Rotavirus NSP4, initially characterized as an endoplasmic reticulum intracellular receptor, is a multifunctional viral enterotoxin that induces diarrhea in murine pups. There have been recent reports of the secretion of a cleaved NSP4 fragment (residues 112 to 175) and of the association of NSP4 with LC3-positive autophagosomes, raft membranes, and microtubules. To determine if NSP4 traffics to a specific subset of rafts at the plasma membrane, we isolated caveolae from plasma membrane-enriched material that yielded caveola membranes free of endoplasmic reticulum and nonraft plasma membrane markers. Analyses of the newly isolated caveolae from rotavirus-infected MDCK cells revealed full-length, highmannose glycosylated NSP4. The lack of Golgi network-specific processing of the caveolar NSP4 glycans supports studies showing that NSP4 bypasses the Golgi apparatus. Confocal imaging showed the colocalization of NSP4 with caveolin-1 early and late in infection, elucidating the temporal and spatial NSP4-caveolin-1 association during infection. These data were extended with fluorescent resonance energy transfer analyses that confirmed the NSP4 and caveolin-1 interaction in that the specific fluorescently tagged antibodies were within 10 nm of each other during infection. Cells transfected with NSP4 showed patterns of staining and colocalization with caveolin-1 similar to those of infected cells. This study presents an endoplasmic reticulum contaminant-free caveola isolation protocol; describes the presence of full-length, endoglycosidase H-sensitive NSP4 in plasma membrane caveolae; provides confirmation of the NSP4-caveolin interaction in the presence and absence of other viral proteins; and provides a final plasma membrane destination for Golgi network-bypassing NSP4 transport.Rotaviruses (RV) are the leading viral etiologic agents of severe pediatric gastroenteritis worldwide, affecting nearly all children before the age of 5, with 2 million cases resulting in 444,000 deaths annually (33,34,40). RV nonstructural protein 4 (NSP4) was initially characterized as an endoplasmic reticulum (ER) transmembrane glycoprotein due to the protein's high-mannose glycosylation and its critical function as an intracellular receptor for the translocation of subviral particles into the ER during virion morphogenesis (2,5,14). However, the identification of NSP4 and NSP4 amino acids (aa) 114 to 135 (NSP4 114-135 ) as enterotoxic and the redistribution of RVencoded proteins upon NSP4 silencing led to a reevaluation of NSP4 function(s) and subcellular localization(s) (4, 31).A cleaved NSP4 fragment, aa 112 to 175, is secreted from RV-infected epithelial cells, indicating that some portion of NSP4 traffics from the ER to the plasma membrane (PM) (65). The colocalization of NSP4 114-135 and the extracellular matrix proteins laminin-3 and fibronectin at the basement membrane of small-intestinal epithelia from RV strain EDIM-infected mouse pups also supports NSP4 transport to the PM during host infection (8). While both findings demonstrat...
Introduction The coronavirus disease 2019 (COVID-19) pandemic is a global public health emergency causing a disparate burden of death and disability around the world. The molecular characteristics of the virus that predict better or worse outcome are largely still being discovered. Methods We downloaded 155,958 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from GISAID. Of these genomes, 3,637 samples included useable metadata on patient outcomes. Using this subset, we evaluated whether SARS-CoV-2 viral genomic variants improved prediction of reported severity beyond age and region. First, we established whether including genomic variants as model features meaningfully increased predictive power of our model. Next, we evaluated specific variants in order to determine the magnitude of association with severity and the frequency of these variants among SARS-CoV-2 genomes. Results Logistic regression models that included viral genomic variants outperformed other models (AUC = 0.91 as compared with 0.68 for age and gender alone; p < 0.001). Among individual variants, we found 17 single nucleotide variants in SARS-CoV-2 have more than two-fold greater odds of being associated with higher severity and 67 variants associated with ≤0.5 times the odds of severity. The median frequency of associated variants was 0.15% (interquartile range 0.09%-0.45%). Altogether 85% of genomes had at least one variant associated with patient outcome. Conclusion Numerous SARS-CoV-2 variants have two-fold or greater association with odds of mild or severe outcome and collectively, these variants are common. In addition to comprehensive mitigation efforts, public health measures should be prioritized to control the more severe manifestations of COVID-19 and the transmission chains linked to these severe cases. Lay Summary This study explores which, if any, SARS-CoV-2 viral genomic variants are associated with mild or severe COVID-19 patient outcomes. Our results suggest that there are common genomic variants in SARS-CoV-2 that are more often associated with negative patient outcomes, which may impact downstream public health measures.
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