Dynamin independent endocytosis is an alternative cell entry mechanism for multiple animal viruses

Abstract

Mammalian receptor-mediated endocytosis (RME) often involves at least one of three isoforms of the large GTPase dynamin (Dyn). Dyn pinches-off vesicles at the plasma membrane and mediates uptake of many viruses, although some viruses directly penetrate the plasma membrane. RME is classically interrogated by genetic and pharmacological interference, but this has been hampered by undesired effects. Here we studied virus entry in conditional genetic knock-out (KO) mouse embryonic fibroblasts lacking expression of all three dynamin isoforms (Dyn-KO-MEFs). The small canine parvovirus known to use a single receptor, transferrin receptor, strictly depended on dynamin. Larger viruses or viruses known to use multiple receptors, including alphaviruses, influenza, vesicular stomatitis, bunya, adeno, vaccinia, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and rhinoviruses infected Dyn-KO-MEFs, albeit at higher dosage than wild-type MEFs. In absence of the transmembrane protease serine subtype 2 (TMPRSS2), which normally activates the SARS-CoV-2 spike protein for plasma membrane fusion, SARS-CoV-2 infected angiotensin-converting enzyme 2 (ACE2)-expressing MEFs predominantly through dynamin- and actin-dependent endocytosis. In presence of TMPRSS2 the ancestral Wuhan-strain bypassed both dynamin-dependent and -independent endocytosis, and was less sensitive to endosome maturation inhibitors than the Omicron B1 and XBB variants, supporting the notion that the Omicron variants do not efficiently use TMPRSS2. Collectively, our study suggests that dynamin function at endocytic pits can be essential for infection with single-receptor viruses, while it is not essential but increases uptake and infection efficiency of multi-receptor viruses that otherwise rely on a functional actin network for infection.

Fig 1. Characterization of MEF dynamin 1,2 conditional KO cells to study virus infection.

A) Western blot analysis of dynamin 1 and 2 levels in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days. Tubulin was used as a loading control. The Dyn1,2 antibody used recognizes both dynamin 1 and 2. B) Quantification of Tf Alexa-647 (Tf) (5 μg/ml) following a 30 min uptake in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days prior fixation and Hoechst DNA staining. Representative fluorescent images on the right show Tf (white) and Hoechst (red) in MEF^DKO cells treated with vehicle control or 4OH-TMX. C-D) Quantification of CTB Alexa-647 (CTB) uptake in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days and incubated with indicated concentrations of CTB for 30 min prior to fixation and Hoechst DNA staining. Representative fluorescent images on right show CTB (white) and Hoechst (red) in MEF^DKO cells treated with vehicle control or 4OH-TMX. E) Quantification of CPV infection in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days and infected with CPV for 24h, and immunostained for non-structural protein 1 (NS1). The inset schematic illustrates the entry mechanism of CPV. F) Quantification of VACV infection in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days and infected with VACS for 6 h. The inset schematic illustrates VACV entry by micropinocytosis. G) Effect of Latrunculin-B (LatB) on VACV infection in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days and infected with VACS for 6 h. H) Quantification of SFV infection in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days and infected with indicated MOIs of SFV-EGFP for 7 h. The inset illustrates the FACS analysis of virus-induced EGFP fluorescence in non-infected (red line) and infected (blue line) vehicle-control treated cells. Values represent the mean of 3 independent experiments. Error bars represent the standard deviation (STDEV). Statistical significance was calculated using a unpaired two-tailed t-test (*p<0,05; ****p<0,0001; n.s. = non-significant).

Fig 2. Dynamin-independent endocytosis is an alternative, efficient virus entry pathway for multiple animal viruses.

Infection of indicated viruses in MEF^DKO cells treated with vehicle control or 4OH-TMX for 6 days and infected for 6 h (VACV), 7 h (SINV, VSV), and 8 h (IAV X31, UUKV). Virus infection was determined by FACS analysis of EGFP (VAVC and VSV), mCherry (SINV), or after immunofluorescence of viral antigens using virus-specific antibodies (IAV X31 and UUKV). Values indicate the mean of three independent experiments and the error bars represent the standard deviation (STDEV). n.i. = non infected. Statistical significance was calculated by unpaired two-tailed t-test (*p<0,05; ** p<0,01; n.s. = non-significant).

Fig 3. Dynamin-independent virus entry is actin dependent.

