possibility that controlling cellular Rab7 activities in infected cells could be a target for new antiviral strategies.

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