SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Scherer, Katharina M.; Mascheroni, Luca; Carnell, George; Wunderlich, Lucia Cs; Makarchuk, Stanislaw; Brockhoff, Marius; Mela, Ioanna; Fernández-Villegas, Ana; Barysevich, Max; Stewart, Hazel; Sans, Maria Suau; George, Charlotte; Lamb, Jacob R.; Schierle, Gabriele S Kaminski; Heeney, Jonathan L.; Kaminski, Clemens F.

doi:10.1126/sciadv.abl4895

Cited by 59 publications

(42 citation statements)

References 63 publications

(87 reference statements)

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“…Dispersion of N transcripts (gRNA and sgRNA) in the cytosol of ACE2 + and CD169 + /ACE2 + THP1/PMA macrophages mirrors the temporal localization phenotype of viral N protein ( Fig. 3C and D ), and might be indicative of formation of N positive viral replication compartments (63). While a majority of virus-exposed CD169 + THP1/MA macrophages expressed fluorescent puncta (gRNA) at 24 hpi (indicated by the white arrowheads showing both ORF1a and N expression, Fig.…”

Section: Resultsmentioning

confidence: 73%

“…To confirm that temporal increases in viral transcripts in CD169 + or ACE2 + THP1/PMA macrophages were generated from ongoing virus transcription, THP1/PMA cells (±CD169 ±ACE2) were pre-treated with remdesivir (RDV), a known inhibitor of SARS-CoV-2 RNA synthesis, prior to infection with SARS-CoV-2 (24, 63). RT-qPCR analysis of both gRNA (N transcripts) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

Jalloh

Olejnik

Berrigan

et al. 2022

Preprint

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Exacerbated and persistent innate immune response marked by pro-inflammatory cytokine expression is thought to be a major driver of chronic COVID-19 pathology. Although macrophages are not the primary target cells of SARS-CoV-2 infection in humans, viral RNA and antigens in activated monocytes and macrophages have been detected in post-mortem samples, and dysfunctional monocytes and macrophages have been hypothesized to contribute to a protracted hyper-inflammatory state in COVID-19 patients. In this study, we demonstrate that CD169, a myeloid cell specific I-type lectin, facilitated ACE2-independent SARS-CoV-2 fusion and entry in macrophages. CD169- mediated SARS-CoV-2 entry in macrophages resulted in expression of viral genomic and sub-genomic (sg) RNAs with minimal viral protein expression and no infectious viral particle release, suggesting a post-entry restriction of the SARS-CoV-2 replication cycle. Intriguingly this post-entry replication block was alleviated by exogenous ACE2 expression in macrophages. Restricted expression of viral gRNA and sgRNA in CD169+ macrophages elicited a pro-inflammatory cytokine expression (TNFα, IL-6 and IL-1β) in a RIG-I, MDA-5 and MAVS-dependent manner, which was suppressed by remdesivir pre- treatment. These findings suggest that de novo expression of SARS-CoV-2 RNA in macrophages contributes to the pro-inflammatory cytokine signature and that blocking CD169-mediated ACE2 independent infection and subsequent activation of macrophages by viral RNA might alleviate COVID-19-associated hyperinflammatory response.Author SummaryOver-exuberant production of pro-inflammatory cytokine expression by macrophages has been hypothesized to contribute to severity of COVID-19 disease. Molecular mechanisms that contribute to macrophage-intrinsic immune activation during SARS- CoV-2 infection are not fully understood. Here we show that CD169, a macrophage- specific sialic-acid binding lectin, facilitates abortive SARS-CoV-2 infection of macrophages that results in innate immune sensing of viral replication intermediates and production of proinflammatory responses. We identify an ACE2-independent, CD169- mediated endosomal viral entry mechanism that results in cytoplasmic delivery of viral capsids and initiation of virus replication, but absence of infectious viral production. Restricted viral replication in CD169+ macrophages and detection of viral genomic and sub-genomic RNAs by cytoplasmic RIG-I-like receptor family members, RIG-I and MDA5, and initiation of downstream signaling via the adaptor protein MAVS, was required for innate immune activation. These studies uncover mechanisms important for initiation of innate immune sensing of SARS-CoV-2 infection in macrophages, persistent activation of which might contribute to severe COVID-19 pathophysiology.

