In addition to members causing milder human infections, the Coronaviridae family includes potentially lethal zoonotic agents causing severe acute respiratory syndrome (SARS) and the recently emerged Middle East respiratory syndrome. The ∼30-kb positive-stranded RNA genome of coronaviruses encodes a replication/transcription machinery that is unusually complex and composed of 16 nonstructural proteins (nsps). SARS-CoV nsp12, the canonical RNA-dependent RNA polymerase (RdRp), exhibits poorly processive RNA synthesis in vitro, at odds with the efficient replication of a very large RNA genome in vivo. Here, we report that SARS-CoV nsp7 and nsp8 activate and confer processivity to the RNA-synthesizing activity of nsp12. Using biochemical assays and reverse genetics, the importance of conserved nsp7 and nsp8 residues was probed. Whereas several nsp7 mutations affected virus replication to a limited extent, the replacement of two nsp8 residues (P183 and R190) essential for interaction with nsp12 and a third (K58) critical for the interaction of the polymerase complex with RNA were all lethal to the virus. Without a loss of processivity, the nsp7/nsp8/nsp12 complex can associate with nsp14, a bifunctional enzyme bearing 3′-5′ exoribonuclease and RNA cap N7-guanine methyltransferase activities involved in replication fidelity and 5′-RNA capping, respectively. The identification of this tripartite polymerase complex that in turn associates with the nsp14 proofreading enzyme sheds light on how coronaviruses assemble an RNA-synthesizing machinery to replicate the largest known RNA genomes. This protein complex is a fascinating example of the functional integration of RNA polymerase, capping, and proofreading activities.

Significance: Emerging coronaviruses (CoVs; severe acute respiratory syndrome-CoV and Middle East respiratory syndrome-CoV) pose serious health threats globally, with no specific antiviral treatments available. These viruses are able to faithfully synthesize their large genomic RNA. We report, however, that their main RNA polymerase, nsp12, is not accurate. To achieve accuracy, CoVs have acquired nsp14, a bifunctional enzyme able to methylate the viral RNA cap [methyltransferase (MTase)] and excise erroneous mutagenic nucleotides inserted by nsp12. Strikingly, ribavirin can be excised from the viral genome, thus showing no antiviral activity. The crystal structure of nsp14 shows that it is unique, having been replaced by other MTase types during evolution. This unprecedented RNA correction machinery has allowed RNA genome size expansion, but also provided potential nucleoside drug resistance to these deadly pathogens.

The replication/transcription complex of severe acute respiratory syndrome coronavirus is composed of at least 16 nonstructural proteins (nsp1–16) encoded by the ORF-1a/1b. This complex includes replication enzymes commonly found in positive-strand RNA viruses, but also a set of RNA-processing activities unique to some nidoviruses. The nsp14 protein carries both exoribonuclease (ExoN) and (guanine-N7)-methyltransferase (N7-MTase) activities. The nsp14 ExoN activity ensures a yet-uncharacterized function in the virus life cycle and must be regulated to avoid nonspecific RNA degradation. In this work, we show that the association of nsp10 with nsp14 stimulates >35-fold the ExoN activity of the latter while playing no effect on N7-MTase activity. Nsp10 mutants unable to interact with nsp14 are not proficient for ExoN activation. The nsp10/nsp14 complex hydrolyzes double-stranded RNA in a 3′ to 5′ direction as well as a single mismatched nucleotide at the 3′-end mimicking an erroneous replication product. In contrast, di-, tri-, and longer unpaired ribonucleotide stretches, as well as 3′-modified RNAs, resist nsp10/nsp14-mediated excision. In addition to the activation of nsp16-mediated 2′-O-MTase activity, nsp10 also activates nsp14 in an RNA processing function potentially connected to a replicative mismatch repair mechanism.

Background: Our understanding of the global scale of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection remains incomplete: Routine surveillance data underestimate infection and cannot infer on population immunity; there is a predominance of asymptomatic infections, and uneven access to diagnostics. We meta-analyzed SARS-CoV-2 seroprevalence studies, standardized to those described in the World Health Organization’s Unity protocol (WHO Unity) for general population seroepidemiological studies, to estimate the extent of population infection and seropositivity to the virus 2 years into the pandemic. Methods and findings: We conducted a systematic review and meta-analysis, searching MEDLINE, Embase, Web of Science, preprints, and grey literature for SARS-CoV-2 seroprevalence published between January 1, 2020 and May 20, 2022. The review protocol is registered with PROSPERO (CRD42020183634). We included general population cross-sectional and cohort studies meeting an assay quality threshold (90% sensitivity, 97% specificity; exceptions for humanitarian settings). We excluded studies with an unclear or closed population sample frame. Eligible studies—those aligned with the WHO Unity protocol—were extracted and critically appraised in duplicate, with risk of bias evaluated using a modified Joanna Briggs Institute checklist. We meta-analyzed seroprevalence by country and month, pooling to estimate regional and global seroprevalence over time; compared seroprevalence from infection to confirmed cases to estimate underascertainment; meta-analyzed differences in seroprevalence between demographic subgroups such as age and sex; and identified national factors associated with seroprevalence using meta-regression. We identified 513 full texts reporting 965 distinct seroprevalence studies (41% low- and middle-income countries [LMICs]) sampling 5,346,069 participants between January 2020 and April 2022, including 459 low/moderate risk of bias studies with national/subnational scope in further analysis. By September 2021, global SARS-CoV-2 seroprevalence from infection or vaccination was 59.2%, 95% CI [56.1% to 62.2%]. Overall seroprevalence rose steeply in 2021 due to infection in some regions (e.g., 26.6% [24.6 to 28.8] to 86.7% [84.6% to 88.5%] in Africa in December 2021) and vaccination and infection in others (e.g., 9.6% [8.3% to 11.0%] in June 2020 to 95.9% [92.6% to 97.8%] in December 2021, in European high-income countries [HICs]). After the emergence of Omicron in March 2022, infection-induced seroprevalence rose to 47.9% [41.0% to 54.9%] in Europe HIC and 33.7% [31.6% to 36.0%] in Americas HIC. In 2021 Quarter Three (July to September), median seroprevalence to cumulative incidence ratios ranged from around 2:1 in the Americas and Europe HICs to over 100:1 in Africa (LMICs). Children 0 to 9 years and adults 60+ were at lower risk of seropositivity than adults 20 to 29 (p < 0.001 and p = 0.005, respectively). In a multivariable model using prevaccination data, stringent public health and social measures were associated with lower seroprevalence (p = 0.02). The main limitations of our methodology include that some estimates were driven by certain countries or populations being overrepresented. Conclusions: In this study, we observed that global seroprevalence has risen considerably over time and with regional variation; however, over one-third of the global population are seronegative to the SARS-CoV-2 virus. Our estimates of infections based on seroprevalence far exceed reported Coronavirus Disease 2019 (COVID-19) cases. Quality and standardized seroprevalence studies are essential to inform COVID-19 response, particularly in resource-limited regions.