scholarly journals Significance in Replication of the Terminal Nucleotides of the Flavivirus Genome

2003 ◽  
Vol 77 (19) ◽  
pp. 10623-10629 ◽  
Author(s):  
Alexander A Khromykh ◽  
Natasha Kondratieva ◽  
Jean-Yves Sgro ◽  
Ann Palmenberg ◽  
Edwin G Westaway
Keyword(s):  
Rna Synthesis ◽  
Point Mutations ◽  
In Vitro Activity ◽  
Rna Dependent Rna Polymerase ◽  
Stem Loop ◽  
Double Stranded Rna ◽  
Positive Strand ◽  
Proposed Model

ABSTRACT Point mutations that resulted in a substitution of the conserved 3′-penultimate cytidine in genomic RNA or the RNA negative strand of the self-amplifying replicon of the Flavivirus Kunjin virus completely blocked in vivo replication. Similarly, substitutions of the conserved 3′-terminal uridine in the RNA negative or positive strand completely blocked replication or caused much-reduced replication, respectively. The same preference for cytidine in the 3′-terminal dinucleotide was noted in reports of the in vitro activity of the RNA-dependent RNA polymerase (RdRp) for the other genera of Flaviviridae that also employ a double-stranded RNA (dsRNA) template to initiate asymmetric semiconservative RNA positive-strand synthesis. The Kunjin virus replicon results were interpreted in the context of a proposed model for initiation of RNA synthesis based on the solved crystal structure of the RdRp of φ6 bacteriophage, which also replicates efficiently using a dsRNA template with conserved 3′-penultimate cytidines and a 3′-terminal pyrimidine. A previously untested substitution of the conserved pentanucleotide at the top of the 3′-terminal stem-loop of all Flavivirus species also blocked detectable in vivo replication of the Kunjin virus replicon RNA.

scholarly journals A Viral Noncoding RNA Generated by cis-Element-Mediated Protection against 5′→3′ RNA Decay Represses both Cap-Independent and Cap-Dependent Translation

2008 ◽  
Vol 82 (20) ◽  
pp. 10162-10174 ◽  
Author(s):  
Hiro-oki Iwakawa ◽  
Hiroyuki Mizumoto ◽  
Hideaki Nagano ◽  
Yuka Imoto ◽  
Kazuma Takigawa ◽  
...  
Keyword(s):  
Noncoding Rna ◽  
Rna Synthesis ◽  
Red Clover ◽  
Stem Loop ◽  
Genomic Rnas ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.

scholarly journals Point mutations in the West Nile virus (Flaviviridae; Flavivirus) RNA-dependent RNA polymerase alter viral fitness in a host-dependent manner in vitro and in vivo

Virology ◽  
2012 ◽  
Vol 427 (1) ◽  
pp. 18-24 ◽  
Author(s):  
Greta A. Van Slyke ◽  
Alexander T. Ciota ◽  
Graham G. Willsey ◽  
Joachim Jaeger ◽  
Pei-Yong Shi ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rna Polymerase ◽  
Point Mutations ◽  
Viral Fitness ◽  
Dependent Manner ◽  
The West ◽  
Rna Dependent Rna Polymerase ◽  
West Nile

scholarly journals Structural and Functional Analysis of the cis-Acting Elements Required for Plus-Strand RNA Synthesis of Bamboo Mosaic Virus

2005 ◽  
Vol 79 (14) ◽  
pp. 9046-9053 ◽  
Author(s):  
Jen-Wen Lin ◽  
Hsiao-Ning Chiu ◽  
I-Hsuan Chen ◽  
Tzu-Chi Chen ◽  
Yau-Heiu Hsu ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Viral Rna ◽  
Minus Strand ◽  
Internal Deletion ◽  
Stem Loop ◽  
Cis Acting ◽  
Rna Accumulation

ABSTRACT Bamboo mosaic virus (BaMV) has a single-stranded positive-sense RNA genome. The secondary structure of the 3′-terminal sequence of the minus-strand RNA has been predicted by MFOLD and confirmed by enzymatic structural probing to consist of a large, stable stem-loop and a small, unstable stem-loop. To identify the promoter for plus-strand RNA synthesis in this region, transcripts of 39, 77, and 173 nucleotides (Ba-39, Ba-77, and Ba-173, respectively) derived from the 3′ terminus of the minus-strand RNA were examined by an in vitro RNA-dependent RNA polymerase assay for the ability to direct RNA synthesis. Ba-77 and Ba-39 appeared to direct the RNA synthesis efficiently, while Ba-173 failed. Ba-77/Δ5, with a deletion of the 3′-terminal UUUUC sequence in Ba-77, directed the RNA synthesis only to 7% that of Ba-77. However, Ba-77/Δ16 and Ba-77/Δ31, with longer deletions but preserving the terminal UUUUC sequence of Ba-77, restored the template activity to about 60% that of the wild type. Moreover, mutations that changed the sequence in the stem of the large stem-loop interfered with the efficiency of RNA synthesis and RNA accumulation in vivo. The mutant with an internal deletion in the region between the terminal UUUUC sequence and the large stem-loop reduced the viral RNA accumulation in protoplasts, but mutants with insertions did not. Taken together, these results suggest that three cis-acting elements in the 3′ end of the minus-strand RNA, namely, the terminal UUUUC sequence, the sequence in the large stem-loop, and the distance between these two regions, are involved in modulating the efficiency of BaMV plus-strand viral RNA synthesis.

