RESUMEN
SARS-CoV-2 infections pose a global threat to human health and an unprecedented research challenge. Among the most urgent tasks is obtaining a detailed understanding of the molecular interactions that facilitate viral replication or contribute to host defense mechanisms in infected cells. While SARS-CoV-2 co-opts cellular factors for viral translation and genome replication, a comprehensive map of the host cell proteome in direct contact with viral RNA has not been elucidated. Here, we use RNA antisense purification and mass spectrometry (RAP-MS) to obtain an unbiased and quantitative picture of the human proteome that directly binds the SARS-CoV-2 RNA in infected human cells. We discover known host factors required for coronavirus replication, regulators of RNA metabolism and host defense pathways, along with dozens of potential drug targets among direct SARS-CoV-2 binders. We further integrate the SARS-CoV-2 RNA interactome with proteome dynamics induced by viral infection, linking interactome proteins to the emerging biology of SARS-CoV-2 infections. Validating RAP-MS, we show that CNBP, a regulator of proinflammatory cytokines, directly engages the SARS-CoV-2 RNA. Supporting the functional relevance of identified interactors, we show that the interferon-induced protein RYDEN suppresses SARS-CoV-2 ribosomal frameshifting and demonstrate that inhibition of SARS-CoV-2-bound proteins is sufficient to manipulate viral replication. The SARS-CoV-2 RNA interactome provides an unprecedented molecular perspective on SARS-CoV-2 infections and enables the systematic dissection of host dependency factors and host defense strategies, a crucial prerequisite for designing novel therapeutic strategies.
RESUMEN
BackgroundTransmission of COVID-19 from people without symptoms poses considerable challenges to public health containment measures. The distribution of viral loads in individuals with and without symptoms remains uncertain. Comprehensive cross-sectional screening of all individuals in a given setting provides an unbiased way to assess viral loads independent of symptoms, which informs transmission risks. COVID-19 cases initially peaked in Massachusetts in mid-April 2020 before declining through June, and congregate living facilities were particularly affected during this early surge. We performed a retrospective analysis of data from a large public health-directed outbreak response initiative that involved comprehensive screening within nursing homes and assisted living facilities in Massachusetts to compare nasopharyngeal (NP) viral loads (as measured by RT-PCR cycle threshold (Ct) levels) in residents and staff to inform our ability to detect SARS-CoV-2 in individuals with or without symptoms in the population. MethodsBetween April 9 and June 9, 2020, we tested NP swabs from 32,480 unique individuals comprising staff and residents of the majority of nursing homes and assisted living facilities in Massachusetts. Under the direction of the MA Department of Public Health (MDPH), symptomatology at the time of sampling and demographic information was provided by each facility for each individual to facilitate reporting to health officials. NP swabs were collected, RNA extracted, and SARS-CoV-2 testing performed using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). ResultsThe nursing home and assisted living facilities resident cohort (N =16,966) was 65% female with a mean age of 82 years (SD 13 yrs). The staff cohort (N = 15,514) was 76% female with a median age of 45 (SD 15 yrs). A total 2654 residents (15.5%) and 624 staff (4.1%) tested positive for SARS-CoV-2. 12.7% of residents and 3.7% of staff without symptoms tested positive for SARS-CoV-2, compared to 53.1% of residents and 18.2% of staff with symptoms. Of the individuals who tested positive, 70.8% of residents and 92.4% of staff lacked symptoms at the time of testing. In aggregate, the distributions of Cts for viral probes used in the qRT-PCR assay were very similar, with a statistically but not meaningfully different mean ({Delta}Ct 0.71 cycles, p = 0.006) and a similar range (12-38 cycles), between populations with and without symptoms over the entire time period, across all sub-categories examined (age, race, ethnicity, sex, resident/staff). Importantly, the Ct mean values and range were indistinguishable between the populations by symptom class during the peak of the outbreak in Massachusetts, with a Ct gap appearing only later in the survey period, reaching >3 cycles (p [≤] 0.001) for facilities sampled during the last two weeks of the study. ConclusionsIn a large cohort of individuals screened for SARS-CoV-2 by qRT-PCR, we found strikingly similar distributions of viral load in patients with or without symptoms at the time of testing during the local peak of the epidemic; as the epidemic waned, individuals without symptoms at the time of testing had lower viral loads. The size of the study population, including both staff and residents spanning a wide range of ages, provides a comprehensive cross-sectional point prevalence measurement of viral burden in a study spanning 2 months. Because the distributions of viral loads in infected individuals irrespective of symptomatology are very similar, existing testing modalities that have been validated for detection of SARS-CoV-2 RNA in symptomatic patients should perform similarly in individuals without symptoms at the time of testing.
