Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells

Ding, Siyuan; Zhu, Shu; Ren, Lili; Feng, Ningguo; Song, Yanhua; Ge, Xiaomei; Li, Bin; Flavell, Richard A.; Greenberg, Harry B.

doi:10.7554/elife.39494

Cited by 63 publications

(71 citation statements)

References 75 publications

(129 reference statements)

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“…1A, left panel). We found that ACE2 was predominantly expressed in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes ( 19, 20 ) (Fig. 1A, right panel).…”

Section: Resultsmentioning

confidence: 87%

“…VSV-SARS-CoV-2-S-GFP and VSV-SARS-CoV-S-GFP were propagated in MA104 cells in T175 flasks and virus sequences can be found in Table S1. Plaque assays were performed in MA104 cells seeded in 6-well plates using an adapted version of the rotavirus plaque assay protocol ( 19 ). TCID50 assays were performed in MA104 cells seeded in flat-bottom 96-well plates using serial dilutions.…”

Section: Methodsmentioning

confidence: 99%

“…Small intestinal IEC isolation by EDTA-DTT extraction method and sorting were performed as previously described ( 19, 52 ). Cells were stained with LIVE/DEAD Aqua Dead Cell Stain Kit (Thermo Fisher) and live cells were sorted using a BD FACSAria II flow cytometer.…”

Section: Methodsmentioning

confidence: 99%

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TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

Zang

Castro

McCune

et al. 2020

Preprint

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Both gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA have been frequently observed in COVID-19 patients. However, whether SARS-CoV-2 replicate in the human intestine and its clinical relevance to potential fecal-oral transmission remain unclear. Here, we demonstrate productive infection of SARS-CoV-2 in ACE2+ mature enterocytes in human small intestinal enteroids. In addition to TMPRSS2, another mucosa-specific serine protease, TMPRSS4, also enhanced SARS-CoV-2 spike fusogenic activity and mediated viral entry into host cells. However, newly synthesized viruses released into the intestinal lumen were rapidly inactivated by human colonic fluids and no infectious virus was recovered from the stool specimens of COVID-19 patients. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression.

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“…1A, left panel). We found that ACE2 was predominantly expressed in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes ( 19, 20 ) (Fig. 1A, right panel).…”

Section: Resultsmentioning

confidence: 87%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

Zang

Castro

McCune

et al. 2020

Preprint

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“…Five-day-old 129/Sv strain suckling mice were obtained from the Greenberg laboratory breeding colony at Stanford University. IECs were isolated from small intestine tissue pooled from two males and one female by EDTA-dithiothreitol extraction method, and cell sorting was performed as previously described (23,59…”

Section: Scrna-seq Analysismentioning

confidence: 99%

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

et al. 2020

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Gastrointestinal symptoms and fecal shedding of SARS-CoV-2 RNA are frequently observed in COVID-19. However, it is unclear whether SARS-CoV-2 replicates in the human intestine and contributes to possible fecal-oral transmission. Here, we report productive infection of SARS-CoV-2 in ACE2 + mature enterocytes in human small intestinal enteroids. Expression of two mucosa-specific serine proteases, TMPRSS2 and TMPRSS4, facilitated SARS-CoV-2 spike fusogenic activity and promoted virus entry into host cells. We also demonstrate that viruses released into the intestinal lumen were inactivated by simulated human colonic fluid, and infectious virus was not recovered from the stool specimens of patients with COVID-19. Our results highlight the intestine as a potential site of SARS-CoV-2 replication, which may contribute to local and systemic illness and overall disease progression. RESULTS SARS-CoV-2 infects human intestinal enteroidsIn the intestine, ACE2 functions as a chaperone for the sodiumdependent neutral amino acid transporter B 0 AT1 (encoded by SLC6A19) on IECs and regulates microbial homeostasis (21,22). ACE2 expression is substantially higher in the small intestine than in all the other organs, including the lung, in both humans and mice (fig. S1, A and B). Given these data, we assessed whether SARS-CoV-2 can infect IECs as a first step for understanding its implications for fecal-oral transmission. We performed single-cell RNA sequencing (scRNA-seq) to capture the global transcriptomics in all IEC subsets in the mouse small intestinal epithelium (Fig. 1A, left). ACE2 mRNA was predominantly seen in Cd26 + Epcam + Cd44 − Cd45 − mature enterocytes (Fig. 1A, right) (23, 24). In addition, bulk RNA-seq revealed that primary human ileum enteroids had substantially higher mRNA levels of all known CoV receptors, including ACE2, than the of 10 that Ace2 high cells are also positive for Cd26 and Epcam but negative for Cd44 and Cd45. (B) Human duodenum enteroids were cultured in the Transwell monolayer system using maintenance (MAINT) or differentiation (DIFF) conditions for 3 days. Monolayers were stained for ACE2 (red) and actin (phalloidin, white). Scale bars, 32 m. (C) Human duodenum enteroids in monolayer, cultured in either maintenance (MAINT) or differentiation (DIFF) conditions, were apically infected with 1.5 × 10 5 plaque-forming units (PFU) of VSV-SARS-CoV-2 [multiplicity of infection (MOI) = 0.3] for 24 hours. The expression of VSV-N was measured by RT-qPCR and normalized to that of GAPDH. p.i., post-infection. (D) Human duodenum enteroids in 3D Matrigel were cultured in maintenance (MAINT) medium or differentiation (DIFF) medium for 3 days and infected with 2.2 × 10 5 PFU of VSV-SARS-CoV-2 for 18 hours. Enteroids were stained for virus (green), actin (phalloidin, white), and nucleus (DAPI, blue). Scale bars, 50 m. (E) Same as (C) except that virus titers were measured using a TCID 50 assay instead of viral RNA levels by qPCR. (F) Same as (D) except that human ileum enteroids were used instead. Scale bars, ...

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“…to increase with repeated infection or immunization (1). However, innate immunity also may contribute to rotavirus disease, and rotaviruses have been shown to antagonize innate signaling pathways using multiple distinct mechanisms (1)(2)(3).…”

mentioning

confidence: 99%

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein

Diller

Parrington

Patton

et al. 2019

J Virol

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Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis. IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

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Rotavirus VP3 targets MAVS for degradation to inhibit type III interferon expression in intestinal epithelial cells

Cited by 63 publications

References 75 publications

TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

TMPRSS2 and TMPRSS4 mediate SARS-CoV-2 infection of human small intestinal enterocytes

TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein

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