Response to “Non-segmented negative-strand RNA virus RNA synthesis in vivo”

ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.

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1-β-d-Arabinofuranosylcytosine Inhibits Borna Disease Virus Replication and Spread

Journal of Virology ◽

10.1128/jvi.76.12.6268-6286.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 6268-6276 ◽

Cited By ~ 14

Author(s):

Jeffrey J. Bajramovic ◽

Sylvie Syan ◽

Michel Brahic ◽

Juan Carlos de la Torre ◽

Daniel Gonzalez-Dunia

Keyword(s):

Disease Virus ◽

Measles Virus ◽

Rna Virus ◽

Neurological Diseases ◽

Borna Disease Virus ◽

Negative Strand ◽

Neuropsychiatric Diseases ◽

Borna Disease

ABSTRACT Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that causes neurological diseases in a variety of warm-blooded animal species. There is general consensus that BDV can also infect humans, being a possible zoonosis. Although the clinical consequences of human BDV infection are still controversial, experimental BDV infection is a well-described model for human neuropsychiatric diseases. To date, there is no effective treatment against BDV. In this paper, we demonstrate that the nucleoside analog 1-β-d-arabinofuranosylcytosine (Ara-C), a known inhibitor of DNA polymerases, inhibits BDV replication. Ara-C treatment inhibited BDV RNA and protein synthesis and prevented BDV cell-to-cell spread in vitro. Replication of other negative-strand RNA viruses such as influenza virus or measles virus was not inhibited by Ara-C, underscoring the particularity of the replication machinery of BDV. Strikingly, Ara-C treatment induced nuclear retention of viral ribonucleoparticles. These findings could not be attributed to known effects of Ara-C on the host cell, suggesting that Ara-C directly inhibits the BDV polymerase. Finally, we show that Ara-C inhibits BDV replication in vivo in the brain of infected rats, preventing persistent infection of the central nervous system as well as the development of clinical disease. These findings open the way to the development of effective antiviral therapy against BDV.

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SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

Journal of Virology ◽

10.1128/jvi.78.23.13153-13162.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13153-13162 ◽

Cited By ~ 37

Author(s):

Keum S. Choi ◽

Akihiro Mizutani ◽

Michael M. C. Lai

Keyword(s):

Mammalian Cells ◽

Rna Synthesis ◽

Rna Binding ◽

Hepatitis Virus ◽

Affinity Purification ◽

Mouse Hepatitis Virus ◽

Viral Rna ◽

Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

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Constraints of Viral RNA Synthesis on Codon Usage of Negative-Strand RNA Virus

Journal of Virology ◽

10.1128/jvi.01775-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 6

Author(s):

Ryan H. Gumpper ◽

Weike Li ◽

Ming Luo

Keyword(s):

Rna Polymerase ◽

Codon Usage ◽

Rna Synthesis ◽

Rna Viruses ◽

Rna Virus ◽

Protein Translation ◽

Viral Rna ◽

Genomic Rna ◽

Negative Strand ◽

Unique Mechanism

ABSTRACTNegative-strand RNA viruses (NSVs) include some of the most pathogenic human viruses known. NSVs completely rely on the host cell for protein translation, but their codon usage bias is often different from that of the host. This discrepancy may have originated from the unique mechanism of NSV RNA synthesis in that the genomic RNA sequestered in the nucleocapsid serves as the template. The stability of the genomic RNA in the nucleocapsid appears to regulate its accessibility to the viral RNA polymerase, thus placing constraints on codon usage to balance viral RNA synthesis. Byin situanalyses of vesicular stomatitis virus RNA synthesis, specific activities of viral RNA synthesis were correlated with the genomic RNA sequence. It was found that by simply altering the sequence and not the amino acid that it encoded, a significant reduction, up to an ∼750-fold reduction, in viral RNA transcripts occurred. Through subsequent sequence analysis and thermal shift assays, it was found that the purine/pyrimidine content modulates the overall stability of the polymerase complex, resulting in alteration of the activity of viral RNA synthesis. The codon usage is therefore constrained by the obligation of the NSV genome for viral RNA synthesis.IMPORTANCENegative-strand RNA viruses (NSVs) include the most pathogenic viruses known. New methods to monitor their evolutionary trends are urgently needed for the development of antivirals and vaccines. The protein translation machinery of the host cell is currently recognized as a main genomic regulator of RNA virus evolution, which works especially well for positive-strand RNA viruses. However, this approach fails for NSVs because it does not consider the unique mechanism of their viral RNA synthesis. For NSVs, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the genome sequestered in the nucleocapsid. Our work suggests a paradigm shift that the interactions between the RNA genome and the nucleocapsid protein regulate the activity of vRdRp, which selects codon usage.

