scholarly journals Viral RNA switch mediates the dynamic control of flavivirus replicase recruitment by genome cyclization

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Zhong-Yu Liu ◽  
Xiao-Feng Li ◽  
Tao Jiang ◽  
Yong-Qiang Deng ◽  
Qing Ye ◽  
...  
Keyword(s):  
Virus Replication ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Virus ◽  
Dynamic Control ◽  
Viral Rna ◽  
Human Pathogens ◽  
Cis Acting ◽  
Viral Replicase ◽  
Rna Switch

Viral replicase recruitment and long-range RNA interactions are essential for RNA virus replication, yet the mechanism of their interplay remains elusive. Flaviviruses include numerous important human pathogens, e.g., dengue virus (DENV) and Zika virus (ZIKV). Here, we revealed a highly conserved, conformation-tunable cis-acting element named 5′-UAR-flanking stem (UFS) in the flavivirus genomic 5′ terminus. We demonstrated that the UFS was critical for efficient NS5 recruitment and viral RNA synthesis in different flaviviruses. Interestingly, stabilization of the DENV UFS impaired both genome cyclization and vRNA replication. Moreover, the UFS unwound in response to genome cyclization, leading to the decreased affinity of NS5 for the viral 5′ end. Thus, we propose that the UFS is switched by genome cyclization to regulate dynamic RdRp binding for vRNA replication. This study demonstrates that the UFS enables communication between flavivirus genome cyclization and RdRp recruitment, highlighting the presence of switch-like mechanisms among RNA viruses.

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 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.

Viral RNA structural equilibria and their interactions with host proteins

2019 ◽  
Author(s):  
◽  
Samantha Elizabeth Brady
Keyword(s):  
Reverse Transcription ◽  
Rna Structure ◽  
Drug Targets ◽  
Rna Viruses ◽  
Rna Virus ◽  
Protein Translation ◽  
Viral Rna ◽  
Primer Binding Site ◽  
In Vitro Techniques

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Understanding viral RNA structure and how it functions is crucial in elucidating new drug targets. There are many kinds of viruses that utilize RNA as a critical component of their life cycle, such as retroviruses, single-stranded plus or minus sense RNA viruses, and double-stranded RNA viruses. Two viruses that are studied in this thesis are human immunodeficiency virus (HIV), which is a retrovirus, and hepatitis C virus (HCV), which is a single-stranded plus sense RNA virus. It has been previously reported that a human host factor, RNA helicase A (RHA), is packaged into HIV virions by binding to the primer binding site (PBS) segment of the 5'untranslated region in the HIV genomic RNA. We determined RHA is required for efficient reverse transcription prior to capsid uncoating by utilizing cell based and in vitro techniques. It has also been suggested that RHA plays other roles during HIV infection besides reverse transcription. Utilizing NMR, we demonstrated that RHA binds to the monomeric 5'UTR at the bottom of the TAR hairpin, which is different from how it binds during viral packaging. Next, we employed NMR techniques to probe the 3'end of the HCV genome called 3'X. We determined that the 3'X is in structural equilibrium between two states: an open conformation and a closed conformation. These two conformations have been suggested to play a role in minus sense synthesis and viral protein translation, respectively. Taken together, my thesis work has elucidated how many viruses manipulate and utilize their RNA structure to modulate their outcome.

scholarly journals Mutational Analysis of the Rubella Virus Nonstructural Polyprotein and Its Cleavage Products in Virus Replication and RNA Synthesis

2000 ◽  
Vol 74 (11) ◽  
pp. 5133-5141 ◽  
Author(s):  
Yuying Liang ◽  
Shirley Gillam
Keyword(s):  
Virus Replication ◽  
Rubella Virus ◽  
Rna Synthesis ◽  
Rnase Protection Assay ◽  
Viral Rna ◽  
Nonstructural Proteins ◽  
Rnase Protection ◽  
Positive Strand ◽  
Negative Strand ◽  
Positive Strand Rna

ABSTRACT Rubella virus nonstructural proteins, translated from input genomic RNA as a p200 polyprotein and subsequently processed into p150 and p90 by an intrinsic papain-like thiol protease, are responsible for virus replication. To examine the effect of p200 processing on virus replication and to study the roles of nonstructural proteins in viral RNA synthesis, we introduced into a rubella virus infectious cDNA clone a panel of mutations that had variable defective effects on p200 processing. The virus yield and viral RNA synthesis of these mutants were examined. Mutations that completely abolished (C1152S and G1301S) or largely abolished (G1301A) cleavage of p200 resulted in noninfectious virus. Mutations that partially impaired cleavage of p200 (R1299A and G1300A) decreased virus replication. An RNase protection assay revealed that all of the mutants synthesized negative-strand RNA as efficiently as the wild type does but produced lower levels of positive-strand RNA. Our results demonstrated that processing of rubella virus nonstructural protein is crucial for virus replication and that uncleaved p200 could function in negative-strand RNA synthesis, whereas the cleavage products p150 and p90 are required for efficient positive-strand RNA synthesis.

scholarly journals Hsp90 is required for snakehead vesiculovirus replication via stabilizing the viral L protein

2021 ◽  
Author(s):  
Yanwei Zhang ◽  
Yong-An Zhang ◽  
Jiagang Tu
Keyword(s):  
Rna Synthesis ◽  
Rna Viruses ◽  
Insoluble Fraction ◽  
Viral Rna ◽  
Cellular Proteins ◽  
Economic Losses ◽  
L Protein ◽  
Hsp90 Activity ◽  
The Stability

