Global S&T Development Trend Analysis Platform of Resources and Environment
Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate inEscherichia coliowing to the size and occasional instability of the genome(1-3). Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of theCoronaviridae,FlaviviridaeandPneumoviridaefamilies. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step inSaccharomyces cerevisiaeusing transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)(4), which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.
A yeast-based synthetic genomics platform is used to reconstruct and characterize large RNA viruses from synthetic DNA fragments
Sea-level histories during the two most recent deglacial-interglacial intervals show substantial differences(1-3) despite both periods undergoing similar changes in global mean temperature(4,5) and forcing from greenhouse gases(6). Although the last interglaciation (LIG) experienced stronger boreal summer insolation forcing than the present interglaciation(7), understanding why LIG global mean sea level may have been six to nine metres higher than today has proven particularly challenging(2). Extensive areas of polar ice sheets were grounded below sea level during both glacial and interglacial periods, with grounding lines and fringing ice shelves extending onto continental shelves(8). This suggests that oceanic forcing by subsurface warming may also have contributed to ice-sheet loss(9-12) analogous to ongoing changes in the Antarctic(13,14) and Greenland(15) ice sheets. Such forcing would have been especially effective during glacial periods, when the Atlantic Meridional Overturning Circulation (AMOC) experienced large variations on millennial timescales(16), with a reduction of the AMOC causing subsurface warming throughout much of the Atlantic basin(9,12,17). Here we show that greater subsurface warming induced by the longer period of reduced AMOC during the penultimate deglaciation can explain the more-rapid sea-level rise compared with the last deglaciation. This greater forcing also contributed to excess loss from the Greenland and Antarctic ice sheets during the LIG, causing global mean sea level to rise at least four metres above modern levels. When accounting for the combined influences of penultimate and LIG deglaciation on glacial isostatic adjustment, this excess loss of polar ice during the LIG can explain much of the relative sea level recorded by fossil coral reefs and speleothems at intermediate- and far-field sites.
