Specifically, we asked if a paramyxovirus and a filovirus polymerase utilized a single or multiple initiation sites, and if there are fundamental differences between these and the RSV polymerase to facilitate different initiation strategies.
We also examined back-priming activity in an attempt to gain more information regarding this enigmatic process. In the case of RSV, previous studies have found evidence that the two initiation sites dictate different outcomes, with initiation at position 1 beginning the process of replication and initiation at position 3 beginning the process of mRNA transcription [ 22 , 23 , 44 ].
Similarly, although the EBOV polymerase initiates at position 2 rather than position 1, it is possible that position 2 is its transcription and replication initiation site. The evolution of promoters with a single initiation site versus two initiation sites could be significant for understanding the mechanisms by which the balance of transcription versus replication is regulated during infection.
A long-standing model of nsNSV transcription and replication is that the polymerase initiates at position 1 of the le promoter region for both processes [ 45 — 47 ]. According to this model, commitment to transcription versus replication occurs depending on whether the polymerase releases the positive sense le transcript and reinitiates at the first gene start signal to begin transcription, or continues to elongate the nascent RNA, to begin replication.
In contrast, in the pneumoviruses, where there are two initiation sites, the commitment to transcription versus replication can occur prior to the polymerase beginning RNA synthesis and does not appear to be governed by N protein concentration [ 25 , 48 ].
The fact that the PIV-3 and MARV polymerases initiate exclusively at position 1 of their own promoters, whereas the RSV and EBOV polymerases can initiate at internal sites, raised the possibility that these different polymerases might possess different structural features to facilitate initiation.
We envisaged that polymerases that can initiate internally might either have a more flexible priming loop positioning or, in the case of the pneumoviruses, might have evolved a different mechanism to enable de novo initiation from position 3. If so, these features would not necessarily be shared with polymerases that initiate exclusively at position 1 of their own promoters. Apo structures are now available for the polymerases of VSV, rabies, RSV, HMPV, and parainfluenza virus type 5 PIV-5 [ 7 — 13 ] but at present it is not possible to relate these structures to the data presented here because the structural features required for RNA synthesis initiation are poorly defined for the pneumoviruses, paramyxoviruses and filoviruses.
The VSV and rabies virus polymerases have a priming loop that extends into the RNA dependent RNA polymerase active site, positioning a priming residue appropriately for de novo initiation [ 10 — 12 , 35 ]. In the other polymerase structures, this loop is retracted and is more integrated with the capping domain in which it also plays a functional role [ 7 — 9 , 13 ]. The priming loop that was identified in VSV and rabies virus polymerases is poorly conserved in primary amino acid sequence between nsNSVs and there is no clearly conserved priming residue [ 13 ].
Furthermore, studies with RSV failed to identify a priming residue in the same position as that of VSV and rabies virus [ 13 ]. Therefore, it is not clear where the priming loop and residue are for the paramyxoviruses or pneumoviruses or filoviruses.
Despite the difference in positioning of the intrusion loop between the paramyxovirus and pneumovirus polymerases, the data presented here show that the PIV-3 polymerase is not constrained to initiate only at position 1 and can initiate efficiently from an internal site Fig 3. Thus, VSV polymerase appears to be exceptional amongst polymerases that have been examined to date in that it cannot initiate efficiently from an internal site, whereas the other polymerases appear to be flexible, regardless of their apo structure conformation.
The results showed that the promoter sequences could play a key role in determining initiation site. It is possible that this U stretch is a polymerase binding site and that when the PIV-3 polymerase binds to the RSV promoter, this is its preferred site positioning the active site at nucleotide 3C.
Although the U-tract appears to play a key role, when the PIV-3 polymerase was provided a non-specific RNA template in which a U tract was not present, it initiated from an internal site, possibly from a UG motif that mimics positions 1 and 2 of its own promoter Fig 3E.
For example, although the PIV-3 polymerase initiated at the same sites as the RSV polymerase on the RSV le promoter, the two polymerases differed in their efficiency at initiating at position 1U, relative to position 3C Fig 3. Likewise, whereas the PIV-3 polymerase appeared to initiate exclusively at position 1U of its own promoter, the RSV polymerase initiated at internal sites on this promoter Fig 4.
