Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)—a new coronavirus that has led to a worldwide pandemic¹—has a furin cleavage site (PRRAR) in its spike protein that is absent in other group-2B coronaviruses². To explore whether the furin cleavage site contributes to infection and pathogenesis in this virus, we generated a mutant SARS-CoV-2 that lacks the furin cleavage site (ΔPRRA). Here we report that replicates of ΔPRRA SARS-CoV-2 had faster kinetics, improved fitness in Vero E6 cells and reduced spike protein processing, as compared to parental SARS-CoV-2. However, the ΔPRRA mutant had reduced replication in a human respiratory cell line and was attenuated in both hamster and K18-hACE2 transgenic mouse models of SARS-CoV-2 pathogenesis. Despite reduced disease, the ΔPRRA mutant conferred protection against rechallenge with the parental SARS-CoV-2. Importantly, the neutralization values of sera from patients with coronavirus disease 2019 (COVID-19) and monoclonal antibodies against the receptor-binding domain of SARS-CoV-2 were lower against the ΔPRRA mutant than against parental SARS-CoV-2, probably owing to an increased ratio of particles to plaque-forming units in infections with the former. Together, our results demonstrate a critical role for the furin cleavage site in infection with SARS-CoV-2 and highlight the importance of this site for evaluating the neutralization activities of antibodies.

CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2- to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4+/CD45RO+/CD62L-true memory cells compared with CD4+/CD45RO+/CD62L+ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitromaturation of PBDC resulted in median 3- and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from ≈5,000 to ≈50,000 ABS) whereas granulocyte-macrophage colony-stimulating factor caused a marked decrease of CXCR4 (from ≈5,000 ABS to <500) while up-regulating CCR5 expression (from ≈5,000 to ≈20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.

DC-SIGN is a C-type lectin, highly expressed on the surface ofimmature dendritic cells (DCs), that mediates efficient infection of Tcells in trans by its ability to bind HIV-1, HIV-2, and SIV. Inaddition, the ability of DC-SIGN to bind adhesion molecules on surfacesof naïve T cells and endothelium also suggests its involvementin T-cell activation and DC trafficking. To gain further insights intothe range of expression and potential functions of DC-SIGN, weperformed a detailed analysis of DC-SIGN expression in adult and fetaltissues and also analyzed its regulated expression on cultured DCs andmacrophages. First, we show that DC-SIGN expression is restricted tosubsets of immature DCs in tissues and on specialized macrophages inthe placenta and lung. There were no overt differences between DC-SIGNexpression in adult and fetal tissues except that DC-SIGN expression inalveolar macrophages was only present after birth. Similarly, intissues, DC-SIGN was observed primarily on immature (CD83-negative)DCs. Secondly, in the peripheral blood, we found expression of DC-SIGNon a small subset of BDCA-2+ plasmacytoid DC precursors (pDC2),concordant with our finding of large numbers of DC-SIGN-positive cellsin allergic nasal polyps (previously shown to be infiltrated by DC2). Triple-label confocal microscopy indicated that DC-SIGN was colocalizedwith BDCA-2 and CD123 on DCs in nasal polyp tissue. Consistent withthis finding is our observation that DC-SIGN can be up-regulated onmonocyte-derived macrophages upon exposure to the Th2 cytokine, IL-13. In summary, our data demonstrate the relevant populations of DC andmacrophages that express DC-SIGN in vivo where it may impact theefficiency of virus infection and indicate that DC-SIGN expression maybe involved in the Th2 axis of immunity.