The P323L substitution in the SARS-CoV-2 polymerase (NSP12) confers a selective advantage during infection
Authors: Goldswain Hannah , Dong Xiaofeng , Penrice-Randal Rebekah , Alruwaili Muhannad , Shawli Ghada T. , Prince Tessa , Williamson Maia Kavanagh , Raghwani Jayna , Randle Nadine , Jones Benjamin , Donovan-Banfield I’ah , Salguero Francisco J. , Tree Julia A. , Hall Yper , Hartley Catherine , Erdmann Maximilian , Bazire James , Jearanaiwitayakul Tuksin , Semple Malcolm G. , Openshaw Peter J. M. , Baillie J. Kenneth ,   ISARIC4C Investigators, Baillie J. Kenneth , Semple Malcolm G. , Openshaw Peter J. M. , Carson Gail , Alex Beatrice , Andrikopoulos Petros , Bach Benjamin , Barclay Wendy S. , Bogaert Debby , Chand Meera , Chechi Kanta , Cooke Graham S. , da Silva Filipe Ana , de Silva Thushan , Docherty Annemarie B. , dos Santos Correia Gonçalo , Dumas Marc-Emmanuel , Dunning Jake , Fletcher Tom , Green Christoper A. , Greenhalf William , Griffin Julian L. , Gupta Rishi K. , Harrison Ewen M. , Hiscox Julian A. , Ho Antonia Ying Wai , Horby Peter W. , Ijaz Samreen , Khoo Saye , Klenerman Paul , Law Andrew , Lewis Matthew R. , Liggi Sonia , Lim Wei Shen , Maslen Lynn , Mentzer Alexander J. , Merson Laura , Meynert Alison M. , Moore Shona C. , Noursadeghi Mahdad , Olanipekun Michael , Osagie Anthonia , Palmarini Massimo , Palmieri Carlo , Paxton William A. , Pollakis Georgios , Price Nicholas , Rambaut Andrew , Robertson David L. , Russell Clark D. , Sancho-Shimizu Vanessa , Sands Caroline J. , Scott Janet T. , Sigfrid Louise , Solomon Tom , Sriskandan Shiranee , Stuart David , Summers Charlotte , Swann Olivia V. , Takats Zoltan , Takis Panteleimon , Tedder Richard S. , Thompson A. A. Roger , Thomson Emma C. , Thwaites Ryan S. , Turtle Lance C. W. , Zambon Maria , Hardwick Hayley , Donohue Chloe , Griffiths Fiona , Oosthuyzen Wilna , Donegan Cara , Spencer Rebecca G. , Norman Lisa , Pius Riinu , Drake Thomas M. , Fairfield Cameron J. , Knight Stephen R. , Mclean Kenneth A. , Murphy Derek , Shaw Catherine A. , Dalton Jo , Girvan Michelle , Saviciute Egle , Roberts Stephanie , Harrison Janet , Marsh Laura , Connor Marie , Halpin Sophie , Jackson Clare , Gamble Carrol , Plotkin Daniel , Lee James , Leeming Gary , Law Andrew , Wham Murray , Clohisey Sara , Hendry Ross , Scott-Brown James , Shaw Victoria , McDonald Sarah E. , Keating Seán , Ahmed Katie A. , Armstrong Jane A. , Ashworth Milton , Asiimwe Innocent G. , Bakshi Siddharth , Barlow Samantha L. , Booth Laura , Brennan Benjamin , Bullock Katie , Catterall Benjamin W. A. , Clark Jordan J. , Clarke Emily A. , Cole Sarah , Cooper Louise , Cox Helen , Davis Christopher , Dincarslan Oslem , Dunn Chris , Dyer Philip , Elliott Angela , Evans Anthony , Finch Lorna , Fisher Lewis W. S. , Foster Terry , Garcia-Dorival Isabel , Gunning Philip , Jensen Rebecca L. , Jones Christopher B. , Jones Trevor R. , Khandaker Shadia , King Katharine , Kiy Robyn T. , Koukorava Chrysa , Lake Annette , Lant Suzannah , Latawiec Diane , Lavelle-Langham Lara , Lefteri Daniella , Lett Lauren , Livoti Lucia A. , Mancini Maria , McDonald Sarah , McEvoy Laurence , McLauchlan John , Metelmann Soeren , Miah Nahida S. , Middleton Joanna , Mitchell Joyce , Moore Shona C. , Murphy Ellen G. , Pilgrim Jack , Reynolds Will , Ridley P. Matthew , Sales Debby , Shaw Victoria E. , Shears Rebecca K. , Small Benjamin , Subramaniam Krishanthi S. , Szemiel Agnieska , Taggart Aislynn , Tanianis-Hughes Jolanta , Thomas Jordan , Trochu Erwan , van Tonder Libby , Wilcock Eve , Zhang J. Eunice , Flaherty Lisa , Maziere Nicole , Cass Emily , Carracedo Alejandra Doce , Carlucci Nicola , Holmes Anthony , Massey Hannah , Murphy Lee , McCafferty Sarah , Clark Richard , Fawkes Angie , Morrice Kirstie , Maclean Alan , Wrobel Nicola , Donnelly Lorna , Coutts Audrey , Hafezi Katarzyna , MacGillivray Louise , Gilchrist Tammy , Adeniji Kayode , Agranoff Daniel , Agwuh Ken , Ail Dhiraj , Aldera Erin L. , Alegria Ana , Allen Sam , Angus Brian , Ashish Abdul , Atkinson Dougal , Bari Shahedal , Barlow Gavin , Barnass Stella , Barrett Nicholas , Bassford Christopher , Basude Sneha , Baxter David , Beadsworth Michael , Bernatoniene Jolanta , Berridge John , Berry Colin , Best Nicola , Bothma Pieter , Chadwick David , Brittain-Long Robin , Bulteel Naomi , Burden Tom , Burtenshaw Andrew , Caruth Vikki , Chadwick David , Chambler Duncan , Chee Nigel , Child Jenny , Chukkambotla Srikanth , Clark Tom , Collini Paul , Cosgrove Catherine , Cupitt Jason , Cutino-Moguel Maria-Teresa , Dark Paul , Dawson Chris , Dervisevic Samir , Donnison Phil , Douthwaite Sam , Drummond Andrew , DuRand Ingrid , Dushianthan Ahilanadan , Dyer Tristan , Evans Cariad , Eziefula Chi , Fegan Chrisopher , Finn Adam , Fullerton Duncan , Garg Sanjeev , Garg Sanjeev , Garg Atul , Gkrania-Klotsas Effrossyni , Godden Jo , Goldsmith Arthur , Graham Clive , Hardy Elaine , Hartshorn Stuart , Harvey Daniel , Havalda Peter , Hawcutt Daniel B. , Hobrok Maria , Hodgson Luke , Hormis Anil , Jacobs Michael , Jain Susan , Jennings Paul , Kaliappan Agilan , Kasipandian Vidya , Kegg Stephen , Kelsey Michael , Kendall Jason , Kerrison Caroline , Kerslake Ian , Koch Oliver , Koduri Gouri , Koshy George , Laha Shondipon , Laird Steven , Larkin Susan , Leiner Tamas , Lillie Patrick , Limb James , Linnett Vanessa , Little Jeff , Lyttle Mark , MacMahon Michael , MacNaughton Emily , Mankregod Ravish , Masson Huw , Matovu Elijah , McCullough Katherine , McEwen Ruth , Meda Manjula , Mills Gary , Minton Jane , Mirfenderesky Mariyam , Mohandas Kavya , Mok Quen , Moon James , Moore Elinoor , Morgan Patrick , Morris Craig , Mortimore Katherine , Moses Samuel , Mpenge Mbiye , Mulla Rohinton , Murphy Michael , Nagel Megan , Nagarajan Thapas , Nelson Mark , Norris Lillian , O’Shea Matthew K. , Otahal Igor , Ostermann Marlies , Pais Mark , Palmieri Carlo , Panchatsharam Selva , Papakonstantinou Danai , Paraiso Hassan , Patel Brij , Pattison Natalie , Pepperell Justin , Peters Mark , Phull Mandeep , Pintus Stefania , Pooni Jagtur Singh , Planche Tim , Post Frank , Price David , Prout Rachel , Rae Nikolas , Reschreiter Henrik , Reynolds Tim , Richardson Neil , Roberts Mark , Roberts Devender , Rose Alistair , Rousseau Guy , Ruge Bobby , Ryan Brendan , Saluja Taranprit , Schmid Matthias L. , Shah Aarti , Shanmuga Prad , Sharma Anil , Shawcross Anna , Sizer Jeremy , Shankar-Hari Manu , Smith Richard , Snelson Catherine , Spittle Nick , Staines Nikki , Stambach Tom , Stewart Richard , Subudhi Pradeep , Szakmany Tamas , Tatham Kate , Thomas Jo , Thompson Chris , Thompson Robert , Tridente Ascanio , Tupper-Carey Darell , Twagira Mary , Vallotton Nick , Vancheeswaran Rama , Vincent-Smith Lisa , Visuvanathan Shico , Vuylsteke Alan , Waddy Sam , Wake Rachel , Walden Andrew , Welters Ingeborg , Whitehouse Tony , Whittaker Paul , Whittington Ashley , Papineni Padmasayee , Wijesinghe Meme , Williams Martin , Wilson Lawrence , Cole Sarah , Winchester Stephen , Wiselka Martin , Wolverson Adam , Wootton Daniel G. , Workman Andrew , Yates Bryan , Young Peter , Emmett Stevan R. , Digard Paul , Matthews David A. , Turtle Lance , Darby Alistair C. , Davidson Andrew D. , Carroll Miles W. , Hiscox Julian A. 
Published: March 2023

Abstract
Background
The mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure.

Results
Investigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323.

Conclusions
These data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.