scholarly journals Mapping of Transcription Termination within the S Segment of SFTS Phlebovirus Facilitated Generation of NSs Deletant Viruses

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Benjamin Brennan ◽  
Veronica V. Rezelj ◽  
Richard M. Elliott
Keyword(s):  
Virus Infection ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transcription Termination ◽  
Severe Disease ◽  
Open Reading Frame ◽  
Coding Region ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT SFTS phlebovirus (SFTSV) is an emerging tick-borne bunyavirus that was first reported in China in 2009. Here we report the generation of a recombinant SFTSV (rHB29NSsKO) that cannot express the viral nonstructural protein (NSs) upon infection of cells in culture. We show that rHB29NSsKO replication kinetics are greater in interferon (IFN)-incompetent cells and that the virus is unable to suppress IFN induced in response to viral replication. The data confirm for the first time in the context of virus infection that NSs acts as a virally encoded IFN antagonist and that NSs is dispensable for virus replication. Using 3′ rapid amplification of cDNA ends (RACE), we mapped the 3′ end of the N and NSs mRNAs, showing that the mRNAs terminate within the coding region of the opposite open reading frame. We show that the 3′ end of the N mRNA terminates upstream of a 5′-GCCAGCC-3′ motif present in the viral genomic RNA. With this knowledge, and using virus-like particles, we could demonstrate that the last 36 nucleotides of the NSs open reading frame (ORF) were needed to ensure the efficient termination of the N mRNA and were required for recombinant virus rescue. We demonstrate that it is possible to recover viruses lacking NSs (expressing just a 12-amino-acid NSs peptide or encoding enhanced green fluorescent protein [eGFP]) or an NSs-eGFP fusion protein in the NSs locus. This opens the possibility for further studies of NSs and potentially the design of attenuated viruses for vaccination studies. IMPORTANCE SFTS phlebovirus (SFTSV) and related tick-borne viruses have emerged globally since 2009. SFTSV has been shown to cause severe disease in humans. For bunyaviruses, it has been well documented that the nonstructural protein (NSs) enables the virus to counteract the human innate antiviral defenses and that NSs is one of the major determinants of virulence in infection. Therefore, the use of reverse genetics systems to engineer viruses lacking NSs is an attractive strategy to rationally attenuate bunyaviruses. Here we report the generation of several recombinant SFTS viruses that cannot express the NSs protein or have the NSs open reading frame replaced with a reporter gene. These viruses cannot antagonize the mammalian interferon (IFN) response mounted to virus infection. The generation of NSs-lacking viruses was achieved by mapping the transcriptional termination of two S-segment-derived subgenomic mRNAs, which revealed that transcription termination occurs upstream of a 5′-GCCAGCC-3′ motif present in the virus genomic S RNA.

scholarly journals Mutation in yaaT Leads to Significant Inhibition of Phosphorelay during Sporulation in Bacillus subtilis

2002 ◽  
Vol 184 (20) ◽  
pp. 5545-5553 ◽  
Author(s):  
Shigeo Hosoya ◽  
Kei Asai ◽  
Naotake Ogasawara ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Fluorescent Protein ◽  
Early Stage ◽  
Response Regulator ◽  
Significant Inhibition ◽  
Open Reading Frame ◽  
Histidine Kinases ◽  
Reading Frame ◽  
Green Fluorescent ◽  
Sporulation Genes

ABSTRACT In the course of a Bacillus subtilis functional genomics project which involved screening for sporulation genes, we identified an open reading frame, yaaT, whose disruptant exhibits a sporulation defect. Twenty-four hours after the initiation of sporulation, most cells of the yaaT mutant exhibited stage 0 of sporulation, indicating that the yaaT mutation blocks sporulation at an early stage. Furthermore, the mutation in yaaT led to a significant decrease in transcription from a promoter controlled by Spo0A, a key response regulator required for the initiation of sporulation. However, neither the level of transcription of spo0A, the activity of σH, which transcribes spo0A, nor the amount of Spo0A protein was severely affected by the mutation in yaaT. Bypassing the phosphorelay by introducing an spo0A mutation (sof-1) into the yaaT mutant suppressed the sporulation defect, suggesting that the yaaT mutation interferes with the phosphorelay process comprising Spo0F, Spo0B, and histidine kinases. We also observed that mutation of spo0E, which encodes the phosphatase that dephosphorylates Spo0A-P, suppressed the sporulation defect in the yaaT mutant. These results strongly suggest that yaaT plays a significant role in the transduction of signals to the phosphorelay for initiation of sporulation. Micrographs indicated that YaaT-green fluorescent protein localizes to the peripheral membrane, as well as to the septum, during sporulation.