A caveat to the interpretation of these results is that it is possible that the PIV-3 polymerase was highly efficient at initiating at internal sites, even on its own promoter, but could not elongate internally initiated products very efficiently. If this were the case, these internally initiated products would not be detectable if they had not incorporated the radiolabeled nucleotide.
While this caveat cannot be excluded, it seems likely that the RSV and PIV-3 polymerases do have inherent properties that help to determine their initiation sites. Another property of the pneumovirus polymerases is their propensity to use the template RNA to engage in a back-priming reaction [ 13 , 21 , 51 ].
One caveat to this experiment is that the PIV-3 polymerase was examined in on-bead assays and it is possible that it was more constrained than the RSV polymerase. However, the PIV-3 polymerase was retained on beads via a tag at the C-terminus of P, which would normally be tethered to the N-RNA template, and it was clearly capable of performing de novo initiation and elongation, indicating that L could adopt different conformations.
Therefore, we think that it is unlikely that this caveat would have affected these experimental results. It should be noted that back-priming on an RSV template has been reported for the polymerase of measles virus [ 52 ], which suggests that there might be differences between different paramyxoviruses.
The role of back-priming in RSV infection is not known, but the dominance of this activity suggests that it might play an important function. It is possible that the conformation adopted by the apo forms of the RSV and HMPV polymerases, with the priming and intrusion loops folded away from the RNA dependent RNA polymerase active site, creates space for the back-priming template structure [ 8 , 9 , 13 ].
However, the polymerases do differ in their ability to engage in back-priming activity, at least under the conditions tested, suggesting that this might reflect a fundamental difference in the structures that different nsNSV polymerases can adopt.
A mutated version of each plasmid was also generated in which the catalytic aspartic residue of the GDNQ motif in the RNA dependent RNA polymerase domain of each of the L proteins was substituted with alanine. The proteins were expressed in Sf21 insect cells. L-P complexes bound to beads were washed three times with lysis buffer containing 60 mM imidazole and once with lysis buffer containing mM imidazole.
L-P complexes were eluted from the agarose resin with lysis buffer containing mM imidazole. All isolated polymerase complexes were analyzed by SDS- PAGE and PageBlue staining Fermentas and the L protein concentration was estimated by comparing its band intensity against bovine serum albumin reference standards. Other NTP concentration variations to modulate initiation from different sites are described in the text.
The RNA was extracted with acid phenol-chloroform and ethanol precipitation. All gels were analyzed using phosphorimaging or autoradiography. Products were subjected to phenol chloroform extraction and ethanol precipitation prior to use as markers. We would like to thank Dr. NIAID had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. PLoS Pathog.
Published online Dec Afzaal M. Sean P. Whelan, Editor. Author information Article notes Copyright and License information Disclaimer. Hoffmann-La Roche Ltd. Received Sep 23; Accepted Nov This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Author summary The non-segmented negative strand RNA viruses are a large group of viruses that includes a number of significant human pathogens. Introduction The order Mononegavirales , otherwise known as the non-segmented, negative strand RNA viruses nsNSVs , is a large and diverse group of viruses that are united by having a genome consisting of a single strand of RNA that is transcribed to produce a series of mRNAs, and replicated via a positive-sense antigenome intermediate to produce new RNA genomes [ 1 ].
Results The RSV and PIV-3 polymerases differ in initiation properties on their respective promoters As noted in the Introduction section, our previous studies have shown that the RSV polymerase initiates at positions 1U and 3C on both the le and tr promoters [ 20 , 22 ].
Open in a separate window. Fig 1. Fig 2. Fig 3. Fig 4. Fig 5. Acknowledgments We would like to thank Dr. Data Availability All relevant data are within the manuscript. References 1. Arch Virol. Liang B. Structures of the Mononegavirales Polymerases. J Virol. Ogino T, Green TJ. Frontiers in microbiology. Fearns R, Deval J.
New antiviral approaches for respiratory syncytial virus and other mononegaviruses: Inhibiting the RNA polymerase. Antiviral research. Fearns R, Plemper RK.
