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 Releasing the Genomic RNA Sequestered in the Mumps Virus Nucleocapsid

2016 ◽  
Vol 90 (22) ◽  
pp. 10113-10119 ◽  
Author(s):  
Chelsea Severin ◽  
James R. Terrell ◽  
James R. Zengel ◽  
Robert Cox ◽  
Richard K. Plemper ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Nucleocapsid Protein ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Mumps Virus ◽  
Viral Rna ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Negative Strand ◽  
Viral Rdrp

ABSTRACT In a negative-strand RNA virus, the genomic RNA is sequestered inside the nucleocapsid when the viral RNA-dependent RNA polymerase uses it as the template for viral RNA synthesis. It must require a conformational change in the nucleocapsid protein (N) to make the RNA accessible to the viral polymerase during this process. The structure of an empty mumps virus (MuV) nucleocapsid-like particle was determined to 10.4-Å resolution by cryo-electron microscopy (cryo-EM) image reconstruction. By modeling the crystal structure of parainfluenza virus 5 into the density, it was shown that the α-helix close to the RNA became flexible when RNA was removed. Point mutations in this helix resulted in loss of polymerase activities. Since the core of N is rigid in the nucleocapsid, we suggest that interactions between this region of the mumps virus N and its polymerase, instead of large N domain rotations, lead to exposure of the sequestered genomic RNA. IMPORTANCE Mumps virus (MuV) infection may cause serious diseases, including hearing loss, orchitis, oophoritis, mastitis, and pancreatitis. MuV is a negative-strand RNA virus, similar to rabies virus or Ebola virus, that has a unique mechanism of viral RNA synthesis. They all make their own RNA-dependent RNA polymerase (RdRp). The viral RdRp uses the genomic RNA inside the viral nucleocapsid as the template to synthesize viral RNAs. Since the template RNA is always sequestered in the nucleocapsid, the viral RdRp must find a way to open it up in order to gain access to the covered template. Our work reported here shows that a helix structural element in the MuV nucleocapsid protein becomes open when the sequestered RNA is released. The amino acids related to this helix are required for RdRp to synthesize viral RNA. We propose that the viral RdRp pulls this helix open to release the genomic RNA.

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 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 Identification of guanylyltransferase activity in the SARS-CoV-2 RNA polymerase

2021 ◽  
Author(s):  
Alexander P Walker ◽  
Haitian Fan ◽  
Jeremy R Keown ◽  
Jonathan M Grimes ◽  
Ervin Fodor
Keyword(s):  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Active Form ◽  
Antiviral Drug ◽  
Viral Rna ◽  
Protein Domain ◽  
Structural Protein ◽  
Viral Mrnas ◽  
Synthesis Mechanism

AbstractSARS-CoV-2 is a positive-sense RNA virus that is responsible for the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors that are involved in RNA proofreading and 5’ capping of viral mRNAs. The formation of the 5’ cap-1 structure is known to require a guanylyltransferase (GTase) as well as 5’ triphosphatase and methyltransferase activities. However, the mechanism of SARS-CoV-2 mRNA capping remains poorly understood. Here we show that the SARS-CoV-2 RNA polymerase nsp12 functions as a GTase. We characterise this GTase activity and find that the nsp12 NiRAN (nidovirus RdRP-associated nucleotidyltransferase) domain is responsible for carrying out the addition of a GTP nucleotide to the 5’ end of viral RNA via a 5’ to 5’ triphosphate linkage. We also show that remdesivir triphosphate, the active form of the antiviral drug remdesivir, inhibits the SARS-CoV-2 GTase reaction as efficiently as RNA polymerase activity. These data improve understanding of coronavirus mRNA cap synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.ImportanceSARS-CoV-2 is a respiratory RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Coronaviruses encode an RNA polymerase which, in combination with other viral proteins, is responsible for synthesising capped viral mRNA. mRNA cap synthesis requires a guanylyltransferase enzyme; here we show that the SARS-CoV-2 guanylyltransferase is located in the viral RNA polymerase, and we identify the protein domain responsible for guanylyltransferase activity. Furthermore we demonstrate that remdesivir triphosphate, the active metabolite of remdesivir, inhibits both the guanylyltransferase and RNA polymerase functions of the SARS-CoV-2 RNA polymerase. These findings improve understanding of the coronavirus mRNA cap synthesis mechanism, in addition to highlighting a new target for the development of therapeutics to treat SARS-CoV-2 infection.

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 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 Nucleolar Targeting of Hepatitis Delta Antigen Abolishes Its Ability To Initiate Viral Antigenomic RNA Replication

2007 ◽  
Vol 82 (2) ◽  
pp. 692-699 ◽  
Author(s):  
Wen-Hung Huang ◽  
Yen-Shun Chen ◽  
Pei-Jer Chen
Keyword(s):  
Subcellular Localization ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Synthesis ◽  
Rna Virus ◽  
Rna Replication ◽  
Genomic Rna ◽  
Hepatitis Delta ◽  
Delta Antigen ◽  
Hepatitis Delta Antigen

ABSTRACT Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.

scholarly journals Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Nidhi Kaushik ◽  
Chandru Subramani ◽  
Saumya Anang ◽  
Rajagopalan Muthumohan ◽  
Shalimar ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Hepatitis E Virus ◽  
Zinc Supplementation ◽  
Hepatitis E ◽  
Viral Rna ◽  
Health Concern ◽  
Healthy Individuals ◽  
Genomic Rna ◽  
Rna Dependent Rna Polymerase ◽  
Zinc Salts

ABSTRACT Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection. IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

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