A) Schematic description of indicated drug treatments in MEF^DKO cells following 6-day treatment with vehicle (ctrl) or 4OH-TMX, and infected with SFV-EGFP. B) Quantification of the experiment described in A. After drug treatment and infection, the percentage of infected cells was determined by FACS analysis of virus-induced expression of EGFP. The red-boxed area indicates the treatments with actin depolymerizing drugs. C) Schematic description of indicated drug treatments in MEF^DKO cells following 6-day treatment with vehicle (ctrl) or 4OH-TMX, and infected with VACV-EGFP. D) Quantification of the experiment described in C. After drug treatment and infection, the percentage of infected cells was determined by FACS analysis of virus induced expression of EGFP. The red-boxed area indicates the treatments with actin depolymerizing drugs. Values indicate the mean of three independent experiments and the error bars represent the standard deviation. Statistical significance was calculated by unpaired two-tailed t-test (** p<0,01; n.s. = non-significant). E) Representative confocal images of virus entry in MEF^TKOcells pretreated with 4OH-TMX for 6 days, incubated with DMSO control, EIPA (80 μM), or Dyngo-4A (25 μM) and viruses (equivalent MOI = 100) 30 minutes before incubation on ice for 120 min. After washes, cells where incubated with DMSO or drugs at 37°C and fixed at indicated times before processing for sequential immunofluorescence as described in S4C Fig. Inset images show a magnification of the area indicated by the white boxes. Virus out = non internalized viruses; virus in = internalized viruses. F) Quantification of non-internalized (virus out) and internalized (virus in) virions using automated image analysis. Values represent the mean of >5 cells from 3 independent experiments, and error bars represent the STDEV. Scale bar = 10 μm. Statistical analysis was performed using ordinary two-way ANOVA multiple comparison test (n.s. = non significant; *** p<0.001). G) Representative confocal images of virus entry and acidification of the E1 envelope protein by sequencial immunofluorescence as described in E using a monoclonal antibody against the acid form of the E1 glycoprotein (E1 acid) and a combination of antibodies agains E1 and e2 to detect virions at the outer side of the plasma membrane (virus out). Scale bar = 10 μm. H). Quantification of non-internalized (virus out) and internalized virues detected by the E1a-1 antibody against the acid form of SFV E1 glycoprotein using image analysis performed with the Imaris software. Values represent the mean of >5 cells from 3 independent experiments, and error bars represent the STDEV. Scale bar = 10 μm. Statistical analysis was performed using ordinary two-way ANOVA multiple comparison test (n.s. = non significant; *** p<0.001).

Fig 4. Ultrastructural analysis of Semliki Forest Virus entry in MEF^DKO cells.

Representative TEM images of MEF^DKO cells treated with vehicle control (A-D) or 4OH-TMX (E-G, and D, 4OH-TMX) for 6 days and infected with SFV (MOI 1000) on ice for 2 h followed by shift to 37°C for 15 min before fixation and processing for TEM. The fractions of total viral particles found in each of the described endocytic processes are quantified in H and I. Scale bar 100 nm. CCP = clathrin-coated profile, Mv = microvilli, PM = plasma membrane. The asterisks (*) in panel A indicate an endocytic process. White arrowheads indicate elongated tubular membranous structures connected to CCP. Boxed areas are magnified at bottom right corner of each figure panel. All values represent the mean and standard deviation of three replicas. Quantification of each treatment (EtOH vehicle ctrl or 4OH-TMX) includes over 120 viral particles. Statistical analysis was performed using ordinary two-way ANOVA multiple comparisons test (* p<0.05; *** p<0.001; **** p<0.0001). Scale bars = 5 μm.

Fig 5. ACE2-mediated endocytosis of SARS-CoV-2 trimeric spike proteins is dynamin-dependent.

A) Representative confocal fluorescence images of MEF^DKO cells non transfected, transiently expressing hACE2, or hACE2 and Rab5-EGFP. B) Quantification of the mean spike fluorescence per cell. A.u. = arbitrary units. C) Representative confocal fluorescence images of MEF^DKO cells transiently expressing hACE2 and Rab5-EGFP and treated with vehicle control or 4OH-TMX for 6 days and incubated for 3h with Alexa Fluor-555-labelled transferrin (Tf, magenta) and Alexa Fluor-647 labeled soluble trimeric SARS-CoV-2 Spike protein (red). The insets in A) and C) show magnified images from the indicated white boxed areas. The images represent a single optical slice of the imaged cell. B) Quantification of colocalization between indicated proteins in cells treated as described in C). The mean ±STDEV of 16 Ctrl cells and 14 4OH-TMX treated cells for 15 min; 16 Ctrl cells and 15 4OH-TMX cells for 45 min; and 17 Ctrl cells and 18 4OH-TMX cells for 3 h are shown. The statistical significance was calculated using a non parametric Mann-Whitney U test (****P<0,0001).