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Section: Resultsmentioning

confidence: 73%

Section: Resultsmentioning

confidence: 99%

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

Jalloh

Olejnik

Berrigan

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…We next tested if specific RNA sequences or regions of the genome promote formation of the vRNP. Four 600-nt genomic regions were transcribed in vitro, individually mixed with N protein, and analyzed by native gel electrophoresis: (1) 5’-600 (nucleotides 1–600), (2) Nsp3 (nucleotides 7,800–8,400), (3) Nsp8/9 (nucleotides 12,250–12,850), (4) Nsp10 (nucleotides 13,200–13,800). All RNAs appeared to form appropriately-sized vRNPs, although the Nsp3 RNA appeared less effective (Fig.…”

Section: Resultsmentioning

confidence: 99%

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation

Carlson

Adly

et al. 2022

Preprint

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The nucleocapsid (N) protein of coronaviruses is responsible for compaction of the ~30-kb RNA genome in the ~100-nm virion. Cryo-electron tomography suggests that each virion contains 35-40 viral ribonucleoprotein (vRNP) complexes, or ribonucleosomes, arrayed along the genome. There is, however, little mechanistic understanding of the vRNP complex. Here, we show that N protein, when combined with viral RNA fragments in vitro, forms cylindrical 15-nm particles similar to the vRNP structures observed within coronavirus virions. These vRNPs form in the presence of stem-loop-containing RNA and depend on regions of N protein that promote protein-RNA and protein-protein interactions. Phosphorylation of N protein in its disordered serine/arginine (SR) region weakens these interactions and disrupts vRNP assembly. We propose that unmodified N binds stem-loop-rich regions in genomic RNA to form compact vRNP complexes within the nucleocapsid, while phosphorylated N maintains uncompacted viral RNA to promote the protein's transcriptional function.

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“…SARS-CoV-2 infection or expression of certain viral proteins induces Golgi fragmentation by modulating the expression level of GRASP55 and TGN46 ( Zhang et al, 2022 ). Interference with the dysregulated GRASP55/TGN46 expression reduces SARS-CoV-2 infection in Huh7-ACE2 cells ( Scherer et al, 2022 ; Zhang et al, 2022 ). Transmission electron microscopy analysis revealed the presence of a large number of small secretory vesicles containing a single virion in SARS-CoV-2-infected Vero cells ( Eymieux et al, 2021 ).…”

Section: The Late Endosome/lysosome Exocytic Pathway Mediates Sars-co...mentioning

confidence: 99%

“…Virions are assembled at the ER or the ER–Golgi intermediate compartment (ERGIC) and are thought to use smooth-wall secretory vesicles for release ( Figure 1 ) ( Knoops et al, 2008 ; Reggiori et al, 2010 ; Snijder et al, 2020 ). Recent studies of SARS-CoV-2 infection, which induces extensive ER remodeling, Golgi fragmentation and mitochondrial network reshaping ( Cortese et al, 2020 ; Scherer et al, 2022 ), have greatly advanced our understanding of the mechanism and function of endomembrane remodeling in coronavirus infection. For example, double-membrane-spanning pores in DMVs transport the viral RNA products from the interior of DMVs to the cytosol for translation or viral assembly ( Wolff et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

Endomembrane remodeling in SARS-CoV-2 infection

2022

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SARS-CoV-2 nucleocapsid protein adheres to replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Abstract: Super-resolution optical imaging reveals the accumulation of SARS-CoV-2 nucleocapsid protein at viral replication organelles.

Cited by 59 publications

References 63 publications

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

CD169-mediated restrictive SARS-CoV-2 infection of macrophages induces pro-inflammatory responses

Reconstitution of the SARS-CoV-2 ribonucleosome provides insights into genomic RNA packaging and regulation by phosphorylation

Endomembrane remodeling in SARS-CoV-2 infection

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