ABSTRACT
Host-viral interactions during SARS-CoV-2 infection are needed to understand COVID-19 pathogenesis and may help to guide the design of novel antiviral therapeutics. N6-methyladenosine modification (m6A), one of the most abundant cellular RNA modifications, regulates key processes in RNA metabolism during a stress response. Gene expression profiles observed post-infection with different SARS-CoV-2 variants show changes in the expression of genes related to RNA catabolism, including m6A readers and erasers. We found that infection with SARS-CoV-2 variants caused a loss of m6A in cellular RNAs, whereas m6A was detected abundantly in viral RNA. METTL3, the m6A methyltransferase, showed an unusual cytoplasmic localization post-infection. The B.1.351 variant had a less pronounced effect on METTL3 localization and loss of m6A than the B.1 and B.1.1.7 variants. We also observed a loss of m6A upon SARS-CoV-2 infection in air/liquid interface cultures of human airway epithelia, confirming that m6A loss is characteristic of SARS-CoV-2 infected cells. Further, transcripts with m6A modification were preferentially down-regulated post-infection. Inhibition of the export protein XPO1 resulted in the restoration of METTL3 localization, recovery of m6A on cellular RNA, and increased mRNA expression. Stress granule formation, which was compromised by SARS-CoV-2 infection, was restored by XPO1 inhibition and accompanied by a reduced viral infection in vitro. Together, our study elucidates how SARS-CoV-2 inhibits the stress response and perturbs cellular gene expression in an m6A-dependent manner.
Subject(s)
Severe Acute Respiratory Syndrome , Virus Diseases , COVID-19ABSTRACT
Throughout the SARS-CoV-2 pandemic, multiple waves of variants of concern have swept across populations, leading to a chain of new and yet more contagious lineages dominating COVID-19 cases. Here, we tracked the remarkably rapid shift from Omicron BA.1 to BA.2 sub-variant dominance in the Swedish population during January-March 2022. By analysis of 174,933 clinical nasopharyngeal swab samples using a custom variant-typing RT-PCR assay, we uncover nearly two-fold higher levels of viral RNA in cases with Omicron BA.2. Importantly, increased viral load in the upper pharynx upon BA.2 infection may provide part of the explanation why Omicron BA.2 is more transmissible and currently outcompetes the BA.1 variant across populations.
Subject(s)
COVID-19ABSTRACT
Understanding innate immune responses in COVID-19 is important for deciphering mechanisms of host responses and interpreting disease pathogenesis. Natural killer (NK) cells are innate effector lymphocytes that respond to acute viral infections, but might also contribute to immune pathology. Here, using 28-color flow cytometry, we describe a state of strong NK cell activation across distinct subsets in peripheral blood of COVID-19 patients, a pattern mirrored in scRNA-seq signatures of lung NK cells. Unsupervised high-dimensional analysis identified distinct immunophenotypes that were linked to disease severity. Hallmarks of these immunophenotypes were high expression of perforin, NKG2C, and Ksp37, reflecting a high presence of adaptive NK cell expansions in circulation of patients with severe disease. Finally, arming of CD56bright NK cells was observed in course of COVID-19 disease states, driven by a defined protein-protein interaction network of inflammatory soluble factors. This provides a detailed map of the NK cell activation-landscape in COVID-19 disease.
Subject(s)
Virus Diseases , COVID-19ABSTRACT
Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The most widely used method of COVID-19 diagnostics is a reverse transcription polymerase chain reaction (RT-PCR) assay, to detect the presence of SARS-CoV-2 RNA in patient samples, typically from nasopharyngeal swabs. RNA extraction is a major bottleneck in current COVID-19 testing, in terms of turn-around, logistics, component availability and cost, which delays or completely precludes COVID-19 diagnostics in many settings. Efforts to simplify the current methods are critical, as increased diagnostic availability and efficiency would benefit patient care and infection control. Here, we describe methods to circumvent RNA extraction in COVID-19 testing by performing RT-PCR directly on heat-inactivated subject samples as well as samples lysed with readily available detergents. Our data, including benchmarking with 597 clinically diagnosed patient samples against a standardised and sensitive diagnostic system, show that direct RT-PCR is a viable option to extraction-based COVID-19 diagnostics. Furthermore, using controlled amounts of active SARS-CoV-2, we evaluated performance of generic buffers as sample medium for the direct RT-PCR assay, identifying several suitable formulations. We also confirmed the effectiveness of heat inactivation of SARS-CoV-2 by plaque assay. Significant savings in terms of time and cost can be achieved by embracing RNA-extraction-free protocols, that feed directly into the established PCR-based testing pipeline. This could aid the expansion of COVID-19 testing.