scholarly journals Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts

2003 ◽  
Vol 77 (10) ◽  
pp. 5703-5711 ◽  
Author(s):  
K. Sivakumaran ◽  
M. Hema ◽  
C. Cheng Kao
Keyword(s):  
Mosaic Virus ◽  
Rna Synthesis ◽  
Previous Analysis ◽  
Brome Mosaic Virus ◽  
Minus Strand ◽  
Stem Loop ◽  
Virus Rna ◽  
Different Levels

ABSTRACT The RNA replicase extracted from Brome mosaic virus (BMV)-infected plants has been used to characterize the cis-acting elements for RNA synthesis and the mechanism of RNA synthesis. Minus-strand RNA synthesis in vitro requires a structure named stem-loop C (SLC) that contains a clamped adenine motif. In vitro, there are several specific requirements for SLC recognition. We examined whether these requirements also apply to BMV replication in barley protoplasts. BMV RNA3s with mutations in SLC were transfected into barley protoplasts, and the requirements for minus- and plus-strand replication were found to correlate well with the requirements in vitro. Furthermore, previous analysis of replicase recognition of the Cucumber mosaic virus (CMV) and BMV SLCs indicates that the requirements in the BMV SLC are highly specific. In protoplasts, we found that BMV RNA3s with their SLCs replaced with two different CMV SLCs were defective for replication. In vitro results generated with the BMV replicase and minimal-length RNAs generally agreed with those of in vivo BMV RNA replication. To extend this conclusion, we determined that, corresponding with the process of infection, the BMV replicases extracted from plants at different times after infection have different levels of recognition of the minimal promoters for plus- and minus-strand RNA syntheses.

scholarly journals Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.

1994 ◽  
Vol 14 (3) ◽  
pp. 1709-1720 ◽  
Author(s):  
N B Pandey ◽  
A S Williams ◽  
J H Sun ◽  
V D Brown ◽  
U Bond ◽  
...  
Keyword(s):  
Base Pair ◽  
Point Mutations ◽  
Histone Mrna ◽  
Base Pairs ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Loop Sequence ◽  
Processing Signal

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.

scholarly journals The RNA Replication Enhancer Element of Tombusviruses Contains Two Interchangeable Hairpins That Are Functional during Plus-Strand Synthesis

2003 ◽  
Vol 77 (1) ◽  
pp. 258-269 ◽  
Author(s):  
T. Panavas ◽  
P. D. Nagy
Keyword(s):  
Rna Synthesis ◽  
Minus Strand ◽  
Stem Loop ◽  
Vitro Assay ◽  
Enhancer Element ◽  
Strand Initiation ◽  
Vitro Data ◽  
In Vitro Data

ABSTRACT Replication of the RNA genomes of tombusviruses, which are small plus-sense RNA viruses of plants, may be regulated by cis-acting elements, including promoters and replication enhancers that are present in the RNA templates. Using a partially purified RNA-dependent RNA polymerase (RdRp) preparation (P. D. Nagy and J. Pogany, Virology 276:279-288, 2000), we demonstrate that the minus-strand templates of tombusviruses contain a replication enhancer, which can upregulate RNA synthesis initiating from the minimal plus-strand initiation promoter by 10- to 20-fold in an in vitro assay. Dissection of the sequence of the replication enhancer element revealed that the two stem-loop structures present within the ∼80-nucleotide-long enhancer region have interchangeable roles in upregulating RNA synthesis. The single-stranded sequence located between the two stem-loops also plays an important role in stimulation of RNA synthesis. We also demonstrate that one of the two hairpins, both of which are similar to the hairpin of the minus-strand initiation promoter, can function as a promoter in vitro in the presence of short cytidylate-containing initiation sites. Overall, the in vitro data presented are consistent with previous in vivo results (D. Ray and K. A. White, Virology 256:162-171, 1999) and they firmly establish the presence of a replication enhancer on the minus-stranded RNA of tombusviruses.

scholarly journals Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation

1994 ◽  
Vol 14 (3) ◽  
pp. 1709-1720
Author(s):  
N B Pandey ◽  
A S Williams ◽  
J H Sun ◽  
V D Brown ◽  
U Bond ◽  
...  
Keyword(s):  
Base Pair ◽  
Point Mutations ◽  
Histone Mrna ◽  
Base Pairs ◽  
Stem Loop ◽  
Histone Mrnas ◽  
Loop Sequence ◽  
Processing Signal

