Up Close and Personal With SARS-CoV-2's Replication Machinery / Da vicino e personale con il macchinario di replica di SARS-CoV-2
Up Close and Personal With SARS-CoV-2's Replication Machinery / Da vicino e personale con il macchinario di replica di SARS-CoV-2
Segnalato dal Dott. Giuseppe Cotellessa / Reported by Dr. Giuseppe Cotellessa
When a viral pathogen invades, it enters the host cells and must rapidly divide to survive. In order to divide, it is required to multiply its genetic material – long strands of RNA. The machinery responsible for performing this task is known as the viral polymerase.
Scientists are working to understand the structure and function of the viral polymerase; block the replication machinery, block the virus's ability to continue to infect the host. This is the premise behind antiviral drugs which bind to and inhibit viral polymerases.
Structural biology can lend a hand here. Advances in techniques such as cryo-electron microscopy (Cryo-EM) are progressing the at a rapid pace, enabling scientists to accurately determine the 3D structures of proteins in a more efficient manner. Of course, in a pandemic, this is key.
A preprint study published via the online server bioRxiv outlines the efforts of a team of scientists, led by Patrick Cramer at the Max Planck Institute for Biophysical Chemistry in Göttingen, in determining the 3D structure of the novel coronavirus polymerase.
Technology Networks spoke with Cramer to learn more about the study, it's relevance in developing therapeutics against SARS-CoV-2 and to gain his insights on the value of preprints in a pandemic.
Molly Campbell (MC): For our readers that may be unfamiliar, please can you tell us about your lab's research focus?
Patrick Cramer: We are combining in vitro and in vivo approaches to study gene transcription and the regulation of genes in human cells. In particular, we develop and use integrated structural biology to analyze the structure of large assemblies of protein that are involved in transcription.
Scientists are working to understand the structure and function of the viral polymerase; block the replication machinery, block the virus's ability to continue to infect the host. This is the premise behind antiviral drugs which bind to and inhibit viral polymerases.
Structural biology can lend a hand here. Advances in techniques such as cryo-electron microscopy (Cryo-EM) are progressing the at a rapid pace, enabling scientists to accurately determine the 3D structures of proteins in a more efficient manner. Of course, in a pandemic, this is key.
A preprint study published via the online server bioRxiv outlines the efforts of a team of scientists, led by Patrick Cramer at the Max Planck Institute for Biophysical Chemistry in Göttingen, in determining the 3D structure of the novel coronavirus polymerase.
Technology Networks spoke with Cramer to learn more about the study, it's relevance in developing therapeutics against SARS-CoV-2 and to gain his insights on the value of preprints in a pandemic.
Molly Campbell (MC): For our readers that may be unfamiliar, please can you tell us about your lab's research focus?
Patrick Cramer: We are combining in vitro and in vivo approaches to study gene transcription and the regulation of genes in human cells. In particular, we develop and use integrated structural biology to analyze the structure of large assemblies of protein that are involved in transcription.
MC: You recently published a preprint study in which you have determined the 3D structures of the corona polymerase. Why it was important to you to determine the polymerase's structure?
PC: In the light of the ongoing pandemic, we wanted to help. We decided to use our experience in polymerase biology and structure-function analysis to investigate how the virus replicates its genetic material and how it transcribes its genes.
It is important that we got the polymerase structure because it is the target of antiviral substances, including remdesivir, which is currently being tested in the clinic. Next, we will investigate the mechanism that remdesivir uses to block the corona polymerase and prevent duplication of its genome.
MC: Can you tell us what you discovered about the corona polymerase through studying its structure?
PC: The core structure was known, but we did not know what the accessory factors, in particular the nsp8 subunits, really do. We resolved them here for the first time in the context of the complete replicating polymerase.
We found that they make very extensive interactions with the RNA that exits from the enzyme. Comparison with the literature suggests these long nsp8 extensions are important for holding onto RNA to make replication processive. This is important to replicate the entire genome, and to prevent premature dissociation of the polymerase from RNA.
MC: What research techniques did you utilize to determine the 3D structure of the corona polymerase?
PC: Cryo-electron microscopy. This technique does not require you to form protein crystals, as is required for X-ray analysis.
MC: What challenges did you encounter in the study, and how did you overcome them?
PC: Other groups also tried to see the entire enzyme but always got to see only its core part. We were lucky in that we accidentally assembled a longer RNA molecule that then stabilizes the newly observed regions. Hard work and making use of our experience in preparing proteins, measuring their activity and solving their structures helped us in this study.
