scholarly journals A Polyamide Inhibits Replication of Vesicular Stomatitis Virus by Targeting RNA in the Nucleocapsid

2018 ◽  
Vol 92 (8) ◽  
pp. e00146-18 ◽  
Author(s):  
Ryan H. Gumpper ◽  
Weike Li ◽  
Carlos H. Castañeda ◽  
M. José Scuderi ◽  
James K. Bashkin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Vesicular Stomatitis Virus ◽  
Ebola Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Viral Rna ◽  
Negative Strand ◽  
Double Stranded Dna ◽  
Vesicular Stomatitis ◽  
Syncytial Virus

ABSTRACTPolyamides have been shown to bind double-stranded DNA by complementing the curvature of the minor groove and forming various hydrogen bonds with DNA. Several polyamide molecules have been found to have potent antiviral activities against papillomavirus, a double-stranded DNA virus. By analogy, we reason that polyamides may also interact with the structured RNA bound in the nucleocapsid of a negative-strand RNA virus. Vesicular stomatitis virus (VSV) was selected as a prototype virus to test this possibility since its genomic RNA encapsidated in the nucleocapsid forms a structure resembling one strand of an A-form RNA duplex. One polyamide molecule, UMSL1011, was found to inhibit infection of VSV. To confirm that the polyamide targeted the nucleocapsid, a nucleocapsid-like particle (NLP) was incubated with UMSL1011. The encapsidated RNA in the polyamide-treated NLP was protected from thermo-release and digestion by RNase A. UMSL1011 also inhibits viral RNA synthesis in the intracellular activity assay for the viral RNA-dependent RNA polymerase. The crystal structure revealed that UMSL1011 binds the structured RNA in the nucleocapsid. The conclusion of our studies is that the RNA in the nucleocapsid is a viable antiviral target of polyamides. Since the RNA structure in the nucleocapsid is similar in all negative-strand RNA viruses, polyamides may be optimized to target the specific RNA genome of a negative-strand RNA virus, such as respiratory syncytial virus and Ebola virus.IMPORTANCENegative-strand RNA viruses (NSVs) include several life-threatening pathogens, such as rabies virus, respiratory syncytial virus, and Ebola virus. There are no effective antiviral drugs against these viruses. Polyamides offer an exceptional opportunity because they may be optimized to target each NSV. Our studies on vesicular stomatitis virus, an NSV, demonstrated that a polyamide molecule could specifically target the viral RNA in the nucleocapsid and inhibit viral growth. The target specificity of the polyamide molecule was proved by its inhibition of thermo-release and RNA nuclease digestion of the RNA bound in a model nucleocapsid, and a crystal structure of the polyamide inside the nucleocapsid. This encouraging observation provided the proof-of-concept rationale for designing polyamides as antiviral drugs against NSVs.

scholarly journals Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

2015 ◽  
Vol 90 (2) ◽  
pp. 715-724 ◽  
Author(s):  
Shihong Qiu ◽  
Minako Ogino ◽  
Ming Luo ◽  
Tomoaki Ogino ◽  
Todd J. Green
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Three Dimensional ◽  
Viral Transcription ◽  
Content Type ◽  
Negative Strand ◽  
Terminal Domain ◽  
Vesicular Stomatitis ◽  
L Proteins

ABSTRACTViruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of theRhabdoviridae,Paramyxoviridae, andFiloviridaeshare sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses.IMPORTANCENegative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses.

scholarly journals Oligomerization of the Vesicular Stomatitis Virus Phosphoprotein Is Dispensable for mRNA Synthesis but Facilitates RNA Replication

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Louis-Marie Bloyet ◽  
Benjamin Morin ◽  
Vesna Brusic ◽  
Erica Gardner ◽  
Robin A. Ross ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Rna Virus ◽  
Rna Replication ◽  
Binding Domain ◽  
Genome Replication ◽  
Mrna Synthesis ◽  
Wild Type ◽  
Vesicular Stomatitis ◽  
Oligomerization Domain