Polymerases of paramyxoviruses and pneumoviruses. Virus research. Transcription and replication of nonsegmented negative-strand RNA viruses. Curr Top Microbiol Immunol. Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation.
Nature communications. Structure of a rabies virus polymerase complex from electron cryo-microscopy. Cell Rep. Structure of the human metapneumovirus polymerase phosphoprotein complex. Mapping the transcription and replication promoters of respiratory syncytial virus. Structure of the vesicular stomatitis virus nucleoprotein-RNA complex.
Mechanism of RNA synthesis initiation by the vesicular stomatitis virus polymerase. The EMBO journal. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Noton SL, Fearns R. Initiation and regulation of paramyxovirus transcription and replication. Evidence that the polymerase of respiratory syncytial virus initiates RNA replication in a nontemplated fashion.
The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter. PLoS pathogens. Respiratory syncytial virus polymerase can initiate transcription from position 3 of the leader promoter. RNA elongation by respiratory syncytial virus polymerase is calibrated by conserved region V.
Roles of the respiratory syncytial virus trailer region: effects of mutations on genome production and stress granule formation. Mechanism for de novo initiation at two sites in the respiratory syncytial virus promoter.
Nucleic acids research. Ebolavirus polymerase uses an unconventional genome replication mechanism. Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase. A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase.
J Biol Chem. A dual-functional priming-capping loop of rhabdoviral RNA polymerases directs terminal de novo initiation and capping intermediate formation. RNA synthesis in a cage—structural studies of reovirus polymerase lambda3. The role of the priming loop in influenza A virus RNA synthesis. Nat Microbiol. Nat Rev Microbiol. Structures of influenza A virus RNA polymerase offer insight into viral genome replication. Precise mapping of the replication and transcription promoters of human parainfluenza virus type 3.
Killing two birds with one stone: How the respiratory syncytial virus polymerase initiates transcription and replication. Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis. Steps may vary depending on your condition and your doctor's practices.
A healthcare professional will connect you to an ECG machine with leads that stick to your skin and place a blood pressure cuff on your arm. You will probably not feel anything when the tracer is given. As another option, a small amount of blood may be withdrawn from your vein so that it can be tagged with the tracer. The tracer will be added to the blood and will be absorbed into the red blood cells, then the blood will be returned into your vein through the IV.
During the test, it will be very important for you to lie as still as possible, as any movement can affect the quality of the scan. Your doctor will position the gamma camera over you as you lie on the table. It will record images of your heart as it pumps the tagged blood cells through your body. You may be asked to change positions during the test; however, once you have changed position, you will need to lie still without talking.
After the scan is done, the IV line will be removed, and you can leave, unless your doctor tells you differently. Be sure to move slowly when getting up from the scanner table to avoid any dizziness or lightheadedness from lying flat for the length of the test. Drink plenty of fluids and empty your bladder often for 24 to 48 hours after the test to help flush the remaining radioactive tracer from your body.
A nurse will check the IV site for any signs of redness or swelling. If you notice any pain, redness, or swelling at the IV site after you go home, tell your doctor as this may be a sign of infection or other type of reaction.
Health Home Treatments, Tests and Therapies. Reasons for your doctor to request a resting radionuclide angiogram RNA include: Chest pain Shortness of breath Dizziness Fatigue extreme tiredness If your doctor thinks you have some type of heart disease, a resting RNA may be done. There may be other reasons for your doctor to recommend this test. What are the risks of an RNA? Certain factors may interfere with or affect the results of this test.
These include: Caffeine intake before the procedure Smoking or using any form of tobacco before the procedure Certain heart medicines How do I prepare for an RNA? Your doctor will explain the test and you can ask questions. You may need to restrict tobacco and caffeine 2 or 3 hours before testing. Tell your doctor if you have a pacemaker or any other implanted cardiac devices. Based on your medical condition, your doctor may request other specific preparation.
What happens during an RNA? Generally, a resting RNA follows this process: You will be asked to remove any jewelry or other objects that may interfere with the test. You may need to change into a hospital gown. A healthcare professional will start an intravenous IV line in your hand or arm.
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