Fig 6. Dynamin-dependent and -independent entry of SARS-CoV-2 Wuh and Delta infection in ACE2-expressing MEF^TKO cells is actin dependent.

B) Representative confocal images of virus entry in MEF^TKOACE2 cells (4OH-TMX) or control cells (Ctrl) fixed at 60 min after virus inoculation and processed for sequential immunofluorescence as described in A. Inset images show a magnification of the area indicated by the white dashed boxes, with merged as well as separated fluorescence images. Virus out = non internalized viruses; virus in = internalized viruses. C) Quantification of non-internalized (virus out) and internalized (virus in) virions using automated image analysis. Values represent the mean of 15 cells from 3 independent experiments, and error bars represent the STDEV. Scale bars = 10 μm Statistical significance was calculated by unpaired two-tailed t-test (**p<0,01; n.s. = non-significant). D) Schematic description of the Lat-B treatment in MEF^TKOACE2. E) Representative fluorescence images of MEF^TKOACE2 treated with indicated compounds 15 minutes before infection and infected with Wuhan or the Delta variant of SARS-CoV-2 for 20 h at 6 days after vehicle control (Ctrl) or 4OH-TMX treatment. Scale bars = 200 μm F-H) Quantification by image analysis of SARS-COV-2 Wuhan or Delta infection in MEF^TKOACE2 cells treated with vehicle control (Ctrl) or 4OH-TMX for 6 days and infected with indicated MOIs for 20 h. Values indicate the mean of at least three independent experiments and the error bars represent the STDEV. Statistical significance was calculated using a non parametric Mann-Whitney U test (*p<0,05; **p<0,01; ***p<0,001).

Fig 7. Ultrastructural analysis of SARS-CoV-2 entry in MEF^TKOACE2 cells.

Representative TEM images of MEF^TKOACE2 cells treated with vehicle control (A-D) or 4OH-TMX (E-G, and D, 4OH-TMX) for 6 days and infected with SARS-CoV-2 Wuhan (MOI 100) on ice for 2 h followed by shift to 37°C for 15 min before fixation and processing for TEM. The fraction of total viral particles found in each of the described endocytic processes is quantified in H and I. Scale bar 100 nm. CCP = clathrin-coated profile, Mv = microvilli, PM = plasma membrane, asterisks (*) in panel A indicate an endocytic process. White arrowheads in panel A and E indicate electrondense filaments connecting the virions to the cell membrane. Boxed areas are magnified at bottom right corner of each figure panel. All values represent the mean and standard deviation of three replicas. Quantification of each treatment (EtOH vehicle ctrl or 4OH-TMX) includes 120 viral particles per condition. Statistical analysis was performed using ordinary two-way ANOVA multiple comparisons test (* p<0.05; ** p<0.01; ns = non significant).

Fig 8. TMPRSS2 expression bypasses the SARS-CoV-2 Wuhan but not Omicron lineage virus need for dynamin-dependent endocytosis and endosome maturation.

A-C) Image analysis quantification of the levels of SARS-CoV-2 Wuhan (A), Omicron B1.4 (B), and (C) Omicron Xbb1.5 infection following immunofluorescence detection of viral NP protein in MEF ^TKOACE2 cells. Cells were pre-treated with vehicle (Ctrl) or 4OH-TMX for 6 days and then further treated with DMSO or 1 μM Apilimod 30 min prior to infection. For each variant, the amount of virus was calibrated to infect approximately 20% of the Ctrl + DMSO treated cells at 20 hpi (here normalized to 1). D-F) The above mentioned viruses and drugs were used to infect MEF^TKOACE2 cells stably expressing low levels of TMPRSS2-GFP (MEF^TKOAT) as described for A-C. All values represent the mean and standard deviation of three independent experiments. Cells were infected using an equivalent MOI 3 (virus titers determined in VeroE6-TMPRSS2 cells). More than 10.000 cells were analysed for each experiment after high content imaging and automated image analysis. Statistical analysis was performed using ordinary two-way ANOVA multiple comparisons test (* p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001; ns = non significant).