ABSTRACT Nonsegmented negative-strand (NNS) RNA viruses possess a ribonucleoprotein template in which the genomic RNA is sequestered within a homopolymer of nucleocapsid protein (N). The viral RNA-dependent RNA polymerase (RdRP) resides within an approximately 250-kDa large protein (L), along with unconventional mRNA capping enzymes: a GDP:polyribonucleotidyltransferase (PRNT) and a dual-specificity mRNA cap methylase (MT). To gain access to the N-RNA template and orchestrate the LRdRP, LPRNT, and LMT, an oligomeric phosphoprotein (P) is required. Vesicular stomatitis virus (VSV) P is dimeric with an oligomerization domain (OD) separating two largely disordered regions followed by a globular C-terminal domain that binds the template. P is also responsible for bringing new N protomers onto the nascent RNA during genome replication. We show VSV P lacking the OD (PΔOD) is monomeric but is indistinguishable from wild-type P in supporting mRNA transcription in vitro. Recombinant virus VSV-PΔOD exhibits a pronounced kinetic delay in progeny virus production. Fluorescence recovery after photobleaching demonstrates that PΔOD diffuses 6-fold more rapidly than the wild type within viral replication compartments. A well-characterized defective interfering particle of VSV (DI-T) that is only competent for RNA replication requires significantly higher levels of N to drive RNA replication in the presence of PΔOD. We conclude P oligomerization is not required for mRNA synthesis but enhances genome replication by facilitating RNA encapsidation. IMPORTANCE All NNS RNA viruses, including the human pathogens rabies, measles, respiratory syncytial virus, Nipah, and Ebola, possess an essential L-protein cofactor, required to access the N-RNA template and coordinate the various enzymatic activities of L. The polymerase cofactors share a similar modular organization of a soluble N-binding domain and a template-binding domain separated by a central oligomerization domain. Using a prototype of NNS RNA virus gene expression, vesicular stomatitis virus (VSV), we determined the importance of P oligomerization. We find that oligomerization of VSV P is not required for any step of viral mRNA synthesis but is required for efficient RNA replication. We present evidence that this likely occurs through the stage of loading soluble N onto the nascent RNA strand as it exits the polymerase during RNA replication. Interfering with the oligomerization of P may represent a general strategy to interfere with NNS RNA virus replication.

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

2018 ◽  
Vol 93 (5) ◽  
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.

scholarly journals Vesicular Stomatitis Virus: From Agricultural Pathogen to Vaccine Vector

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1092
Author(s):  
Guodong Liu ◽  
Wenguang Cao ◽  
Abdjeleel Salawudeen ◽  
Wenjun Zhu ◽  
Karla Emeterio ◽  
...  
Keyword(s):  
Vesicular Stomatitis Virus ◽  
Reverse Genetics ◽  
Ebola Virus ◽  
Rna Virus ◽  
Research Tool ◽  
Vaccine Vector ◽  
The Family ◽  
Vesicular Stomatitis ◽  
Negative Sense ◽  
Family Rhabdoviridae

Vesicular stomatitis virus (VSV), which belongs to the Vesiculovirus genus of the family Rhabdoviridae, is a well studied livestock pathogen and prototypic non-segmented, negative-sense RNA virus. Although VSV is responsible for causing economically significant outbreaks of vesicular stomatitis in cattle, horses, and swine, the virus also represents a valuable research tool for molecular biologists and virologists. Indeed, the establishment of a reverse genetics system for the recovery of infectious VSV from cDNA transformed the utility of this virus and paved the way for its use as a vaccine vector. A highly effective VSV-based vaccine against Ebola virus recently received clinical approval, and many other VSV-based vaccines have been developed, particularly for high-consequence viruses. This review seeks to provide a holistic but concise overview of VSV, covering the virus’s ascension from perennial agricultural scourge to promising medical countermeasure, with a particular focus on vaccines.