scholarly journals Mapping the Rubella Virus Subgenomic Promoter

2002 ◽  
Vol 76 (7) ◽  
pp. 3189-3201 ◽  
Author(s):  
Wen-Pin Tzeng ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Infectious Clone ◽  
Start Site ◽  
Structural Protein ◽  
Reading Frame ◽  
Gfp Gene ◽  
Green Fluorescent ◽  
Positive Strand Rna

ABSTRACT Rubella virus (RUB), the sole member of the Rubivirus genus in the Togaviridae family of positive-strand RNA viruses, synthesizes a single subgenomic (SG) RNA containing sequences from the 3′ end of the genomic RNA including the open reading frame (ORF) that encodes the virion proteins. The synthesis of SG RNA is initiated internally on a negative-strand, genome-length template at a site known as the SG promoter (SGP). Mapping the RUB SGP was initiated by using an infectious cDNA vector, dsRobo402/GFP, in which the region containing the SGP was duplicated (K. V. Pugachev, W.-P. Tzeng, and T. K. Frey, J. Virol. 74:10811-10815, 2000). In dsRobo402/GFP, the 5′-proximal nonstructural protein ORF (NS-ORF) is followed by the first SGP (SGP-1), the green fluorescent protein (GFP) gene, the second SGP (SGP-2), and the structural protein ORF. The duplicated SGP, SGP-2, contained nucleotides (nt) −175 to +76 relative to the SG start site, including the 3′ 127 nt of the NS-ORF and 47 nt between the NS-ORF and the SG start site. 5′ Deletions of SGP-2 to nt −40 (9 nt beyond the 3′ end of the NS-ORF) resulted in a wild-type (wt) phenotype in terms of virus replication and RNA synthesis. Deletions beyond this point impaired viability; however, the analysis was complicated by homologous recombination between SGP-1 and SGP-2 that resulted in deletion of the GFP gene and resurrection of viable virus with one SGP. Since the NS-ORF region was not necessary for SGP activity, subsequent mapping was done by using both replicon vectors, RUBrep/GFP and RUBrep/CAT, in which the SP-ORF is replaced with the reporter GFP and chloramphenical acetyltransferase genes, respectively, and the wt infectious clone, Robo402. In the replicon vectors, 5′ deletions to nt −26 resulted in the synthesis of SG RNA. In the infectious clone, deletions through nt −28 gave rise to viable virus. A series of short internal deletions confirmed that the region between nt −28 and the SG start site was essential for viability and showed that the repeated UCA triplet at the 5′ end of SG RNA was also required. Thus, the minimal SGP maps from nt −26 through the SG start site and appears to extend to at least nt +6, although a larger region is required for the generation of virus with a wt phenotype. Interestingly, while the positioning of the RUB SGP immediately adjacent the SG start site is thus similar to that of members of the genus Alphavirus, the other genus in the Togaviridae family, it does not include a region of nucleotide sequence homology with the alphavirus SGP that is located between nt −48 and nt −23 with respect to the SG start site in the RUB genome.

scholarly journals Reverse Genetics System for Uukuniemi Virus (Bunyaviridae): RNA Polymerase I-Catalyzed Expression of Chimeric Viral RNAs

2001 ◽  
Vol 75 (4) ◽  
pp. 1643-1655 ◽  
Author(s):  
Ramon Flick ◽  
Ralf F. Pettersson
Keyword(s):  
Rna Polymerase ◽  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Rna Polymerase I ◽  
Gfp Expression ◽  
Pol I ◽  
Green Fluorescent ◽  
Polymerase I ◽  
Expression Plasmids

ABSTRACT We describe here the development of a reverse genetics system for the phlebovirus Uukuniemi virus, a member of theBunyaviridae family, by using RNA polymerase I (pol I)-mediated transcription. Complementary DNAs containing the coding sequence for either chloramphenicol acetyltransferase (CAT) or green fluorescent protein (GFP) (both in antisense orientation) were flanked by the 5′- and 3′-terminal untranslated regions of the Uukuniemi virus sense or complementary RNA derived from the medium-sized (M) RNA segment. This chimeric cDNA (pol I expression cassette) was cloned between the murine pol I promoter and terminator and the plasmid transfected into BHK-21 cells. When such cells were either superinfected with Uukuniemi virus or cotransfected with expression plasmids encoding the L (RNA polymerase), N (nucleoprotein), and NSs (nonstructural protein) viral proteins, strong CAT activity or GFP expression was observed. CAT activity was consistently stronger in cells expressing L plus N than following superinfection. No activity was seen without superinfection, nor was activity detected when either the L or N expression plasmid was omitted. Omitting NSs expression had no effect on CAT activity or GFP expression, indicating that this protein is not needed for viral RNA replication or transcription. CAT activity could be serially passaged to fresh cultures by transferring medium from CAT-expressing cells, indicating that recombinant virus containing the reporter construct had been produced. In summary, we demonstrate that the RNA pol I system, originally developed for influenza virus, which replicates in the nucleus, has strong potential for the development of an efficient reverse genetics system also for Bunyaviridae members, which replicate in the cytoplasm.