MC: In the press release, you say: "The determination of the polymerase structure will not be the last contribution of the Göttingen researchers to tackling the pandemic" – Can you tell us about your next steps in this research space?
PC: We will investigate biochemically how the drug candidate remdesivir, an antiviral compound, will block replication. Then we try to visualize the polymerase with bound remdesivir, to understand its detailed mechanism. This will hopefully lead to new insights that can be used to try to improve antiviral substances.
MC: How has the COVID-19 outbreak impacted your laboratory's research?
PC: Most of us are still working in a home office. Many can process data from home and thus can to some extent be productive, others look forward to return to the lab, but – safety first.
MC: The study is published in its current format as a preprint. In your opinion, what is the value of sharing research prior to peer-review in the current climate?
PC: It is essential. We have already got requests for materials and data that others need for progressing in their research. I was surprised that the journal where we submitted the work for consideration even asked us to post a preprint online. Science moves at an incredible pace these days.
PC: In the light of the ongoing pandemic, we wanted to help. We decided to use our experience in polymerase biology and structure-function analysis to investigate how the virus replicates its genetic material and how it transcribes its genes.
It is important that we got the polymerase structure because it is the target of antiviral substances, including remdesivir, which is currently being tested in the clinic. Next, we will investigate the mechanism that remdesivir uses to block the corona polymerase and prevent duplication of its genome.
MC: Can you tell us what you discovered about the corona polymerase through studying its structure?
PC: The core structure was known, but we did not know what the accessory factors, in particular the nsp8 subunits, really do. We resolved them here for the first time in the context of the complete replicating polymerase.
We found that they make very extensive interactions with the RNA that exits from the enzyme. Comparison with the literature suggests these long nsp8 extensions are important for holding onto RNA to make replication processive. This is important to replicate the entire genome, and to prevent premature dissociation of the polymerase from RNA.
MC: What research techniques did you utilize to determine the 3D structure of the corona polymerase?
PC: Cryo-electron microscopy. This technique does not require you to form protein crystals, as is required for X-ray analysis.
MC: What challenges did you encounter in the study, and how did you overcome them?
PC: Other groups also tried to see the entire enzyme but always got to see only its core part. We were lucky in that we accidentally assembled a longer RNA molecule that then stabilizes the newly observed regions. Hard work and making use of our experience in preparing proteins, measuring their activity and solving their structures helped us in this study.
MC: In the press release, you say: "The determination of the polymerase structure will not be the last contribution of the Göttingen researchers to tackling the pandemic" – Can you tell us about your next steps in this research space?
PC: We will investigate biochemically how the drug candidate remdesivir, an antiviral compound, will block replication. Then we try to visualize the polymerase with bound remdesivir, to understand its detailed mechanism. This will hopefully lead to new insights that can be used to try to improve antiviral substances.
MC: How has the COVID-19 outbreak impacted your laboratory's research?
PC: Most of us are still working in a home office. Many can process data from home and thus can to some extent be productive, others look forward to return to the lab, but – safety first.
MC: The study is published in its current format as a preprint. In your opinion, what is the value of sharing research prior to peer-review in the current climate?
PC: It is essential. We have already got requests for materials and data that others need for progressing in their research. I was surprised that the journal where we submitted the work for consideration even asked us to post a preprint online. Science moves at an incredible pace these days.
ITALIANO
Quando un patogeno virale invade, entra nelle cellule ospiti e deve rapidamente dividersi per sopravvivere. Per dividere, è necessario moltiplicare il suo materiale genetico - lunghi filamenti di RNA. Il macchinario responsabile dell'esecuzione di questo compito è noto come la polimerasi virale.
Gli scienziati stanno lavorando per comprendere la struttura e la funzione della polimerasi virale; bloccare il meccanismo di replica, bloccare la capacità del virus di continuare a infettare l'host. Questa è la premessa alla base dei farmaci antivirali che si legano e inibiscono le polimerasi virali.
La biologia strutturale può dare una mano qui. I progressi in tecniche come la microscopia crioelettronica (Cryo-EM) stanno procedendo rapidamente, consentendo agli scienziati di determinare con precisione le strutture 3D delle proteine in modo più efficiente. Naturalmente, in una pandemia, questa è la chiave.
Uno studio di prestampa pubblicato tramite il server online bioRxiv delinea gli sforzi di un team di scienziati, guidato da Patrick Cramer del Max Planck Institute for Biophysical Chemistry di Göttingen, nel determinare la struttura 3D della nuova coronavirus polimerasi.