Snakehead vesiculovirus (SHVV), a kind of fish rhabdovirus isolated from diseased hybrid snakehead fish, has caused great economic losses in snakehead fish culture in China. The large (L) protein, together with its cofactor phosphoprotein (P), forms a P/L polymerase complex and catalyzes the transcription and replication of viral genomic RNA. In this study, the cellular heat shock protein 90 (Hsp90) was identified as an interacting partner of SHVV L protein. The Hsp90 activity was required for the stability of SHVV L because Hsp90 dysfunction by using its inhibitor destabilized SHVV L and thereby suppressed SHVV replication via reducing viral RNA synthesis. SHVV L expressed alone was detected mainly in the insoluble fraction and the insoluble L was degraded by Hsp90 dysfunction through the proteasomal pathway, while the presence of SHVV P promoted the solubility of SHVV L and the soluble L was degraded by Hsp90 dysfunction through the autophagy pathway. Collectively, our data suggest that Hsp90 contributes to the maturation of SHVV L and ensure the effective replication of SHVV, which exhibits an important anti-SHVV target. This study will help understand the role of Hsp90 in stabilizing the L protein and regulating the replication of negative-stranded RNA viruses. Importance It has long been proposed that cellular proteins are involved in viral RNA synthesis via interacting with the viral polymerase protein. This study focused on identifying cellular proteins interacting with the SHVV L protein, studying the effects of their interactions on SHVV replication, and revealing the underlying mechanisms. We identified Hsp90 as an interacting partner of SHVV L and found that Hsp90 activity was required for SHVV replication. Hsp90 functioned in maintaining the stability of SHVV L. Inhibition of Hsp90 activity with its inhibitor degraded SHVV L through different pathways based on the solubility of SHVV L due to the presence or absence of SHVV P. Our data provide important insights into the role of Hsp90 in SHVV polymerase maturation, which will help understand the polymerase function of negative-stranded RNA viruses.

scholarly journals A Genotype-to-Phenotype Modeling Framework to Predict Human Pathogenicity of Novel Coronaviruses

2021 ◽  
Author(s):  
Phillip Davis ◽  
Joseph A Russell
Keyword(s):  
Rna Viruses ◽  
Rna Virus ◽  
Human Pathogens ◽  
Sequencing Data ◽  
Genome Sequences ◽  
Modeling Framework ◽  
Strong Argument ◽  
Genomic Epidemiology ◽  
Phenotype Model ◽  
Level Information

Leveraging prior viral genome sequencing data to make predictions on whether an unknown, emergent virus harbors a phenotype-of-concern has been a long-sought goal of genomic epidemiology. A predictive phenotype model built from nucleotide-level information alone has previously been considered un-tenable with respect to RNA viruses due to the ultra-high intra-sequence variance of their genomes, even within closely related clades. Building from our prior work developing a degenerate k-mer method to accommodate this high intra-sequence variation of RNA virus genomes for modeling frameworks, and leveraging a taxonomic group-shuffle-split paradigm on complete coronavirus assemblies from prior to October 2018, we trained multiple regularized logistic regression classifiers at the nucleotide k-mer level capable of accurately predicting withheld SARS-CoV-2 genome sequences as human pathogens and accurately predicting withheld Swine Acute Diarrhea Syndrome coronavirus (SADS-CoV) genome sequences as non-human pathogens. LASSO feature selection identified several degenerate nucleotide predictor motifs with high model coefficients for the human pathogen class that were present across widely disparate classes of coronaviruses. However, these motifs differed in which genes they were present in, what specific codons were used to encode them, and what the translated amino acid motif was. This emphasizes the importance of a phenetic view of emerging pathogenic RNA viruses, as opposed to the canonical phylogenetic interpretations most-commonly used to track and manage viral zoonoses. Applying our model to more recent Orthocoronavirinae genomes deposited since October 2018 yields a novel contextual view of pathogen-potential across bat-related, canine-related, porcine-related, and rodent-related coronaviruses and critical adaptations which may have contributed to the emergence of the pandemic SARS-CoV-2 virus. Finally, we discuss the utility of these predictive models (and their associated predictor motifs) to novel biosurveillance protocols that substantially increase the pound-for-pound information content of field-collected sequencing data and make a strong argument for the necessity of routine collection and sequencing of zoonotic viruses.

scholarly journals Determination of host proteins composing the microenvironment of coronavirus replicase complexes by proximity-labeling

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Philip V'kovski ◽  
Markus Gerber ◽  
Jenna Kelly ◽  
Stephanie Pfaender ◽  
Nadine Ebert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Host Factors ◽  
Viral Rna ◽  
Host Proteins ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Starting Point ◽  
Positive Strand Rna

Positive-sense RNA viruses hijack intracellular membranes that provide niches for viral RNA synthesis and a platform for interactions with host proteins. However, little is known about host factors at the interface between replicase complexes and the host cytoplasm. We engineered a biotin ligase into a coronaviral replication/transcription complex (RTC) and identified >500 host proteins constituting the RTC microenvironment. siRNA-silencing of each RTC-proximal host factor demonstrated importance of vesicular trafficking pathways, ubiquitin-dependent and autophagy-related processes, and translation initiation factors. Notably, detection of translation initiation factors at the RTC was instrumental to visualize and demonstrate active translation proximal to replication complexes of several coronaviruses. Collectively, we establish a spatial link between viral RNA synthesis and diverse host factors of unprecedented breadth. Our data may serve as a paradigm for other positive-strand RNA viruses and provide a starting point for a comprehensive analysis of critical virus-host interactions that represent targets for therapeutic intervention.

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 In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Dongdong Cao ◽  
Yunrong Gao ◽  
Claire Roesler ◽  
Samantha Rice ◽  
Paul D'Cunha ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fine Mapping ◽  
Rna Synthesis ◽  
De Novo ◽  
Rna Viruses ◽  
Rna Virus ◽  
Promoter Sequence ◽  
Mechanistic Understanding ◽  
Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis. IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

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