scholarly journals Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’

2001 ◽  
Vol 82 (5) ◽  
pp. 1013-1025 ◽  
Author(s):  
Michelle L. L. Donnelly ◽  
Garry Luke ◽  
Amit Mehrotra ◽  
Xuejun Li ◽  
Lorraine E. Hughes ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Foot And Mouth Disease ◽  
Peptide Bond ◽  
Open Reading Frame ◽  
Translation Product ◽  
Reading Frame ◽  
C Terminus ◽  
Mouth Disease Virus ◽  
Green Fluorescent

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.

scholarly journals Rotavirus NSP5: Mapping Phosphorylation Sites and Kinase Activation and Viroplasm Localization Domains

2002 ◽  
Vol 76 (7) ◽  
pp. 3461-3470 ◽  
Author(s):  
Catherine Eichwald ◽  
Fulvia Vascotto ◽  
Elsa Fabbretti ◽  
Oscar R. Burrone
Keyword(s):  
Virus Infection ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Phosphorylation Sites ◽  
Kinase Activation ◽  
Infected Cells ◽  
Green Fluorescent

ABSTRACT Rotavirus NSP5 is a nonstructural protein that localizes in cytoplasmic viroplasms of infected cells. NSP5 interacts with NSP2 and undergoes a complex posttranslational hyperphosphorylation, generating species with reduced polyacrylamide gel electrophoresis mobility. This process has been suggested to be due in part to autophosphorylation. We developed an in vitro phosphorylation assay using as a substrate an in vitro-translated NSP5 deletion mutant that was phosphorylated by extracts from MA104 cells transfected with NSP5 mutants but not by extracts from mock-transfected cells. The phosphorylated products obtained showed shifts in mobility similar to what occurs in vivo. From these and other experiments we concluded that NSP5 activates a cellular kinase(s) for its own phosphorylation. Three NSP5 regions were found to be essential for kinase(s) activation. Glutathione S-transferase-NSP5 mutants were produced in Escherichia coli and used to determine phosphoacceptor sites. These were mapped to four serines (Ser153, Ser155, Ser163, and Ser165) within an acidic region with homology to casein kinase II (CKII) phosphorylation sites. CKII was able to phosphorylate NSP5 in vitro. NSP5 and its mutants fused to enhanced green fluorescent protein were used in transfection experiments followed by virus infection and allowed the determination of the domains essential for viroplasm localization in the context of virus infection.

scholarly journals Analysis of the 3′ cis-Acting Elements of Rubella Virus by Using Replicons Expressing a Puromycin Resistance Gene

2004 ◽  
Vol 78 (5) ◽  
pp. 2553-2561 ◽  
Author(s):  
Min-Hsin Chen ◽  
Ilya Frolov ◽  
Joseph Icenogle ◽  
Teryl K. Frey
Keyword(s):  
Rubella Virus ◽  
Nonstructural Protein ◽  
Relative Survival ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Minus Strand ◽  
Structural Protein ◽  
Coding Region ◽  
Reading Frame

ABSTRACT A rubella virus (RUB) replicon, RUBrep/PAC, was constructed and used to map the 3′ cis-acting elements (3′ CSE) of the RUB genome required for RUB replication. The RUBrep/PAC replicon had the structural protein open reading frame partially replaced by a puromycin acetyltransferase (PAC) gene. Cells transfected with RUBrep/PAC transcripts expressed the PAC gene from the subgenomic RNA, were rendered resistant to puromycin, and thus survived selection with this drug. The relative survival following puromycin selection of cells transfected with transcripts from RUBrep/PAC constructs with mutations in the 3′ CSE varied. The 3′ region necessary for optimal relative survival consisted of the 3′ 305 nucleotides (nt), a region conserved in RUB defective-interfering RNAs, and thus this region constitutes the 3′ CSE. Within the 3′ CSE, deletions in the ∼245 nt that overlap the 3′ end of the E1 gene resulted in reduced relative survivals, ranging from 20 to <1% of the parental replicon survival level while most mutations within the ∼60-nt 3′ untranslated region (UTR) were lethal. None of the 3′ CSE mutations affected in vitro translation of the nonstructural protein open reading frame (which is 5′ proximal in the genome and encodes the enzymes involved in virus RNA replication). In cells transfected with replicons with 3′ CSE mutations that survived antibiotic selection (i.e., those with mutations in the region of the 3′ CSE that overlaps the E1 coding region), the amount of replicon-specific minus-strand RNA was uniform; however, the accumulation of both plus-strand RNA species, genomic and subgenomic, varied widely, indicating that this region of the RUB 3′ CSE affects plus-strand RNA accumulation rather than minus-strand RNA synthesis.