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.

scholarly journals Efficient and Specific Initiation of Subgenomic RNA Synthesis by Cucumber Mosaic Virus Replicase In Vitro Requires an Upstream RNA Stem-Loop

2000 ◽  
Vol 74 (23) ◽  
pp. 11201-11209 ◽  
Author(s):  
M.-H. Chen ◽  
M. J. Roossinck ◽  
C. C. Kao
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Rna Synthesis ◽  
Core Promoter ◽  
Brome Mosaic Virus ◽  
Stem Loop ◽  
Promoter Sequences ◽  
Promoter Recognition

ABSTRACT We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions −28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions −28 to −5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.

scholarly journals Comparison of Polymerase Subunits from Double-Stranded RNA Bacteriophages

2001 ◽  
Vol 75 (22) ◽  
pp. 11088-11095 ◽  
Author(s):  
Hongyan Yang ◽  
Eugene V. Makeyev ◽  
Dennis H. Bamford
Keyword(s):  
Molecular Basis ◽  
Rna Synthesis ◽  
Regulatory Mechanism ◽  
Biochemical Properties ◽  
Double Stranded Rna ◽  
The Family ◽  
Dsrna Viruses ◽  
Template Specificity

ABSTRACT The family Cystoviridae comprises several bacteriophages with double-stranded RNA (dsRNA) genomes. We have previously purified the catalytic polymerase subunit (Pol) of one of the Cystoviridae members, bacteriophage φ6, and shown that the protein can catalyze RNA synthesis in vitro. In this reaction, both bacteriophage-specific and heterologous RNAs can serve as templates, but those containing 3′ termini from the φ6 minus strands are favored. This provides a molecular basis for the observation that only plus strands, not minus strands, are transcribed from φ6 dsRNA segments in vivo. To test whether such a regulatory mechanism is also found in other dsRNA viruses, we purified recombinant Pol subunits from the φ6-related bacteriophages φ8 and φ13 and assayed their polymerase activities in vitro. The enzymes catalyze template-dependent RNA synthesis using both single-stranded-RNA (ssRNA) and dsRNA templates. However, they differ from each other as well as from φ6 Pol in certain biochemical properties. Notably, each polymerase demonstrates a distinct preference for ssRNAs bearing short 3′-terminal sequences from the virus-specific minus strands. This suggests that, in addition to other factors, RNA transcription inCystoviridae is controlled by the template specificity of the polymerase subunit.

scholarly journals Mutational Analysis of the GB Virus B Internal Ribosome Entry Site

2000 ◽  
Vol 74 (2) ◽  
pp. 773-783 ◽  
Author(s):  
René Rijnbrand ◽  
Geoffrey Abell ◽  
Stanley M. Lemon
Keyword(s):  
Internal Ribosome Entry Site ◽  
Mutational Analysis ◽  
General Structure ◽  
Point Mutations ◽  
Structural Domain ◽  
Entry Site ◽  
Stem Loop ◽  
Internal Ribosome Entry

ABSTRACT GB virus B (GBV-B) is a recently discovered hepatotropic flavivirus that is distantly related to hepatitis C virus (HCV). We show here that translation of its polyprotein is initiated by internal entry of ribosomes on GBV-B RNA. We analyzed the translational activity of dicistronic RNA transcripts containing wild-type or mutated 5′ nontranslated GBV-B RNA (5′NTR) segments, placed between the coding sequences of two reporter proteins, in vitro in rabbit reticulocyte lysate and in vivo in transfected BT7-H cells. We related these results to a previously proposed model of the secondary structure of the GBV-B 5′NTR (M. Honda, et al. RNA 2:955–968, 1996). We identified an internal ribosome entry site (IRES) bounded at its 5′ end by structural domain II, a location analogous to the 5′ limit of the IRES in both the HCV and pestivirus 5′NTRs. Mutational analysis confirmed the structure proposed for domain II of GBV-B RNA, and demonstrated that optimal IRES-mediated translation is dependent on each of the putative RNA hairpins in this domain, including two stem-loops not present in the HCV or pestivirus structures. IRES activity was also absolutely dependent on (i) phylogenetically conserved, adenosine-containing bulge loops in domain III and (ii) the primary nucleotide sequence of stem-loop IIIe. IRES-directed translation was inhibited by a series of point mutations predicted to stabilize stem-loop IV, which contains the initiator AUG codon in its loop segment. A reporter gene was translated most efficiently when fused directly to the initiator AUG codon, with no intervening downstream GBV-B sequence. This finding indicates that the 3′ limit of the GBV-B IRES is at the initiator AUG and that it does not require downstream polyprotein-coding sequence as suggested for the HCV IRES. These results show that the GBV-B IRES, while sharing a common general structure, differs both structurally and functionally from other flavivirus IRES elements.

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