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The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis

Journal of Virology ◽

10.1128/jvi.77.20.11284-11289.2003 ◽

2003 ◽

Vol 77 (20) ◽

pp. 11284-11289 ◽

Cited By ~ 12

Author(s):

A. Corina Vlot ◽

John F. Bol

Keyword(s):

Mosaic Virus ◽

Untranslated Region ◽

Rna Synthesis ◽

Alfalfa Mosaic Virus ◽

Loop Structure ◽

Stem Loop ◽

Genomic Rnas ◽

Negative Strand ◽

Stem Loop Structure ◽

Virus Rna

ABSTRACT The three genomic RNAs of alfalfa mosaic virus each contain a unique 5′ untranslated region (5′ UTR). Replacement of the 5′ UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5′ stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5′ stem-loop structure is present in RNA 2 but not in RNA 3.

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Brome Mosaic Virus RNA Syntheses In Vitro and in Barley Protoplasts

Journal of Virology ◽

10.1128/jvi.77.10.5703-5711.2003 ◽

2003 ◽

Vol 77 (10) ◽

pp. 5703-5711 ◽

Cited By ~ 15

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.

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Mechanistic Consequences of hnRNP C Binding to Both RNA Termini of Poliovirus Negative-Strand RNA Intermediates

Journal of Virology ◽

10.1128/jvi.02198-09 ◽

2010 ◽

Vol 84 (9) ◽

pp. 4229-4242 ◽

Cited By ~ 40

Author(s):

Kenneth J. Ertel ◽

Jo Ellen Brunner ◽

Bert L. Semler

Keyword(s):

Rna Synthesis ◽

Cultured Cells ◽

Wild Type Virus ◽

Positive Strand ◽

Negative Strand ◽

Virus Rna ◽

Hnrnp C ◽

C Proteins ◽

Positive Strand Rna

ABSTRACT The poliovirus 3′ noncoding region (3′ NCR) is necessary for efficient virus replication. A poliovirus mutant, PVΔ3′NCR, with a deletion of the entire 3′ NCR, yielded a virus that was capable of synthesizing viral RNA, albeit with a replication defect caused by deficient positive-strand RNA synthesis compared to wild-type virus. We detected multiple ribonucleoprotein (RNP) complexes in extracts from poliovirus-infected HeLa cells formed with a probe corresponding to the 5′ end of poliovirus negative-strand RNA (the complement of the genomic 3′ NCR), and the levels of these RNP complexes increased during the course of viral infection. Previous studies have identified RNP complexes formed with the 3′ end of poliovirus negative-strand RNA, including one that contains a 36-kDa protein later identified as heterogeneous nuclear ribonucleoprotein C (hnRNP C). We report here that the 5′ end of poliovirus negative-strand RNA is capable of interacting with endogenous hnRNP C, as well as with poliovirus nonstructural proteins. Further, we demonstrate that the addition of recombinant purified hnRNP C proteins can stimulate virus RNA synthesis in vitro and that depletion of hnRNP C proteins in cultured cells results in decreased virus yields and a correspondingly diminished accumulation of positive-strand RNAs. We propose that the association of hnRNP C with poliovirus negative-strand termini acts to stabilize or otherwise promote efficient positive-strand RNA synthesis.

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Comparison of RNA synthesis initiation properties of non-segmented negative strand RNA virus polymerases

PLoS Pathogens ◽

10.1371/journal.ppat.1010151 ◽

2021 ◽

Vol 17 (12) ◽

pp. e1010151

Author(s):

Afzaal M. Shareef ◽

Barbara Ludeke ◽

Paul Jordan ◽

Jerome Deval ◽

Rachel Fearns

Keyword(s):

Rna Synthesis ◽

De Novo ◽

Rna Viruses ◽

Rna Virus ◽

Promoter Sequence ◽

Structural Features ◽

Marburg Virus ◽

Initiation Mechanism ◽

Negative Strand ◽

Syncytial Virus

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming.

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