scholarly journals SAT: a Late NS Protein of Porcine Parvovirus

2005 ◽  
Vol 79 (20) ◽  
pp. 13129-13138 ◽  
Author(s):  
Zoltán Zádori ◽  
József Szelei ◽  
Peter Tijssen
Keyword(s):  
Fluorescent Protein ◽  
Open Reading Frame ◽  
Start Codon ◽  
Knockout Mutant ◽  
Reading Frame ◽  
Knockout Mutants ◽  
Slow Spreading ◽  
Membrane Spanning ◽  
Green Fluorescent ◽  
Small Open Reading Frame

ABSTRACT The genomes of all members of the Parvovirus genus were found to contain a small open reading frame (ORF), designated SAT, with a start codon four or seven nucleotides downstream of the VP2 initiation codon. Green fluorescent protein or FLAG fusion constructs of SAT demonstrated that these ORFs were expressed. Although the SAT proteins of the different parvoviruses are not particularly conserved, they were all predicted to contain a membrane-spanning helix, and mutations in this hydrophobic stretch affected the localization of the SAT protein. SAT colocalized with calreticulin in the membranes of the endoplasmic reticulum and the nucleus. A knockout mutant (SAT−), with an unmodified VP sequence, showed a “slow-spreading” phenotype. These knockout mutants could be complemented with VP2− SAT+ mutant. The SAT protein is a late nonstructural (NS) protein, in contrast to previously identified NS proteins, since it is expressed from the same mRNA as VP2.

scholarly journals Entirely plasmid-based reverse genetics system for rotaviruses

2017 ◽  
Vol 114 (9) ◽  
pp. 2349-2354 ◽  
Author(s):  
Yuta Kanai ◽  
Satoshi Komoto ◽  
Takahiro Kawagishi ◽  
Ryotaro Nouda ◽  
Naoko Nagasawa ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Nonstructural Protein ◽  
Transmembrane Protein ◽  
Gene Segment ◽  
Severe Diarrhea ◽  
Nsp1 Gene ◽  
Capping Enzyme ◽  
Green Fluorescent

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

scholarly journals Requirement of the N-Terminal Region of Orthobunyavirus Nonstructural Protein NSm for Virus Assembly and Morphogenesis

2006 ◽  
Vol 80 (16) ◽  
pp. 8089-8099 ◽  
Author(s):  
Xiaohong Shi ◽  
Alain Kohl ◽  
Vincent H. J. Léonard ◽  
Ping Li ◽  
Angela McLees ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Fluorescent Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
Nonstructural Protein ◽  
Intracellular Localization ◽  
Integral Membrane Protein ◽  
Terminal Region ◽  
Coding Region ◽  
Hydrophobic Domain

ABSTRACT The nonstructural protein NSm of Bunyamwera virus (BUNV), the prototype of the Bunyaviridae family, is encoded by the M segment in a polyprotein precursor, along with the virion glycoproteins, in the order Gn-NSm-Gc. As little is known of its function, we examined the intracellular localization, membrane integrality, and topology of NSm and its role in virus replication. We confirmed that NSm is an integral membrane protein and that it localizes in the Golgi complex, together with Gn and Gc. Coimmunoprecipitation assays and yeast two-hybrid analysis demonstrated that NSm was able to interact with other viral proteins. NSm is predicted to contain three hydrophobic (I, III, and V) and two nonhydrophobic (II and IV) domains. The N-terminal nonhydrophobic domain II was found in the lumen of an intracellular compartment. A novel BUNV assembly assay was developed to monitor the formation of infectious virus-like-particles (VLPs). Using this assay, we showed that deletions of either the complete NSm coding region or domains I, II, and V individually seriously compromised VLP production. Consistently, we were unable to rescue viable viruses by reverse genetics from cDNA constructs that contained the same deletions. However, we could generate mutant BUNV with deletions in NSm domains III and IV and also a recombinant virus with the green fluorescent protein open reading frame inserted into NSm domain IV. The mutant viruses displayed differences in their growth properties. Overall, our data showed that the N-terminal region of NSm, which includes domain I and part of domain II, is required for virus assembly and that the C-terminal hydrophobic domain V may function as an internal signal sequence for the Gc glycoprotein.

scholarly journals An RNA motif advances transcription by preventing Rho-dependent termination

2015 ◽  
Vol 112 (50) ◽  
pp. E6835-E6843 ◽  
Author(s):  
Anastasia Sevostyanova ◽  
Eduardo A. Groisman
Keyword(s):  
Transcription Termination ◽  
Open Reading Frame ◽  
Leader Region ◽  
Nucleic Acid Binding ◽  
Coding Region ◽  
Reading Frame ◽  
Rna Conformation ◽  
Transcription Termination Factor ◽  
Nucleic Acid Binding Proteins ◽  
Rna Motif

The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity.

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