scholarly journals Adding Genes to the RNA Genome of Vesicular Stomatitis Virus: Positional Effects on Stability of Expression

2002 ◽  
Vol 76 (15) ◽  
pp. 7642-7650 ◽  
Author(s):  
Gail W. Wertz ◽  
Robin Moudy ◽  
L. Andrew Ball
Keyword(s):  
Gene Expression ◽  
Vesicular Stomatitis Virus ◽  
Rna Viruses ◽  
Transcriptional Unit ◽  
N Gene ◽  
Wild Type ◽  
Control Of Gene Expression ◽  
Negative Strand ◽  
Transcriptional Termination ◽  
Vesicular Stomatitis

ABSTRACT Gene expression of the nonsegmented negative strand (NNS) RNA viruses is controlled primarily at the level of transcription by the position of the genes relative to the single transcriptional promoter. We tested this principle by generating engineered variants of vesicular stomatitis virus in which an additional, identical, transcriptional unit was added to the genome at each of the viral gene junctions. Analysis of transcripts confirmed that the level of transcription was determined by the position of the gene relative to the promoter. However, the position at which a gene was inserted affected the replication potential of the viruses. Adding a gene between the first two genes, N and P, reduced replication by over an order of magnitude, whereas addition of a gene at the other gene junctions had no effect on replication levels. All genes downstream of the inserted gene had decreased levels of expression, since transcription of the extra gene introduced an additional transcriptional attenuation event. The added gene was stably maintained in the genome upon repeated passage in all cases. However, expression of the added gene was stable at only three of the four positions. In the case of insertion between the N and P genes, a virus population arose within two passages that had restored replication to wild-type levels. In this population, expression of the additional gene as a monocistronic mRNA was suppressed by mutations at the end of the upstream (N) gene that abolished transcriptional termination. Because transcription is obligatorily sequential, this prevented transcription of the inserted downstream gene as a monocistronic mRNA and resulted instead in polymerase reading through the gene junction to produce a bicistronic mRNA. This eliminated the additional attenuation step and restored expression of all downstream genes and viral replication to wild-type levels. These data show that transcriptional termination is a key element in control of gene expression of the negative strand RNA viruses and a means by which expression of individual genes may be regulated within the framework of a single transcriptional promoter. Further, these results are directly relevant to the use of NNS viruses as vectors and vaccine delivery agents, as they show that the level of expression of an added gene can be controlled by its insertion position but that not all positions of insertion yield stable expression of the added gene.

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

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.

scholarly journals Nucleocapsid Structure of Negative Strand RNA Virus

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 835 ◽  
Author(s):  
Ming Luo ◽  
James Ross Terrell ◽  
Shelby Ashlyn Mcmanus
Keyword(s):  
Nucleocapsid Protein ◽  
Rna Synthesis ◽  
Ebola Virus ◽  
Rna Virus ◽  
Viral Rna ◽  
Human Pathogens ◽  
Negative Strand ◽  
Rna Genome ◽  
Rna Complex ◽  
Unique Mechanism

Negative strand RNA viruses (NSVs) include many important human pathogens, such as influenza virus, Ebola virus, and rabies virus. One of the unique characteristics that NSVs share is the assembly of the nucleocapsid and its role in viral RNA synthesis. In NSVs, the single strand RNA genome is encapsidated in the linear nucleocapsid throughout the viral replication cycle. Subunits of the nucleocapsid protein are parallelly aligned along the RNA genome that is sandwiched between two domains composed of conserved helix motifs. The viral RNA-dependent-RNA polymerase (vRdRp) must recognize the protein–RNA complex of the nucleocapsid and unveil the protected genomic RNA in order to initiate viral RNA synthesis. In addition, vRdRp must continuously translocate along the protein–RNA complex during elongation in viral RNA synthesis. This unique mechanism of viral RNA synthesis suggests that the nucleocapsid may play a regulatory role during NSV replication.

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