Technology Networks ha parlato con Cramer per saperne di più sullo studio, la sua rilevanza nello sviluppo di terapie contro la SARS-CoV-2 e per ottenere le sue conoscenze sul valore delle prestampe in una pandemia.
Molly Campbell (MC): Per i nostri lettori che potrebbero non avere familiarità, puoi parlarci del focus di ricerca del tuo laboratorio?
Patrick Cramer: Stiamo combinando approcci in vitro e in vivo per studiare la trascrizione genica e la regolazione dei geni nelle cellule umane. In particolare, sviluppiamo e utilizziamo la biologia strutturale integrata per analizzare la struttura di grandi assiemi di proteine coinvolti nella trascrizione.
MC: Hai recentemente pubblicato uno studio di prestampa in cui hai determinato le strutture 3D della corona polimerasi. Perché è stato importante per te determinare la struttura della polimerasi?
PC: Alla luce della pandemia in corso, volevamo aiutare. Abbiamo deciso di utilizzare la nostra esperienza nella biologia della polimerasi e nell'analisi struttura-funzione per studiare come il virus replica il suo materiale genetico e come trascrive i suoi geni.
È importante che abbiamo ottenuto la struttura della polimerasi perché è il bersaglio di sostanze antivirali, incluso remdesivir, che è attualmente in fase di test in clinica. Successivamente, esamineremo il meccanismo che remdesivir utilizza per bloccare la corona polimerasi e prevenire la duplicazione del suo genoma.
MC: Puoi dirci cosa hai scoperto sulla corona polimerasi studiando la sua struttura?
PC: La struttura principale era nota, ma non sapevamo quali fattori accessori, in particolare le subunità nsp8, facessero davvero. Li abbiamo risolti qui per la prima volta nel contesto della completa polimerasi replicante.
Abbiamo scoperto che fanno interazioni molto estese con l'RNA che esce dall'enzima. Il confronto con la letteratura suggerisce che queste lunghe estensioni nsp8 sono importanti per trattenere l'RNA per rendere la replicazione processuale. Questo è importante per replicare l'intero genoma e prevenire la dissociazione prematura della polimerasi dall'RNA.
MC: Quali tecniche di ricerca hai utilizzato per determinare la struttura 3D della corona polimerasi?
PC: microscopia crioelettronica. Questa tecnica non richiede la formazione di cristalli proteici, come è richiesto per l'analisi dei raggi X.
MC: Quali sfide hai incontrato nello studio e come le hai superate?
PC: Anche altri gruppi hanno provato a vedere l'intero enzima ma hanno sempre visto solo la sua parte principale. Siamo stati fortunati a aver assemblato accidentalmente una molecola di RNA più lunga che stabilizzava le regioni appena osservate. Il duro lavoro e l'utilizzo della nostra esperienza nella preparazione delle proteine, nella misurazione della loro attività e nella risoluzione delle loro strutture ci hanno aiutato in questo studio.
MC: Nel comunicato stampa, dici: "La determinazione della struttura della polimerasi non sarà l'ultimo contributo dei ricercatori di Gottinga per affrontare la pandemia" - Puoi parlarci dei tuoi prossimi passi in questo spazio di ricerca?
PC: Indagheremo biochimicamente su come il remdesivir candidato al farmaco un composto antivirale, bloccherà la replicazione. Quindi proviamo a visualizzare la polimerasi con remdesivir legato, per comprenderne il meccanismo dettagliato. Si spera che questo porti a nuove intuizioni che possono essere utilizzate per cercare di migliorare le sostanze antivirali.
MC: In che modo l'epidemia COVID-19 ha influito sulla ricerca del tuo laboratorio?
PC: la maggior parte di noi lavora ancora in un ufficio. Molti possono elaborare i dati da casa e quindi in una certa misura possono essere produttivi, altri non vedono l'ora di tornare in laboratorio, ma prima la sicurezza.
MC: Lo studio è pubblicato nel suo formato attuale come prestampa. Secondo te, qual è il valore della condivisione della ricerca prima della revisione tra pari nell'attuale clima?
PC: è essenziale. Abbiamo già ricevuto richieste di materiali e dati di cui altri hanno bisogno per progredire nella loro ricerca. Sono rimasto sorpreso dal fatto che la rivista in cui abbiamo presentato il lavoro in esame ci abbia persino chiesto di pubblicare una prestampa online. La scienza si muove a un ritmo incredibile in questi giorni.
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